The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production

PubMed Central

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-01-01

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased. PMID:25959098

The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production.

PubMed

Ju, Huiming; Zhang, Jiaqing; Bai, Lijing; Mu, Yulian; Du, Yutao; Yang, Wenxian; Li, Yong; Sheng, Anzhi; Li, Kui

2015-05-11

Sustained expression of the GH gene has been shown to have detrimental effects on the health of animals. In the current study, transgenic founder pigs, with controllable pig growth hormone (pGH) expression, were cloned via the handmade cloning method (HMC), and pGH expression levels were examined at the cellular and organismal levels. The serum pGH levels in 3 founder male pigs were found to be significantly higher after induction with intramuscular injection of doxycycline (DOX) compared to baseline. A daily dose of DOX was administered via feed to these animals for a period of 65 to 155 days. The growth rate, feed efficiency and pGH serum concentration increased in the DOX-induced transgenic group compared with the other groups. 8 numbers of animals were euthanized and the dressing percentage, loin muscle and lean meat percentage were significantly higher in the DOX-induced F1 transgenic group compared with the other groups. In this study a large population of transgenic pigs, with integrated controllable expression of a transgene, was obtained. The transgenic pigs were healthy and normal in terms of reproductive capability. At the same time, feed efficiency was improved, production processes were accelerated and meat yield was increased.

Oviduct-Specific Expression of Human Neutrophil Defensin 4 in Lentivirally Generated Transgenic Chickens

PubMed Central

Liu, Tongxin; Wu, Hanyu; Cao, Dainan; Li, Qingyuan; Zhang, Yaqiong; Li, Ning; Hu, Xiaoxiang

2015-01-01

The expression of oviduct-specific recombinant proteins in transgenic chickens is a promising technology for the production of therapeutic biologics in eggs. In this study, we constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. After the antimicrobial activity of the recombinant HNP4 protein was tested at the cellular level, a 2.8-kb ovalbumin promoter was used to drive HNP4 expression specifically in oviduct tissues. From 669 injected eggs, 218 chickens were successfully hatched. Ten G0 roosters, with semens identified as positive for the transgene, were mated with wild-type hens to generate G1 chickens. From 1,274 total offspring, fifteen G1 transgenic chickens were positive for the transgene, which was confirmed by PCR and Southern blotting. The results of the Southern blotting and genome walking indicated that a single copy of the HNP4 gene was integrated into chromosomes 1, 2, 3, 4, 6 and 24 of the chickens. As expected, HNP4 expression was restricted to the oviduct tissues, and the levels of both transcriptional and translational HNP4 expression varied greatly in transgenic chickens with different transgene insertion sites. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml. These results indicated that the production of transgenic chickens that expressed HNP4 protein in egg whites was successful. PMID:26020529

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1

PubMed Central

Ihemere, Uzoma E.; Narayanan, Narayanan N.; Sayre, Richard T.

2012-01-01

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants. PMID:22993514

Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

PubMed

Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun

2016-01-01

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

PubMed

Smith, A F; Bigsby, R M; Word, R A; Herring, B P

1998-05-01

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

PubMed

Tyč, Dimitrij; Nocarová, Eva; Sikorová, Lenka; Fischer, Lukáš

2017-08-01

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

PubMed

Li, Runzhi; Yu, Keshun; Wu, Yongmei; Tateno, Mizuki; Hatanaka, Tomoko; Hildebrand, David F

2012-01-01

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds. Copyright © 2011 Elsevier Inc. All rights reserved.

Expression of the G72/G30 gene in transgenic mice induces behavioral changes

PubMed Central

Cheng, Lijun; Hattori, Eiji; Nakajima, Akira; Woehrle, Nancy S.; Opal, Mark D.; Zhang, Chunling; Grennan, Kay; Dulawa, Stephanie C.; Tang, Ya-Ping; Gershon, Elliot S.; Liu, Chunyu

2012-01-01

The G72/G30 gene complex is a candidate gene for schizophrenia and bipolar disorder. However, G72 and G30 mRNAs are expressed at very low levels in human brain, with only rare splicing forms observed. We report here G72/G30 expression profiles and behavioral changes in a G72/G30 transgenic mouse model. A human BAC clone containing the G72/G30 genomic region was used to establish the transgenic mouse model, on which gene expression studies, Western blot and behavioral tests were performed. Relative to their minimal expression in humans, G72 and G30 mRNAs were highly expressed in the transgenic mice, and had a more complex splicing pattern. The highest G72 transcript levels were found in testis, followed by cerebral cortex, with very low or undetectable levels in other tissues. No LG72 (the long putative isoform of G72) protein was detected in the transgenic mice. Whole-genome expression profiling identified 361 genes differentially-expressed in transgenic mice compared to wild-type, including genes previously implicated in neurological and psychological disorders. Relative to wild-type mice, the transgenic mice exhibited fewer stereotypic movements in the open field test, higher baseline startle responses in the course of the prepulse inhibition test, and lower hedonic responses in the sucrose preference test. The transcriptome profile changes and multiple mouse behavioral effects suggest that the G72 gene may play a role in modulating behaviors relevant to psychiatric disorders. PMID:23337943

Production of Multiple Transgenic Yucatan Miniature Pigs Expressing Human Complement Regulatory Factors, Human CD55, CD59, and H-Transferase Genes

PubMed Central

Jang, Gun-Hyuk; Jeong, Yeun-Ik; Hwang, In-Sung; Jeong, Yeon-woo; Kim, Yu-Kyung; Shin, Taeyoung; Kim, Nam-Hyung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Hwang, Woo-Suk

2013-01-01

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications. PMID:23704897

The high-level expression of human tissue plasminogen activator in the milk of transgenic mice with hybrid gene locus strategy.

PubMed

Zhou, Yanrong; Lin, Yanli; Wu, Xiaojie; Xiong, Fuyin; Lv, Yuemeng; Zheng, Tao; Huang, Peitang; Chen, Hongxing

2012-02-01

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco.

PubMed

Cidade, Luciana C; de Oliveira, Tahise M; Mendes, Amanda F S; Macedo, Amanda F; Floh, Eny I S; Gesteira, Abelmon S; Soares-Filho, Walter S; Costa, Marcio G C

2012-12-01

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

A comprehensive glycome profiling of Huntington's disease transgenic mice.

PubMed

Gizaw, Solomon T; Koda, Toshiaki; Amano, Maho; Kamimura, Keiko; Ohashi, Tetsu; Hinou, Hiroshi; Nishimura, Shin-Ichiro

2015-09-01

Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. We estimated the structure and expression levels of 87 and 58N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. Total glycome expression levels are significantly different between HD transgenic and control group mice. Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era. Copyright © 2015 Elsevier B.V. All rights reserved.

An E8 promoter-HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin.

PubMed

Kurokawa, Natsuko; Hirai, Tadayoshi; Takayama, Mariko; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi

2013-04-01

The E8 promoter-HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8-MIR-HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30-630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8-MIR-HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113-124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.

Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice.

PubMed

Glasser, S W; Korfhagen, T R; Wert, S E; Bruno, M D; McWilliams, K M; Vorbroker, D K; Whitsett, J A

1991-10-01

Transgenic mice bearing chimeric genes consisting of 5'-sequences derived from the human surfactant protein C (SP-C) gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were generated. Analysis of CAT activity was utilized to demonstrate tissue-specific and developmental expression of chimeric genes containing 3.7 kb of sequences from the human SP-C gene. Lung-specific expression of the 3.7 SP-C-CAT transgene was observed in eight distinct transgenic mouse lines. Expression of the 3.7 SP-C-CAT transgene was first detected in fetal lung on day 11 of gestation and increased dramatically with advancing gestational age, reaching adult levels of activity before birth. In situ hybridization demonstrated that expression of 3.7 SP-C-CAT mRNA was confined to the distal respiratory epithelium. Antisense CAT hybridization was detected in bronchiolar and type II epithelial cells in the adult lung of the 3.7 SP-C-CAT transgenic mice. In situ hybridization of four distinct 3.7 SP-C-CAT transgenic mouse lines demonstrated bronchiolar-alveolar expression of the chimeric CAT gene, although the relative intensity of expression at each site varied within the lines studied. Glucocorticoids increased murine SP-C mRNA in fetal lung organ culture. Likewise, expression of 3.7 SP-C-CAT transgene increased during fetal lung organ or explant culture and was further enhanced by glucocorticoid in vitro. The 5'-regions of human SP-C conferred developmental, lung epithelial, and glucocorticoid-enhanced expression of bacterial CAT in transgenic mice. The increased expression of SP-C accompanying prenatal lung development and exposure to glucocorticoid is mediated, at least in part, at the transcriptional level, being influenced by cis-active elements contained within the 5'-flanking region of the human SP-C gene.

Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize.

PubMed

Johnson, Eric T; Berhow, Mark A; Dowd, Patrick F

2007-04-18

Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Effects of growth hormone over-expression on reproduction in the common carp Cyprinus carpio L.

PubMed

Cao, Mengxi; Chen, Ji; Peng, Wei; Wang, Yaping; Liao, Lanjie; Li, Yongming; Trudeau, Vance L; Zhu, Zuoyan; Hu, Wei

2014-01-01

To study the complex interaction between growth and reproduction we have established lines of transgenic common carp (Cyprinus carpio) carrying a grass carp (Ctenopharyngodon idellus) growth hormone (GH) transgene. The GH-transgenic fish showed delayed gonadal development compared with non-transgenic common carp. To gain a better understanding of the phenomenon, we studied body growth, gonad development, changes of reproduction related genes and hormones of GH-transgenic common carp for 2years. Over-expression of GH elevated peripheral gh transcription, serum GH levels, and inhibited endogenous GH expression in the pituitary. Hormone analyses indicated that GH-transgenic common carp had reduced pituitary and serum level of luteinizing hormone (LH). Among the tested genes, pituitary lhβ was inhibited in GH-transgenic fish. Further analyses in vitro showed that GH inhibited lhβ expression. Localization of ghr with LH indicates the possibility of direct regulation of GH on gonadotrophs. We also found that GH-transgenic common carp had reduced pituitary sensitivity to stimulation by co-treatments with a salmon gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. Together these results suggest that the main cause of delayed reproductive development in GH transgenic common carp is reduced LH production and release. Copyright © 2013 Elsevier Inc. All rights reserved.

Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

PubMed

Unzu, C; Melero, I; Hervás-Stubbs, S; Sampedro, A; Mancheño, U; Morales-Kastresana, A; Serrano-Mendioroz, I; de Salamanca, R E; Benito, A; Fontanellas, A

2015-11-01

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

PubMed

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Influence of human lactoferrin expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco.

PubMed

Kumar, Vinay; Gill, Tejpal; Grover, Sunita; Ahuja, Paramvir Singh; Yadav, Sudesh Kumar

2013-02-01

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms

PubMed Central

Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

2015-01-01

RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control. PMID:26435695

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit.

PubMed

Ruf, S; Hermann, M; Berger, I J; Carrer, H; Bock, R

2001-09-01

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.

PubMed

Markstein, Michele; Pitsouli, Chrysoula; Villalta, Christians; Celniker, Susan E; Perrimon, Norbert

2008-04-01

A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.

Molecular and functional characterization of cry1Ac transgenic pea lines.

PubMed

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-10-01

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Molecular and functional characterization of cry1Ac transgenic pea lines

PubMed Central

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-01-01

ABSTRACT Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses. PMID:27764552

A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters.

PubMed

Javaid, Shaista; Amin, Imran; Jander, Georg; Mukhtar, Zahid; Saeed, Nasir A; Mansoor, Shahid

2016-10-06

The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests.

Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in transgenic elite wheat (Triticum aestivum L.).

PubMed

Cong, Ling; Wang, Cheng; Chen, Ling; Liu, Huijuan; Yang, Guangxiao; He, Guangyuan

2009-09-23

Dietary micronutrient deficiencies, such as the lack of vitamin A, are a major source of morbidity and mortality worldwide. Carotenoids in food can function as provitamin A in humans, while grains of Chinese elite wheat cultivars generally have low carotenoid contents. To increase the carotenoid contents in common wheat endosperm, transgenic wheat has been generated by expressing the maize y1 gene encoding phytoene synthase driven by a endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with the bacterial phytoene desaturase crtI gene from Erwinia uredovora under the constitutive CaMV 35S promoter control. A clear increase of the carotenoid content was detected in the endosperms of transgenic wheat that visually showed a light yellow color. The total carotenoids content was increased up to 10.8-fold as compared with the nontransgenic EM12 cultivar. To test whether the variability of total carotenoid content in different transgenic lines was due to differences in the transgene copy number or expression pattern, Southern hybridization and semiquantitative reverse transcriptase polymerase chain reaction analyses were curried out. The results showed that transgene copy numbers and transcript levels did not associate well with carotenoid contents. The expression patterns of endogenous carotenoid genes, such as the phytoene synthases and carotene desaturases, were also investigated in wild-type and transgenic wheat lines. No significant changes in expression levels of these genes were detected in the transgenic endosperms, indicating that the increase in carotenoid transgenic wheat endosperms resulted from the expression of transgenes.

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

PubMed

Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

2015-01-01

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids.

PubMed

Yao, Jianhong; Pang, Yongzhen; Qi, Huaxiong; Wan, Bingliang; Zhao, Xiuyun; Kong, Weiwen; Sun, Xiaofen; Tang, Kexuan

2003-12-01

Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.

The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells.

PubMed

Nagaya, Shingo; Kawamura, Kazue; Shinmyo, Atsuhiko; Kato, Ko

2010-02-01

To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

PubMed

Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

2000-06-01

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

NASA Technical Reports Server (NTRS)

Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

1993-01-01

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

One- and two-photon states for quantum information

NASA Astrophysics Data System (ADS)

Peters, Nicholas A.

To find expression stability among transgenic lines, the Recombinase Mediated Transgene Integration (RMTI) technology using the Cre/ lox-mediated site-specific gene integration system was used. The objectives were to develop an efficient method of site-specific transgene integration and to test the effectiveness of this method by assaying transgene expression in the RMTI lines. The RMTI technology allows the precise integration of a transgene in a previously placed target genomic location containing a lox site. The efficiency of CRE-mediated site-specific integration in rice by particle bombardment was found to vary from 3 to 28% in nine different experiments. Some hemizygous site-specific integration plants that were derived from homozygous target locus were found to undergo CRE-mediated reversion of the integration locus. No reversion was observed in callus; however, reverting cells may have been excluded due to selection pressure. The expression of the transgene gus was studied in all 40 callus lines, 12 regenerated T0 plants and the T1 and T2 progenies of 5 lines. The isogenic SC lines had an average expression level based on the activity of beta-glucuronidase of 158 +/- 9 units/mg protein (mean +/- SEM; n=3; variance within SC lines are expressed as standard error of the mean SEM) indicating a significantly higher level of expression, as compared to MC lines that had a much lower expression level 44 +/- 8 units/mg protein (mean +/- SEM; n=3) and the imprecise lines that had 22 +/- 8 units/mg protein (mean +/- SEM; n=3). Transgene expression in the callus cells of precise single copy lines varied by ˜3 fold, whereas that in multi-copy lines varied by ˜30 fold. Furthermore, precise single copy lines, on an average, contained ˜3.5 fold higher expression than multi-copy lines. Transgene expression in the plants of precise single-copy lines was highly variable, which was found to be due to the loss of the integration because of CRE-mediated reversion in the locus. (Abstract shortened by UMI.)

Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects

PubMed Central

Laible, Götz; Smolenski, Grant; Wheeler, Thomas; Brophy, Brigid

2016-01-01

We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional β- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics. PMID:27876865

Pyramiding of transgenic Pm3 alleles in wheat results in improved powdery mildew resistance in the field.

PubMed

Koller, Teresa; Brunner, Susanne; Herren, Gerhard; Hurni, Severine; Keller, Beat

2018-04-01

The combined effects of enhanced total transgene expression level and allele-specificity combination in transgenic allele-pyramided Pm3 wheat lines result in improved powdery mildew field resistance without negative pleiotropic effects. Allelic Pm3 resistance genes of wheat confer race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and encode nucleotide-binding domain, leucine-rich repeat (NLR) receptors. Transgenic wheat lines overexpressing alleles Pm3a, b, c, d, f, and g have previously been generated by transformation of cultivar Bobwhite and tested in field trials, revealing varying degrees of powdery mildew resistance conferred by the transgenes. Here, we tested four transgenic lines each carrying two pyramided Pm3 alleles, which were generated by crossbreeding of lines transformed with single Pm3 alleles. All four allele-pyramided lines showed strongly improved powdery mildew resistance in the field compared to their parental lines. The improved resistance results from the two effects of enhanced total transgene expression levels and allele-specificity combinations. In contrast to leaf segment tests on greenhouse-grown seedlings, no allelic suppression was observed in the field. Plant development and yield scores of the pyramided lines were similar to the mean scores of the corresponding parental lines, and thus, the allele pyramiding did not cause any negative effects. On the contrary, in pyramided line, Pm3b × Pm3f normal plant development was restored compared to the delayed development and reduced seed set of parental line Pm3f. Allele-specific RT qPCR revealed additive transgene expression levels of the two Pm3 alleles in the pyramided lines. A positive correlation between total transgene expression level and powdery mildew field resistance was observed. In summary, allele pyramiding of Pm3 transgenes proved to be successful in enhancing powdery mildew field resistance.

Exogenous expression of Drp1 plays neuroprotective roles in the Alzheimer's disease in the Aβ42 transgenic Drosophila model.

PubMed

Lv, Fengshou; Yang, Xiaopeng; Cui, Chuanju; Su, Chunhe

2017-01-01

Alzheimer's disease (AD) is one of the most common neurodegenerative disorders. Recent studies have shown that mitochondrial dysfunction is a causative factor of AD. Drp1 (Dynamin-related protein 1), a regulator of mitochondrial fission, shows neuroprotective effects on Parkinson's disease. In this study, we investigate the effect and mechanism of Drp1 on Aβ42 transgenic Drosophila. Elav-gal4/UAS>Aβ42 transgenic Drosophila model was constructed using Elav-gal4 promoter. The effects of Drp1 on the lifespan, motor ability and neuronal degeneration of the transgenic Drosophila were explored by over-expressing Drp1 in the Aβ42 transgenic Drosophila. ATP levels in the brain tissues of Aβ42 transgenic Drosophila were detected using high performance liquid chromatography (HPLC). Exogenous expression of Drp1 promoted crawling ability, reduced the levels of ATP in Drosophila brain and suppressed the neuronal degeneration. The protective effect of Drp1 on the Aβ42 transgenic Drosophila was achieved by protecting the mitochondrial function, suggesting that Drp1 may be a potential therapeutic strategies for AD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, S.D.; Cooper, P.; Fung, J.

Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression ofmore » human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.« less

Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design.

PubMed

Kosovac, D; Wild, J; Ludwig, C; Meissner, S; Bauer, A P; Wagner, R

2011-02-01

Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA- and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3- to 4-fold increase of mEPO production sustained for >1 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPOΔCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2- to 3-fold increased (mEPOopt) and 50% decreased (mEPOΔCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses <0.5 μg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.

Overexpression of the A-FABP gene facilitates intermuscular fat deposition in transgenic mice.

PubMed

Liu, Z W; Fan, H L; Liu, X F; Ding, X B; Wang, T; Sui, G N; Li, G P; Guo, H

2015-03-31

Adipocyte fatty acid-binding protein (A-FABP), the most abundant FABP in adipocytes, controls fatty acid uptake, transport, and metabolism in fat cells. We constructed a transgenic mice model that overexpressed the cattle A-FABP gene to investigate the relationship between A-FABP expression and intermuscular fat deposition. There was no significant difference in body weight and serum biochemical indexes between transgenic and wild-type mice. Further, there were no significant differences in intermuscular triglyceride content and A-FABP expression levels over three generations of transgenic mice. However, abdominal adipose rate, A-FABP protein content, and intermuscular triglyceride levels of transgenic mice were significantly higher than those of wild-type mice. In addition, triglycerides were remarkably higher in the skeletal muscle but lower in the myocardium of transgenic mice. Thus, overexpression of cattle A-FABP gene promoted fat deposition in the skeletal muscle of transgenic mice.

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

PubMed

Clough, Richard C; Pappu, Kameshwari; Thompson, Kevin; Beifuss, Katherine; Lane, Jeff; Delaney, Donna E; Harkey, Robin; Drees, Carol; Howard, John A; Hood, Elizabeth E

2006-01-01

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations

PubMed Central

Wagaba, Henry; Beyene, Getu; Aleu, Jude; Odipio, John; Okao-Okuja, Geoffrey; Chauhan, Raj Deepika; Munga, Theresia; Obiero, Hannington; Halsey, Mark E.; Ilyas, Muhammad; Raymond, Peter; Bua, Anton; Taylor, Nigel J.; Miano, Douglas; Alicai, Titus

2017-01-01

Cassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96–100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle. PMID:28127301

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene.

PubMed

Panter, S; Chu, P G; Ludlow, E; Garrett, R; Kalla, R; Jahufer, M Z Z; de Lucas Arbiza, A; Rochfort, S; Mouradov, A; Smith, K F; Spangenberg, G

2012-06-01

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum.

PubMed

Han, Jigang; Lakshman, Dilip K; Galvez, Leny C; Mitra, Sharmila; Baenziger, Peter Stephen; Mitra, Amitava

2012-03-09

The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality. A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.

Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR.

PubMed

Zhu, Jin-Qi; Liu, Shumin; Ma, Yao; Zhang, Jia-Qi; Qi, Hai-Sheng; Wei, Zhao-Jun; Yao, Qiong; Zhang, Wen-Qing; Li, Sheng

2012-01-01

The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA) targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E), predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.

Transgenic nude mice ubiquitously expressing fluorescent proteins for color-coded imaging of the tumor microenvironment.

PubMed

Hoffman, Robert M

2014-01-01

We have developed a transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the β-actin promoter drives GFP expression in essentially all tissues. In the adult mice, many organs brightly expressed GFP, including the spleen, heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum as well as the circulatory system. The liver expressed GFP at a lesser level. The red fluorescent protein (RFP) transgenic nude mouse was obtained by crossing non-transgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives RFP (DsRed2) expression in essentially all tissues. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, liver, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. The cyan fluorescent protein (CFP) nude mouse was developed by crossing nontransgenic nude mice with the transgenic CK/ECFP mouse in which the β-actin promoter drives expression of CFP in almost all tissues. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescence signals of all internal organs, which vary in intensity. The GFP, RFP, and CFP nude mice when transplanted with cancer cells of another color are powerful models for color-coded imaging of the tumor microenvironment (TME) at the cellular level.

A transgenic approach to study argininosuccinate synthetase gene expression

PubMed Central

2014-01-01

Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. Conclusions We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene. PMID:24884799

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver.

PubMed

Adrian, G S; Rivera, E V; Adrian, E K; Lu, Y; Buchanan, J; Herbert, D C; Weaker, F J; Walter, C A; Bowman, B H

1993-01-01

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea1[W][OPEN

PubMed Central

Reinecke, Dennis M.; Wickramarathna, Aruna D.; Ozga, Jocelyn A.; Kurepin, Leonid V.; Jin, Alena L.; Good, Allen G.; Pharis, Richard P.

2013-01-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth. PMID:23979969

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

PubMed

Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

2013-10-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

Salicylate and catechol levels are maintained in nahG transgenic poplar

Treesearch

Alison M. Morse; Timothy J. Tschaplinski; Christopher Dervinis; Paula M. Pijut; Eric A. Schmelz; Wendy Day; John M. Davis

2007-01-01

Metabolic profiling was used to investigate the molecular phenotypes of a transgenic Populus tremula × P. alba hybrid expressing the nahG transgene, a bacterial gene encoding salicylate hydroxylase that converts salicylic acid to catechol. Despite the efficacy of this transgenic approach to reduce...

Production of transgenic pigs over-expressing the antiviral gene Mx1.

PubMed

Yan, Quanmei; Yang, Huaqiang; Yang, Dongshan; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Fan, Nana; Ouyang, Hongsheng; Gu, Weiwang; Lai, Liangxue

2014-01-01

The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype.

PubMed

Hasunuma, Tomohisa; Fukusaki, Ei-ichiro; Kobayashi, Akio

2004-08-05

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.

Polycythemia in transgenic mice expressing the human erythropoietin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semenza, G.L.; Traystman, M.D.; Gearhart, J.D.

1989-04-01

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5{prime} flanking sequence and 0.7 kilobase of 3{prime} flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver,more » adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.« less

α-Synuclein transgenic mice reveal compensatory increases in Parkinson's disease-associated proteins DJ-1 and parkin and have enhanced α-synuclein and PINK1 levels after rotenone treatment.

PubMed

George, Sonia; Mok, Su San; Nurjono, Milawaty; Ayton, Scott; Finkelstein, David I; Masters, Colin L; Li, Qiao-Xin; Culvenor, Janetta G

2010-10-01

Parkinson's disease (PD) is a severe neurodegenerative disorder characterised by loss of dopaminergic neurons of the substantia nigra. The pathological hallmarks are cytoplasmic inclusions termed Lewy bodies consisting primarily of aggregated alpha-synuclein (alphaSN). Different lines of transgenic mice have been developed to model PD but have failed to recapitulate the hallmarks of this disease. Since treatment of rodents with the pesticide rotenone can reproduce nigrostriatal cell loss and other features of PD, we aimed to test chronic oral administration of rotenone to transgenic mice over-expressing human alphaSN with the A53T mutation. Initial assessment of this transgenic line for compensatory molecular changes indicated decreased brain beta-synuclein expression and significantly increased levels of the PD-associated oxidative stress response protein, DJ-1, and the E3 ubiquitin ligase enzyme, Parkin. Rotenone treatment of 30 mg/kg for 25 doses over a 35-day period was tolerated in the transgenic mice and resulted in decreased spontaneous locomotor movement and increased cytoplasmic alphaSN expression. The mitochondrial Parkinson's-associated PTEN-induced kinase 1 protein levels were also increased in transgenic mouse brain after rotenone treatment; there was no change in brain dopamine levels or nigrostriatal cell loss. These hA53T alphaSN transgenic mice provide a useful model for presymptomatic Parkinson's features and are valuable for study of associated compensatory changes in early Parkinson's disease stages.

Expression of hsrω-RNAi transgene prior to heat shock specifically compromises accumulation of heat shock-induced Hsp70 in Drosophila melanogaster.

PubMed

Singh, Anand K; Lakhotia, Subhash C

2016-01-01

A delayed organismic lethality was reported in Drosophila following heat shock when developmentally active and stress-inducible noncoding hsrω-n transcripts were down-regulated during heat shock through hs-GAL4-driven expression of the hsrω-RNAi transgene, despite the characteristic elevation of all heat shock proteins (Hsp), including Hsp70. Here, we show that hsrω-RNAi transgene expression prior to heat shock singularly prevents accumulation of Hsp70 in all larval tissues without affecting transcriptional induction of hsp70 genes and stability of their transcripts. Absence of the stress-induced Hsp70 accumulation was not due to higher levels of Hsc70 in hsrω-RNAi transgene-expressing tissues. Inhibition of proteasomal activity during heat shock restored high levels of the induced Hsp70, suggesting very rapid degradation of the Hsp70 even during the stress when hsrω-RNAi transgene was expressed ahead of heat shock. Unexpectedly, while complete absence of hsrω transcripts in hsrω (66) homozygotes (hsrω-null) did not prevent high accumulation of heat shock-induced Hsp70, hsrω-RNAi transgene expression in hsrω-null background blocked Hsp70 accumulation. Nonspecific RNAi transgene expression did not affect Hsp70 induction. These observations reveal that, under certain conditions, the stress-induced Hsp70 can be selectively and rapidly targeted for proteasomal degradation even during heat shock. In the present case, the selective degradation of Hsp70 does not appear to be due to down-regulation of the hsrω-n transcripts per se; rather, this may be an indirect effect of the expression of hsrω-RNAi transgene whose RNA products may titrate away some RNA-binding proteins which may also be essential for stability of the induced Hsp70.

Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum

PubMed Central

2012-01-01

Background The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality. Results A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Conclusions Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum. PMID:22405032

Over-expression of bacterial gamma-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula x Populus alba) dependent on the resulting gamma-glutamylcysteine and glutathione levels.

PubMed

Herschbach, Cornelia; Rizzini, Luca; Mult, Susanne; Hartmann, Tanja; Busch, Florian; Peuke, Andreas D; Kopriva, Stanislav; Ensminger, Ingo

2010-07-01

We compared three transgenic poplar lines over-expressing the bacterial gamma-glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of gamma-glutamylcysteine (gamma-EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1-qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest gamma-EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (F(v)/F(m)) indicated down-regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light-harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole-plant sulphur nutrition are discussed for each line.

Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

DOEpatents

Coruzzi, Gloria [New York, NY; Gutierrez, Rodrigo A [Santiago, CL; Nero, Damion C [Woodside, NY

2012-04-10

Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

PubMed

Arsov, I; Li, X; Matthews, G; Coradin, J; Hartmann, B; Simon, A K; Sealfon, S C; Yue, Z

2008-09-01

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene

PubMed Central

Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C.; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K.

2014-01-01

Background Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Methodology/Principal Findings Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Conclusions/Significance Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops. PMID:24595215

Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

PubMed Central

Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

2011-01-01

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and in limiting Cd availability in the food chain. PMID:21283689

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

PubMed

Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

2000-08-01

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

Transgenic bovine as bioreactors: Challenges and perspectives

PubMed Central

Monzani, Paulo S.; Adona, Paulo R.; Ohashi, Otávio M.; Meirelles, Flávio V.; Wheeler, Matthew B.

2016-01-01

ABSTRACT The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry. PMID:27166649

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

A highly tunable system for the simultaneous expression of multiple enzymes in Saccharomyces cerevisiae.

PubMed

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Matsuyama, Takashi

2015-01-16

Control of the expression levels of multiple enzymes in transgenic yeasts is essential for the effective production of complex molecules through fermentation. Here, we propose a tunable strategy for the control of expression levels based on the design of terminator regions and other gene-expression control elements in Saccharomyces cerevisiae. Our genome-integrated system, which is capable of producing high expression levels over a wide dynamic range, will broadly enable metabolic engineering and synthetic biology. We demonstrated that the activities of multiple cellulases and the production of ethanol were doubled in a transgenic yeast constructed with our system compared with those achieved with a standard expression system.

High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

PubMed

Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

2006-07-13

Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease.

PubMed

Aronovich, Elena L; Hyland, Kendra A; Hall, Bryan C; Bell, Jason B; Olson, Erik R; Rusten, Myra Urness; Hunter, David W; Ellinwood, N Matthew; McIvor, R Scott; Hackett, Perry B

2017-07-01

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl 3 ) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl 3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl 3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl 3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

Discovery of Antibiotics-derived Polymers for Gene Delivery using Combinatorial Synthesis and Cheminformatics Modeling

PubMed Central

Potta, Thrimoorthy; Zhen, Zhuo; Grandhi, Taraka Sai Pavan; Christensen, Matthew D.; Ramos, James; Breneman, Curt M.; Rege, Kaushal

2014-01-01

We describe the combinatorial synthesis and cheminformatics modeling of aminoglycoside antibiotics-derived polymers for transgene delivery and expression. Fifty-six polymers were synthesized by polymerizing aminoglycosides with diglycidyl ether cross-linkers. Parallel screening resulted in identification of several lead polymers that resulted in high transgene expression levels in cells. The role of polymer physicochemical properties in determining efficacy of transgene expression was investigated using Quantitative Structure-Activity Relationship (QSAR) cheminformatics models based on Support Vector Regression (SVR) and ‘building block’ polymer structures. The QSAR model exhibited high predictive ability, and investigation of descriptors in the model, using molecular visualization and correlation plots, indicated that physicochemical attributes related to both, aminoglycosides and diglycidyl ethers facilitated transgene expression. This work synergistically combines combinatorial synthesis and parallel screening with cheminformatics-based QSAR models for discovery and physicochemical elucidation of effective antibiotics-derived polymers for transgene delivery in medicine and biotechnology. PMID:24331709

Recombinant avian adeno-associated virus: transgene expression in vivo and enhancement of expression in vitro.

PubMed

Estevez, Carlos; Villegas, Pedro

2006-06-01

Recombinant avian adeno-associated viruses coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at various time intervals in the embryos inoculated with the recombinant virus in comparison with the mock-inoculated controls. At the embryo level, significant differences were evident at 120 hr postinoculation; hatched chicks showed transgene expression up to 14 days of age. In a second experiment, different cell-line cultures were transfected with plasmids encoding for a reporter gene flanked by the avian adeno-associated virus inverted terminal repeats (ITR), either alone or in the presence of the major nonstructural proteins of the virus (Rep 78/68) to assess the ability of these proteins and DNA elements to enhance gene expression. Results indicate that the inclusion of the viral ITR alone or during coexpression of the Rep proteins significantly enhances the expression of the transgene in all cell lines tested, as evidenced by the detection of the beta-galacrosidase protein through chemiluminescence reactions and staining of transfected monolayers.

Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility

DOE PAGES

Baxter, Holly L.; Mazarei, Mitra; Dumitrache, Alexandru; ...

2017-04-24

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156more » overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.« less

Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

PubMed Central

Spencer, M. J.; Guyon, J. R.; Sorimachi, H.; Potts, A.; Richard, I.; Herasse, M.; Chamberlain, J.; Dalkilic, I.; Kunkel, L. M.; Beckmann, J. S.

2002-01-01

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development. PMID:12084932

Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baxter, Holly L.; Mazarei, Mitra; Dumitrache, Alexandru

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156more » overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.« less

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

PubMed Central

Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J.; Kim, Hyunil; Surh, Charles D.; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation. PMID:23071605

Generation and characterization of human heme oxygenase-1 transgenic pigs.

PubMed

Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

PubMed

Buzina, Alla; Lo, Mandy Y M; Moffett, Angela; Hotta, Akitsu; Fussner, Eden; Bharadwaj, Rikki R; Pasceri, Peter; Garcia-Martinez, J Victor; Bazett-Jones, David P; Ellis, James

2008-04-11

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

Transgenic cells with increased plastoquinone levels and methods of use

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector

PubMed Central

Werner, Stefan; Breus, Oksana; Symonenko, Yuri; Marillonnet, Sylvestre; Gleba, Yuri

2011-01-01

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 104-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing. PMID:21825158

Transformation of an edible crop with the pagA gene of Bacillus anthracis.

PubMed

Aziz, Mohammad Azhar; Sikriwal, Deepa; Singh, Samer; Jarugula, Sridhar; Kumar, P Anand; Bhatnagar, Rakesh

2005-09-01

Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here.

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

PubMed Central

Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

2001-01-01

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

PubMed Central

Kakitani, Makoto; Oshima, Takeshi; Horikoshi, Kaori; Yoshitome, Tetsuo; Ueda, Akiko; Kajikawa, Miwa; Iba, Yumi; Ozone, Yoshinao; Ijima, Yuki; Yoshino, Tohko; Itoh, Mikiko; Seki, Sachiko; Aoki, Ayako; Ishihara, Toshie; Shionoya, Michiyo; Makino, Utako; Kitada, Rina; Ohguma, Atsuko; Ohta, Takami; Yoshida, Yoshimasa; Kudoh, Hiroe; Hanaoka, Kazunori; Sibuya, Kazunori; Ishida, Isao; Kakeda, Minoru; Yagi, Mikio; Yoneya, Takashi; Tomizuka, Kazuma

2005-01-01

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras. PMID:15914664

Field performance of transgenic citrus trees: assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics.

PubMed

Pons, Elsa; Peris, Josep E; Peña, Leandro

2012-07-15

The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.

Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

PubMed Central

2012-01-01

Background The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. Results The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Conclusions Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest. PMID:22794278

Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice.

PubMed Central

Morello, D; Moore, G; Salmon, A M; Yaniv, M; Babinet, C

1986-01-01

Transgenic mice carrying the H-2K/human growth hormone (hGH) fusion gene were produced by microinjecting into the pronucleus of fertilized eggs DNA molecules containing 2 kb of the 5' flanking sequences (including promoter) of the class I H-2Kb gene joined to the coding sequences of the hGH gene. Thirteen transgenic mice were obtained which all contained detectable levels of hGH hormone in their blood. Nine grew larger than their control litter-mates. Endogenous H-2Kb and exogenous hGH mRNA levels were analysed by S1 nuclease digestion experiments. hGH transcripts were found in all the tissues examined and the pattern of expression paralleled that of endogenous H-2K gene expression, being high in liver and lymphoid organs and low in muscle and brain. Thus 2 kb of the 5' promoter/regulatory region of the H-2K gene are sufficient to ensure regulated expression of hGH in transgenic mice. This promoter may therefore be of use to target the expression of different exogenous genes in most tissues of transgenic mice and to study the biological role of the corresponding proteins in different cellular environments. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3019667

Forebrain-specific expression of monoamine oxidase A reduces neurotransmitter levels, restores the brain structure, and rescues aggressive behavior in monoamine oxidase A-deficient mice.

PubMed

Chen, Kevin; Cases, Olivier; Rebrin, Igor; Wu, Weihua; Gallaher, Timothy K; Seif, Isabelle; Shih, Jean Chen

2007-01-05

Previous studies have established that abrogation of monoamine oxidase (MAO) A expression leads to a neurochemical, morphological, and behavioral specific phenotype with increased levels of serotonin (5-HT), norepinephrine, and dopamine, loss of barrel field structure in mouse somatosensory cortex, and an association with increased aggression in adults. Forebrain-specific MAO A transgenic mice were generated from MAO A knock-out (KO) mice by using the promoter of calcium-dependent kinase IIalpha (CaMKIIalpha). The presence of human MAO A transgene and its expression were verified by PCR of genomic DNA and reverse transcription-PCR of mRNA and Western blot, respectively. Significant MAO A catalytic activity, autoradiographic labeling of 5-HT, and immunocytochemistry of MAO A were found in the frontal cortex, striatum, and hippocampus but not in the cerebellum of the forebrain transgenic mice. Also, compared with MAO A KO mice, lower levels of 5-HT, norepinephrine, and DA and higher levels of MAO A metabolite 5-hydroxyindoleacetic acid were found in the forebrain regions but not in the cerebellum of the transgenic mice. These results suggest that MAO A is specifically expressed in the forebrain regions of transgenic mice. This forebrain-specific differential expression resulted in abrogation of the aggressive phenotype. Furthermore, the disorganization of the somatosensory cortex barrel field structure associated with MAO A KO mice was restored and became morphologically similar to wild type. Thus, the lack of MAO A in the forebrain of MAO A KO mice may underlie their phenotypes.

The dynamics of long-term transgene expression in engrafted neural stem cells.

PubMed

Lee, Jean-Pyo; Tsai, David J; In Park, Kook; Harvey, Alan R; Snyder, Evan Y

2009-07-01

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages. Copyright 2009 Wiley-Liss, Inc.

Markedly Increased Susceptibility to Natural Sheep Scrapie of Transgenic Mice Expressing Ovine PrP

PubMed Central

Vilotte, Jean-Luc; Soulier, Solange; Essalmani, Rachid; Stinnakre, Marie-George; Vaiman, Daniel; Lepourry, Laurence; Da Silva, Jose Costa; Besnard, Nathalie; Dawson, Mike; Buschmann, Anne; Groschup, Martin; Petit, Stephanie; Madelaine, Marie-Francoise; Rakatobe, Sabine; Le Dur, Annick; Vilette, Didier; Laude, Hubert

2001-01-01

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrPVRQ-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrPVRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie. PMID:11390599

Breakthrough in chloroplast genetic engineering of agronomically important crops

PubMed Central

Daniell, Henry; Kumar, Shashi; Dufourmantel, Nathalie

2012-01-01

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development. PMID:15866001

Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection.

PubMed

Lipsky, Alexander; Joshi, Janak Raj; Carmi, Nir; Yedidia, Iris

2016-11-20

The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95-100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacteriophage 5' untranslated regions for control of plastid transgene expression.

PubMed

Yang, Huijun; Gray, Benjamin N; Ahner, Beth A; Hanson, Maureen R

2013-02-01

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5' untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5' UTRs and the bacteriophage T7 gene 10 (T7g10) 5' UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5' UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3'-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5' UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5' UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5' UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5' UTR. However, transplastomic plants with the bacteriophage 5' UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5' UTR. Furthermore, expression of the T7g1.3 and T4g23 5' UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

Regulation of expression of transgenes in developing fish.

PubMed

Moav, B; Liu, Z; Caldovic, L D; Gross, M L; Faras, A J; Hackett, P B

1993-05-01

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds.

PubMed

Tan, Helin; Yang, Xiaohui; Zhang, Fengxia; Zheng, Xiu; Qu, Cunmin; Mu, Jinye; Fu, Fuyou; Li, Jiana; Guan, Rongzhan; Zhang, Hongsheng; Wang, Guodong; Zuo, Jianru

2011-07-01

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

PubMed

Ghosh, Gourab; Ganguly, Shreeparna; Purohit, Arnab; Chaudhuri, Rituparna Kundu; Das, Sampa; Chakraborti, Dipankar

2017-07-01

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T 0 transformants were selected on the basis of PCR and protein expression profile. T 1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T 1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T 2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T 3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T 0 , T 1, and T 2 ). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase.

PubMed

Ruiz, Oscar N; Alvarez, Derry; Gonzalez-Ruiz, Gloriene; Torres, Cesar

2011-08-12

The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

PubMed Central

2011-01-01

Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. Results In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. Conclusion The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications. PMID:21838857

Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

PubMed

Hawley, Catherine A; Rojo, Rocio; Raper, Anna; Sauter, Kristin A; Lisowski, Zofia M; Grabert, Kathleen; Bain, Calum C; Davis, Gemma M; Louwe, Pieter A; Ostrowski, Michael C; Hume, David A; Pridans, Clare; Jenkins, Stephen J

2018-03-15

CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r -mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Δ Csf1- enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r- driven reporter lines, Csf1r -mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Δ Csf1r -ECFP- Csf1r -mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r- mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines. Copyright © 2018 The Authors.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice

NASA Technical Reports Server (NTRS)

Bedalov, A.; Salvatori, R.; Dodig, M.; Kronenberg, M. S.; Kapural, B.; Bogdanovic, Z.; Kream, B. E.; Woody, C. O.; Clark, S. H.; Mack, K.;

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs.

PubMed

Bähr, Andrea; Käser, Tobias; Kemter, Elisabeth; Gerner, Wilhelm; Kurome, Mayuko; Baars, Wiebke; Herbach, Nadja; Witter, Kirsti; Wünsch, Annegret; Talker, Stephanie C; Kessler, Barbara; Nagashima, Hiroshi; Saalmüller, Armin; Schwinzer, Reinhard; Wolf, Eckhard; Klymiuk, Nikolai

2016-01-01

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

PubMed

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

2016-11-17

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

PubMed Central

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

2016-01-01

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

Overexpression of porcine lipoprotein-associated phospholipase A2 in swine.

PubMed

Tang, Xiaochun; Wang, Gangqi; Liu, Xingxing; Han, Xiaolei; Li, Zhuang; Ran, Guangyao; Li, Zhanjun; Song, Qi; Ji, Yuan; Wang, Haijun; Wang, Yuhui; Ouyang, Hongsheng; Pang, Daxin

2015-09-25

Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is associated with the risk of vascular disease. It circulates in human blood predominantly in association with low-density lipoprotein cholesterol (LDL-C) and hydrolyses oxidized phospholipids into pro-inflammatory products. However, in the mouse circulation, it predominantly binds to high-density lipoprotein cholesterol (HDL-C) and exhibits anti-inflammatory properties. To further investigate the effects of Lp-PLA2 in the circulation, we generated over-expressed Lp-PLA2 transgenic swine. The eukaryotic expression plasmid of porcine Lp-PLA2 which driven by EF1α promoter was constructed and generate transgenic swine via SCNT. The expression and activity of Lp-PLA2 in transgenic swine were evaluated, and the total cholesterol (TC), HDL-C, LDL-C and triglyceride (TG) levels in the fasting and fed states were also assessed. Compared with wild-type swine controls, the transgenic swine exhibited elevated Lp-PLA2 mRNA levels and activities, and the activity did not depend on the feeding state. The TC, HDL-C and LDL-C levels were not significantly increased. There was no change in the TG levels in the fasting state between transgenic and control pigs. However, in the fed state, the TG levels of transgenic swine were slightly increased compared with the control pigs and were significantly elevated compared with the fasting state. In addition, inflammatory gene (interleukin [IL]-6, monocyte chemotactic protein [MCP]-1 and tumor necrosis factor [TNF]-α) mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly increased. The results demonstrated that Lp-PLA2 is associated with triglycerides which may be helpful for understanding the relationship of this protein with cardiovascular disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Transgenic Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) Overexpressing S-Adenosylmethionine Decarboxylase (SAMDC) Gene for Improved Cold Tolerance Through Involvement of H2O2 and NO Signaling.

PubMed

Luo, Jianhao; Liu, Mingxi; Zhang, Chendong; Zhang, Peipei; Chen, Jingjing; Guo, Zhenfei; Lu, Shaoyun

2017-01-01

Centipedegrass ( Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass species. Transgenic centipedgrass plants overexpressing S-adenosylmethionine decarboxylase from bermudagrass ( CdSAMDC1 ) that was induced in response to cold were generated in this study. Higher levels of CdSAMDC1 transcript and sperimidine (Spd) and spermin (Spm) concentrations and enhanced freezing and chilling tolerance were observed in transgenic plants as compared with the wild type (WT). Transgenic plants had higher levels of polyamine oxidase (PAO) activity and H 2 O 2 than WT, which were blocked by pretreatment with methylglyoxal bis (guanylhydrazone) or MGBG, inhibitor of SAMDC, indicating that the increased PAO and H 2 O 2 were a result of expression of CdSAMDC1 . In addition, transgenic plants had higher levels of nitrate reductase (NR) activity and nitric oxide (NO) concentration. The increased NR activity were blocked by pretreatment with MGBG and ascorbic acid (AsA), scavenger of H 2 O 2 , while the increased NO level was blocked by MGBG, AsA, and inhibitors of NR, indicating that the enhanced NR-derived NO was dependent upon H 2 O 2 , as a result of expression CdSAMDC1 . Elevated superoxide dismutase (SOD) and catalase (CAT) activities were observed in transgenic plants than in WT, which were blocked by pretreatment with MGBG, AsA, inhibitors of NR and scavenger of NO, indicating that the increased activities of SOD and CAT depends on expression of CdSAMDC1 , H 2 O 2 , and NR-derived NO. Our results suggest that the elevated cold tolerance was associated with PAO catalyzed production of H 2 O 2 , which in turn led to NR-derived NO production and induced antioxidant enzyme activities in transgenic plants.

Regulated expression of the Ren-2 gene in transgenic mice derived from parental strains carrying only the Ren-1 gene.

PubMed Central

Tronik, D; Dreyfus, M; Babinet, C; Rougeon, F

1987-01-01

The Ren-2 gene encoding the mouse submaxillary gland (SMG) renin was microinjected into the pronuclei of fertilized eggs from mice carrying only the Ren-1 gene. In addition to the whole transcription unit, the injected DNA contained 2.5 and 3 kb of upstream and downstream flanking sequences, respectively. Three independent transgenic mice lines were obtained; two of them had integrated one copy of the Ren-2 gene, the last one had integrated five and eleven copies at two independent sites. Independently of the number of Ren-2 copies integrated, the pattern of Ren-2 gene expression in all the transgenic mice was identical to that observed in wild-type animals in which Ren-1 and Ren-2 are closely linked on chromosome 1. In particular, the exogenous Ren-2 gene was only transcribed in the kidney and in the SMG. In the kidney, Ren-1 and Ren-2 mRNAs were present at a comparable level, whereas in the SMG Ren-2 mRNA was at least 100-fold more abundant than Ren-1 mRNA. Moreover, Ren-2 expression in the SMG was positively regulated by androgens. Only one difference between transgenic mice and wild-type mice carrying the Ren-2 gene has been observed: the basal level of Ren-2 transcription in the SMG of transgenic females was lower than in two-gene strain females. Androgen treatment of transgenic females induced SMG renin mRNA to a level identical to that of transgenic males. This suggests that the basal level of SMG renin mRNA is dependent upon cis-acting elements which are not present in the microinjected fragment. Images Fig. 1. Fig. 2. Fig. 3. PMID:3297677

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans.

PubMed

Grishok, Alla; Sinskey, Jina L; Sharp, Phillip A

2005-03-15

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

High-efficiency Agrobacterium rhizogenes-mediated transformation of heat inducible sHSP18.2-GUS in Nicotiana tabacum.

PubMed

Chen, Shih-Cheng; Liu, Hui-Wen; Lee, Kung-Ta; Yamakawa, Takashi

2007-01-01

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 microM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42 degrees C heat treatment, and the expressed GUS specific activity was 7-26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.

Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

USDA-ARS?s Scientific Manuscript database

Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, w...

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

PubMed Central

Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

2017-01-01

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

Over-expression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase 1 (hmgr1) gene under super-promoter for enhanced latex biosynthesis in rubber tree (Hevea brasiliensis Muell. Arg.).

PubMed

Jayashree, R; Nazeem, P A; Rekha, K; Sreelatha, S; Thulaseedharan, A; Krishnakumar, R; Kala, R G; Vineetha, M; Leda, P; Jinu, U; Venkatachalam, P

2018-06-01

Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8 kb hmgr1 and 0.6 kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Expansion of the gateway multisite recombination cloning toolkit.

PubMed

Shearin, Harold K; Dvarishkis, Alisa R; Kozeluh, Craig D; Stowers, R Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

Expansion of the Gateway MultiSite Recombination Cloning Toolkit

PubMed Central

Shearin, Harold K.; Dvarishkis, Alisa R.; Kozeluh, Craig D.; Stowers, R. Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters. PMID:24204935

The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo.

PubMed

Ochiai, Hiroshi; Sakamoto, Naoaki; Suzuki, Kenichi; Akasaka, Koji; Yamamoto, Takashi

2008-09-01

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

PubMed

Narvaez, Isabel; Khayreddine, Titouh; Pliego, Clara; Cerezo, Sergio; Jiménez-Díaz, Rafael M; Trapero-Casas, José L; López-Herrera, Carlos; Arjona-Girona, Isabel; Martín, Carmen; Mercado, José A; Pliego-Alfaro, Fernando

2018-01-01

The antifungal protein (AFP) produced by Aspergillus giganteus , encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix , was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae .

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

PubMed Central

Soares, V da C; Gubits, R M; Feigelson, P; Costantini, F

1987-01-01

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene. Images PMID:2446121

Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

2015-10-01

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity.

PubMed

Tomlinson, Elizabeth; Fu, Ling; John, Linu; Hultgren, Bruce; Huang, Xiaojian; Renz, Mark; Stephan, Jean Philippe; Tsai, Saio Ping; Powell-Braxton, Lyn; French, Dorothy; Stewart, Timothy A

2002-05-01

The fibroblast growth factors (FGFs), and the corresponding receptors, are implicated in more than just the regulation of epithelial cell proliferation and differentiation. Specifically, FGF23 is a regulator of serum inorganic phosphate levels, and mice deficient in FGF receptor-4 have altered cholesterol metabolism. The recently described FGF19 is unusual in that it is nonmitogenic and appears to interact only with FGF receptor-4. Here, we report that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure. Further, the FGF19 transgenic mice did not become obese or diabetic on a high fat diet. The FGF19 transgenic mice had increased brown adipose tissue mass and decreased liver expression of acetyl coenzyme A carboxylase 2, providing two mechanisms by which FGF19 may increase energy expenditure. Consistent with the reduction in expression of acetyl CoA carboxylase 2, liver triglyceride levels were reduced.

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

PubMed

Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

2006-10-01

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

Repression of gibberellin biosynthesis or signaling produces striking alterations in poplar growth, morphology, and flowering.

PubMed

Zawaski, Christine; Kadmiel, Mahita; Pickens, Jim; Ma, Cathleen; Strauss, Steven; Busov, Victor

2011-12-01

We modified gibberellin (GA) metabolism and signaling in transgenic poplars using dominant transgenes and studied their effects for 3 years under field conditions. The transgenes that we employed either reduced the bioactive GAs, or attenuated their signaling. The majority of transgenic trees had significant and in many cases dramatic changes in height, crown architecture, foliage morphology, flowering onset, floral structure, and vegetative phenology. Most transgenes elicited various levels of height reduction consistent with the roles of GA in elongation growth. Several other growth traits were proportionally reduced, including branch length, internode distance, and leaf length. In contrast to elongation growth, stem diameter growth was much less affected, suggesting that semi-dwarf trees in dense stands might provide high levels of biomass production and carbon sequestration. The severity of phenotypic effects was strongly correlated with transgene expression among independent transgenic events, but often in a non-linear manner, the form of which varied widely among constructs. The majority of semi-dwarfed, transgenic plants showed delayed bud flush and early bud set, and expression of a native GAI transgene accelerated first time flowering in the field. All of the phenotypic changes observed in multiple years were stable over the 3 years of field study. Our results suggest that transgenic modification of GA action may be useful for producing semi-dwarf trees with modified growth and morphology for horticulture and other uses.

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

PubMed

Gothilf, Yoav; Toyama, Reiko; Coon, Steven L; Du, Shao-Jun; Dawid, Igor B; Klein, David C

2002-11-01

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

Heterologous expression of key C and N metabolic enzymes improves re-assimilation of photorespired CO2 and NH3, and growth.

PubMed

Kaachra, Anish; Vats, Surender Kumar; Kumar, Sanjay

2018-06-11

We investigated the effect of heterologous expression of phosphoenolpyruvate carboxylase (ZmPepcase), aspartate aminotransferase (GmAspAT), and glutamine synthetase (NtGS) on carbon (C) and nitrogen (N) metabolism in Arabidopsis (Arabidopsis thaliana). These transgenes were expressed either separately or in different combinations. The highest gains in shoot dry weight were observed in transgenic lines co-expressing all three genes. Tracer experiments using NaH14CO3 suggested that the co-expression of ZmPepcase, GmAspAT,and NtGS resulted in a higher flux of assimilated CO2 towards sugars and amino acids. Upon feeding the leaf discs with glycine-1-14C, transgenic lines evolved significantly lower 14CO2 levels than the WT, suggesting a higher re-assimilation of CO2 evolved during photorespiration. Leaves of transgenic plants accumulated significantly lower ammonium without any significant difference in the levels of photorespiratory ammonium relative to the WT, suggesting higher re-assimilation of photorespired NH3. Transgenic lines also showed improved photosynthetic rates, higher shoot biomass accumulation, and improved seed yield in comparison to WT plants under both optimum and limiting N conditions. The present work demonstrates that the heterologous co-expression of ZmPepcase, GmAspAT, and NtGS reduced the photorespiratory loss of C and N with concomitant enhancements in shoot biomass and seed yield. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.

PubMed Central

Kwan, H; Pecenka, V; Tsukamoto, A; Parslow, T G; Guzman, R; Lin, T P; Muller, W J; Lee, F S; Leder, P; Varmus, H E

1992-01-01

The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene. Images PMID:1530875

Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA.

PubMed

Carlson, Bradley A; Xu, Xue-Ming; Gladyshev, Vadim N; Hatfield, Dolph L

2005-02-18

Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2'-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N(6)-isopentenyladenosine at position 37 (i(6)A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i(6)A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

PubMed

Qin, Lina; Jiang, Xianzhang; Dong, Zhiyang; Huang, Jianzhong; Chen, Xiuzhen

2018-01-01

The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei . Currently, only the locus of exocellobiohydrolases I encoding gene ( cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei . Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Age and lesion-induced increases of GDNF transgene expression in brain following intracerebral injections of DNA nanoparticles.

PubMed

Yurek, D M; Hasselrot, U; Cass, W A; Sesenoglu-Laird, O; Padegimas, L; Cooper, M J

2015-01-22

In previous studies that used compacted DNA nanoparticles (DNP) to transfect cells in the brain, we observed higher transgene expression in the denervated striatum when compared to transgene expression in the intact striatum. We also observed that long-term transgene expression occurred in astrocytes as well as neurons. Based on these findings, we hypothesized that the higher transgene expression observed in the denervated striatum may be a function of increased gliosis. Several aging studies have also reported an increase of gliosis as a function of normal aging. In this study we used DNPs that encoded for human glial cell line-derived neurotrophic factor (hGDNF) and either a non-specific human polyubiquitin C (UbC) or an astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The DNPs were injected intracerebrally into the denervated or intact striatum of young, middle-aged or aged rats, and glial cell line-derived neurotrophic factor (GDNF) transgene expression was subsequently quantified in brain tissue samples. The results of our studies confirmed our earlier finding that transgene expression was higher in the denervated striatum when compared to intact striatum for DNPs incorporating either promoter. In addition, we observed significantly higher transgene expression in the denervated striatum of old rats when compared to young rats following injections of both types of DNPs. Stereological analysis of GFAP+ cells in the striatum confirmed an increase of GFAP+ cells in the denervated striatum when compared to the intact striatum and also an age-related increase; importantly, increases in GFAP+ cells closely matched the increases in GDNF transgene levels. Thus neurodegeneration and aging may lay a foundation that is actually beneficial for this particular type of gene therapy while other gene therapy techniques that target neurons are actually targeting cells that are decreasing as the disease progresses. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult.

PubMed

Rodgers, Shaefali P; Born, Heather A; Das, Pritam; Jankowsky, Joanna L

2012-06-18

Transgenic mice expressing disease-associated proteins have become standard tools for studying human neurological disorders. Transgenes are often expressed using promoters chosen to drive continuous high-level expression throughout life rather than temporal and spatial fidelity to the endogenous gene. This approach has allowed us to recapitulate diseases of aging within the two-year lifespan of the laboratory mouse, but has the potential for creating aberrant phenotypes by mechanisms unrelated to the human disorder. We show that overexpression of the Alzheimer's-related amyloid precursor protein (APP) during early postnatal development leads to severe locomotor hyperactivity that can be significantly attenuated by delaying transgene onset until adulthood. Our data suggest that exposure to transgenic APP during maturation influences the development of neuronal circuits controlling motor activity. Both when matched for total duration of APP overexpression and when matched for cortical amyloid burden, animals exposed to transgenic APP as juveniles are more active in locomotor assays than animals in which APP overexpression was delayed until adulthood. In contrast to motor activity, the age of APP onset had no effect on thigmotaxis in the open field as a rough measure of anxiety, suggesting that the interaction between APP overexpression and brain development is not unilateral. Our findings indicate that locomotor hyperactivity displayed by the tet-off APP transgenic mice and several other transgenic models of Alzheimer's disease may result from overexpression of mutant APP during postnatal brain development. Our results serve as a reminder of the potential for unexpected interactions between foreign transgenes and brain development to cause long-lasting effects on neuronal function in the adult. The tet-off APP model provides an easy means of avoiding developmental confounds by allowing transgene expression to be delayed until the mice reach adulthood.

Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc12

PubMed Central

Ray, Heather; Yu, Min; Auser, Patricia; Blahut-Beatty, Laureen; McKersie, Brian; Bowley, Steve; Westcott, Neil; Coulman, Bruce; Lloyd, Alan; Gruber, Margaret Y.

2003-01-01

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5′-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5′-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass. PMID:12857826

Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp.] plants.

PubMed

Satyavathi, V V; Prasad, V; Khandelwal, Abha; Shaila, M S; Sita, G Lakshmi

2003-03-01

Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia. The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination. This paper reports the development of transgenic pigeon pea [ Cajanus cajun (L.) Millsp.] expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus. A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciensstrain EHA105. Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation. The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction. Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization. The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein. The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein. The transgenic plants were fertile and the transgene expressed in the progeny.

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.

PubMed

Squire, S; Raymackers, J M; Vandebrouck, C; Potter, A; Tinsley, J; Fisher, R; Gillis, J M; Davies, K E

2002-12-15

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer's disease.

PubMed

Youm, Jung Won; Jeon, Jae Heung; Kim, Hee; Kim, Young Ho; Ko, Kisung; Joung, Hyouk; Kim, Hyunsoon

2008-10-01

Human beta-amyloid (Abeta) is believed to be one of the main components of Alzheimer's disease, so reduction of Abeta is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Abeta with tandem repeats. Integration of the human Abeta gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Abeta protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Abeta antigen.

FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa.

PubMed

Shimada, Takashi; Urakawa, Itaru; Yamazaki, Yuji; Hasegawa, Hisashi; Hino, Rieko; Yoneya, Takashi; Takeuchi, Yasuhiro; Fujita, Toshiro; Fukumoto, Seiji; Yamashita, Takeyoshi

2004-02-06

Fibroblast growth factor (FGF)-23 was identified as a causative factor of tumor-induced osteomalacia and also as a responsible gene for autosomal dominant hypophosphatemic rickets. To clarify the pathophysiological roles of FGF-23 in these diseases, we generated its transgenic mice. The transgenic mice expressing human FGF-23 reproduced the common clinical features of these diseases such as hypophosphatemia probably due to increased renal phosphate wasting, inappropriately low serum 1,25-dihydroxyvitamin D level, and rachitic bone. The renal phosphate wasting in the transgenic mice was accompanied by the reduced expression of sodium phosphate cotransporter type IIa in renal proximal tubules. These results reinforce the notion that the excessive action of FGF-23 plays a causative role in the development of several hypophosphatemic rickets/osteomalacia.

Degeneration of oxidative muscle fibers in HTLV-1 tax transgenic mice.

PubMed

Nerenberg, M I; Wiley, C A

1989-12-01

The HTLV-1 tax gene under control of the HTLV-1 long terminal repeat (LTR) was introduced into transgenic mice. Previously tax protein expression in the muscle and peripheral nerves of three independent mouse lines was reported. Here the localization of this transgenic protein at a cellular and subcellular level is described. Tax protein was expressed in oxidative muscle fibers that developed severe progressive atrophy. It localized to the cytoplasma where it was associated with structures resembling degenerating Z bands. This pattern of muscle fiber involvement is similar to that observed in human retroviral associated myopathy. This transgenic mouse model suggests that preferential expression of the HTLV-1 viral promoter in oxidative muscle fibers may explain the productive infection of these fibers in HTLV-1 myopathy.

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

PubMed

Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

2016-08-01

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

Transgenic soybean plants expressing Spb18S dsRNA exhibit enhanced resistance to the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae).

PubMed

Wang, Zhanchun; Li, Tianyu; Ni, Hejia; Wang, Guoyue; Liu, Xinxin; Cao, Yingxue; Li, Wenbin; Meng, Fanli

2018-06-01

The soybean pod borer [SPB; Leguminivora glycinivorella (Mats.) Obraztsov] is a major soybean pest in northeastern Asia. A useful method for addressing this problem is the generation of transgenic plants producing double-stranded RNA (dsRNA) that target essential insect genes. In this study, we confirmed that 18S ribosomal RNA is critical for SPB development. Downregulated Spb18S expression induced by dsRNA injection increased larval mortality rates and resulted in early pupation. We also assessed whether Spb18S is silenced in SPB larvae fed on transgenic soybean expressing Spb18S dsRNA. Transgenic plants downregulated Spb18S expression levels and second-instar larval survival rates. Moreover, such plants were less damaged by SPB larvae than control plants under field conditions. © 2018 Wiley Periodicals, Inc.

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

PubMed Central

Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa MF; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

2010-01-01

Summary Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due to few attempts or to limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, greater than 50% expressed at levels sufficient for commercial production. Three expressed at 2% to 3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivty achieved demonstrate the utility of C. reinhardtii as a robust platform for human therapeutic protein production. PMID:20230484

The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

PubMed

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

PubMed Central

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O.; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells. PMID:23110238

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz)

PubMed Central

2014-01-01

Background Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Results Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H2O2, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H2O2 synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. Conclusions These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress. PMID:25091029

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

PubMed

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-08-05

Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

PubMed

Rawat, Preeti; Singh, Amarjeet Kumar; Ray, Krishna; Chaudhary, Bhupendra; Kumar, Sanjeev; Gautam, Taru; Kanoria, Shaveta; Kaur, Gurpreet; Kumar, Paritosh; Pental, Deepak; Burma, Pradeep Kumar

2011-06-01

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

Osteoblasts are target cells for transformation in c-fos transgenic mice

PubMed Central

1993-01-01

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype. PMID:8335693

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

PubMed

Bandeira, Vanessa S; Tomás, Hélio A; Alici, Evren; Carrondo, Manuel J T; Coroadinha, Ana S

2017-04-01

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na + ,K + -ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 10 7 infectious particles·ml -1 ). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

PubMed

Verma, Ajay D; Parnaik, Veena K

2017-07-01

Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

PubMed Central

Zhang, Shengxue; Yang, Xuewen; Chen, Rongrong; Zhang, Yuwen; Lu, Wei; Liu, Yan; Wang, Jianhua; Lin, Min; Wang, Guoying

2012-01-01

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops. PMID:22715408

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

PubMed Central

Zhang, Daoxiang; Xiang, Taihe; Li, Peihan; Bao, Lumin

2011-01-01

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants. PMID:22215968

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

PubMed Central

Brough, Rachel; Papanastasiou, Antigoni M; Porter, Andrew CG

2007-01-01

Background The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites. Results To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 104-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-SceI transgenes. Conclusion Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes. PMID:17493262

[Construction of transgenic tobacco expressing popW and analysis of its biological phenotype].

PubMed

Wang, Cui; Liu, Hongxia; Cao, Jing; Wang, Chao; Guo, Jianhua

2014-04-01

In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a new harpin protein. The procaryotically expressed PopW can induce resistance to Tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. Here, we constructed an expression vector pB- popW by cloning popW into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciens strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens. After screening on MS medium containing kanamycin, PCR and RT-PCR analysis, 21 T3 lines were identified as positive transgenic. Genomic intergration and expression of the transferred gene were determined by PCR and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Disease bioassays showed that the transgenic tobacco had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. Transgenic tobacco also exhibited enhanced plant growth, the root length of 15 d old seedlings was 1.7 times longer than that of wild type tobacco. 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.4, 1.7, 1.8 times larger than that of wild type tobacco, respectively.

A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene β glucuronidase in tobacco and cotton.

PubMed

Agarwal, Parul; Garg, Varsha; Gautam, Taru; Pillai, Beena; Kanoria, Shaveta; Burma, Pradeep Kumar

2014-04-01

Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene β glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum.

PubMed

Schnippenkoetter, Wendelin; Lo, Clive; Liu, Guoquan; Dibley, Katherine; Chan, Wai Lung; White, Jodie; Milne, Ricky; Zwart, Alexander; Kwong, Eunjung; Keller, Beat; Godwin, Ian; Krattinger, Simon G; Lagudah, Evans

2017-11-01

The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low-expressing single copy Lr34res genotype that conferred partial resistance. Pathogen-induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24-72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4-reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24-h post-inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3-deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Skeletal Muscle-Specific Overexpression of PGC-1α Induces Fiber-Type Conversion through Enhanced Mitochondrial Respiration and Fatty Acid Oxidation in Mice and Pigs.

PubMed

Zhang, Lin; Zhou, Ying; Wu, Wangjun; Hou, Liming; Chen, Hongxing; Zuo, Bo; Xiong, Yuanzhu; Yang, Jinzeng

2017-01-01

Individual skeletal muscles in the animal body are heterogeneous, as each is comprised of different fiber types. Type I muscle fibers are rich with mitochondria, and have high oxidative metabolisms while type IIB fibers have few mitochondria and high glycolytic metabolic capacity. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcriptional co-activator that regulates mitochondrial biogenesis and respiratory function, is implicated in muscle fiber-type switching. Over-expression of PGC-1α in transgenic mice increased the proportion of red/oxidative type I fiber. During pig muscle growth, an increased number of type I fibers can give meat more red color. To explore the roles of PGC-1α in regulation of muscle fiber type conversion, we generated skeletal muscle-specific PGC-1α transgenic mice and pig. Ectopic over-expression of PGC-1α was detected in both fast and slow muscle fibers. The transgenic animals displayed a remarkable amount of red/oxidative muscle fibers in major skeletal muscle tissues. Skeletal muscles from transgenic mice and pigs have increased expression levels of oxidative fiber markers such as MHC1, MHC2x, myoglobin and Tnni1, and decreased expressions of glycolytic fiber genes (MHC2a, MHC2b, CASQ-1 and Tnni2). The genes responsible for the TCA cycle and oxidative phosphorylation, cytochrome coxidase 2 and 4, and citrate synthase were also increased in the transgenic mice and pigs. These results suggested that transgenic over-expressed PGC-1α significantly increased muscle mitochondrial biogenesis, resulting in qualitative changes from glycolytic to oxidative energy generation. The transgenic animals also had elevated levels of PDK4 and PPARγ proteins in muscle tissue, which can lead to increased glycogen deposition and fatty acid oxidation. Therefore, the results support a significant role of PGC-1α in conversion of fast glycolytic fibers to slow and oxidative fiber through enhanced mitochondrial respiration and fatty acid oxidation, and transgenic over-expression of PGC-1α in skeletal muscle leads to more red meat production in pigs.

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Pigeonpea Hybrid-Proline-Rich Protein (CcHyPRP) Confers Biotic and Abiotic Stress Tolerance in Transgenic Rice

PubMed Central

Mellacheruvu, Sunitha; Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2016-01-01

In this study, we report the overexpression of Cajanus cajan hybrid-proline-rich protein encoding gene (CcHyPRP) in rice which resulted in increased tolerance to both abiotic and biotic stresses. Compared to the control plants, the transgenic rice lines, expressing CcHyPRP, exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity, and heat, as evidenced by increased biomass, chlorophyll content, survival rate, root, and shoot growth. Further, transgenic rice lines showed increased panicle size and grain number compared to the control plants under different stress conditions. The CcHyPRP transgenics, as compared to the control, revealed enhanced activities of catalase and superoxide dismutase (SOD) enzymes and reduced malondialdehyde (MDA) levels. Expression pattern of CcHyPRP::GFP fusion-protein confirmed its predominant localization in cell walls. Moreover, the CcHyPRP transgenics, as compared to the control, exhibited increased resistance to the fungal pathogen Magnaporthe grisea which causes blast disease in rice. Higher levels of bZIP and endochitinase transcripts as well as endochitinase activity were observed in transgenic rice compared to the control plants. The overall results demonstrate the intrinsic role of CcHyPRP in conferring multiple stress tolerance at the whole-plant level. The multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants for enhanced tolerance/resistance to different stress factors. PMID:26834756

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV.

PubMed Central

Cohen, R D; Castellani, L W; Qiao, J H; Van Lenten, B J; Lusis, A J; Reue, K

1997-01-01

Transgenic mouse lines carrying several copies of the mouse apo A-IV gene were produced. Lipoprotein composition and function, and aortic lesion development were examined. Apo A-IV levels in the plasma of transgenic mice were elevated threefold compared with nontransgenic littermates on a chow diet, and sixfold in mice fed an atherogenic diet. Plasma concentrations of total cholesterol, HDL cholesterol, triglycerides, and free fatty acids were similar in transgenic and control mice fed a chow diet. However, with the atherogenic diet, male transgenic mice exhibited significantly higher levels of plasma triglycerides (P < 0.05), total cholesterol (P < 0.01), HDL cholesterol (P < 0.0001), and free fatty acids (P < 0.05), and lower levels of unesterified cholesterol (P < 0.05), than nontransgenic littermates. Expression of the apo A-IV transgene had a protective effect against the formation of diet-induced aortic lesions, with transgenics exhibiting lesion scores of approximately 30% those seen in control mice. HDL-sized lipoproteins isolated from transgenic mice fed the atherogenic diet promoted cholesterol efflux from cholesterol-loaded human monocytes more efficiently than comparable lipoproteins from nontransgenic counterparts. Plasma from transgenics also exhibited higher endogenous cholesterol esterification rates. Taken together, these results suggest that apo A-IV levels influence the metabolism and antiatherogenic properties of HDL. PMID:9109435

Development and Evaluation of Transgenic Nude Mice Expressing Ubiquitous Green Fluorescent Protein.

PubMed

Iyer, Srikanth; Arindkar, Shailendra; Mishra, Alaknanda; Manglani, Kapil; Kumar, Jerald Mahesh; Majumdar, Subeer S; Upadhyay, Pramod; Nagarajan, Perumal

2015-08-01

Researchers had developed and characterized transgenic green/red fluorescent protein (GFP/RFP) nude mouse with ubiquitous RFP or GFP expression, but none has evaluated the level of immune cells and expression levels of GFP in this model. The nude GFP mice were evaluated by imaging, hematological indices, and flow cytometry to compare the proportion of immune T cells. Quantitative real-time PCR (qRT-PCR) was done for evaluating the relative expression of GFP transcripts in few organs of the nude GFP mice. The hematological and immune cells of nude GFP were within the range of nude mice. However, the gene expression levels were relatively less in various tissues compared with B6 GFP mice. These findings suggest that nude GFP is an ideal model resembling normal nude mice; however, GFP expression in various tissues by fluorescence should be considered, as the expression of GFP differs in various organs.

Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.

PubMed

Reichenbach, Myriam; Lim, Tiongti; Reichenbach, Horst-Dieter; Guengoer, Tuna; Habermann, Felix A; Matthiesen, Marieke; Hofmann, Andreas; Weber, Frank; Zerbe, Holm; Grupp, Thomas; Sinowatz, Fred; Pfeifer, Alexander; Wolf, Eckhard

2010-08-01

Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

NASA Technical Reports Server (NTRS)

Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

2003-01-01

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.

PubMed

Ranathunge, Kosala; El-Kereamy, Ashraf; Gidda, Satinder; Bi, Yong-Mei; Rothstein, Steven J

2014-03-01

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

Epithelial reticulon 4B (Nogo-B) is an endogenous regulator of Th2-driven lung inflammation

PubMed Central

Wright, Paulette L.; Yu, Jun; Di, Y.P. Peter; Homer, Robert J.; Chupp, Geoffrey; Elias, Jack A.; Cohn, Lauren

2010-01-01

Nogo-B is a member of the reticulon family of proteins (RTN-4B) that is highly expressed in lung tissue; however, its function remains unknown. We show that mice with Th2-driven lung inflammation results in a loss of Nogo expression in airway epithelium and smooth muscle compared with nonallergic mice, a finding which is replicated in severe human asthma. Mice lacking Nogo-A/B (Nogo-KO) display an exaggerated asthma-like phenotype, and epithelial reconstitution of Nogo-B in transgenic mice blunts Th2-mediated lung inflammation. Microarray analysis of lungs from Nogo-KO mice reveals a marked reduction in palate lung and nasal clone (PLUNC) gene expression, and the levels of PLUNC are enhanced in epithelial Nogo-B transgenic mice. Finally, transgenic expression of PLUNC into Nogo-KO mice rescues the enhanced asthmatic-like responsiveness in these KO mice. These data identify Nogo-B as a novel protective gene expressed in lung epithelia, and its expression regulates the levels of the antibacterial antiinflammatory protein PLUNC. PMID:20975041

Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development.

PubMed

Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

2016-01-01

E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive

PubMed Central

Narvaez, Isabel; Khayreddine, Titouh; Pliego, Clara; Cerezo, Sergio; Jiménez-Díaz, Rafael M.; Trapero-Casas, José L.; López-Herrera, Carlos; Arjona-Girona, Isabel; Martín, Carmen; Mercado, José A.; Pliego-Alfaro, Fernando

2018-01-01

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae. PMID:29875785

Development of high-lysine rice via endosperm-specific expression of a foreign LYSINE RICH PROTEIN gene.

PubMed

Liu, Xin; Zhang, Cuicui; Wang, Xiurong; Liu, Qiaoquan; Yuan, Dingyang; Pan, Gang; Sun, Samuel S M; Tu, Jumin

2016-06-29

Lysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date. We expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain. Endosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.

Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

PubMed

Kendziorek, Maria; Klimecka, Maria; Barabasz, Anna; Borg, Sören; Rudzka, Justyna; Szczęsny, Paweł; Antosiewicz, Danuta Maria

2016-08-12

To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma). Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 μM Zn, no change at 0.5 μM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 μM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the mesophyll of "Zn accumulating cells". In transgenic tomato plants, the export activity of ectopically expressed AtHMA4 changes the cellular Zn status, which induces coordinated tissue-specific responses of endogenous ethylene-related genes and metal transporters. These changes constitute an important mechanism involved in the generation of the metal-related phenotype of transgenic tomato expressing AtHMA4.

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

PubMed

Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.

Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction, Transgene Expression, and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

PubMed Central

Thomas, Michael A.; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A.; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector. PMID:24143187

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

PubMed

Rentsendorj, Otgonchimeg; Nagy, Andrea; Sinkó, Ildikó; Daraba, Andreea; Barta, Endre; Kiss, Ibolya

2005-08-01

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.

Effects of APC De-targeting and GAr modification on the duration of luciferase expression from plasmid DNA delivered to skeletal muscle.

PubMed

Subang, Maria C; Fatah, Rewas; Wu, Ying; Hannaman, Drew; Rice, Jason; Evans, Claire F; Chernajovsky, Yuti; Gould, David

2015-01-01

Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3'UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Genomic localization of the Z/EG transgene in the mouse genome.

PubMed

Colombo, Sophie; Kumasaka, Mayuko; Lobe, Corrinne; Larue, Lionel

2010-02-01

The Z/EG transgenic mouse line, produced by Novak et al., displays tissue-specific EGFP expression after Cre-mediated recombination. The autofluorescence of EGFP allows the visualization of cells of interest displaying Cre recombination. The initial construct was designed such that cells without Cre recombination express the beta-galactosidase marker, facilitating counterselection. We used inverse PCR to identify the site of integration of the Z/EG transgene, to improve the efficiency of homozygous Z/EG mouse production. Recombined cells produced large amounts of EGFP protein, resulting in higher levels of fluorescence and therefore greater contrast with nonrecombined cells. We mapped the transgene to the G1 region of chromosome 5. This random insertion was found to have occurred 230-bp upstream from the start codon of the Rasa4 gene. The insertion of the Z/EG transgene in the C57BL/6 genetic background had no effect on Rasa4 expression. Homozygous Z/EG mice therefore had no obvious phenotype. (c) 2009 Wiley-Liss, Inc.

High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

PubMed

Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

2016-01-01

Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Cardiac hypertrophy elevates serum levels of fibroblast growth factor 23.

PubMed

Matsui, Isao; Oka, Tatsufumi; Kusunoki, Yasuo; Mori, Daisuke; Hashimoto, Nobuhiro; Matsumoto, Ayumi; Shimada, Karin; Yamaguchi, Satoshi; Kubota, Keiichi; Yonemoto, Sayoko; Higo, Tomoaki; Sakaguchi, Yusuke; Takabatake, Yoshitsugu; Hamano, Takayuki; Isaka, Yoshitaka

2018-05-08

Several experimental studies have shown that fibroblast growth factor 23 (FGF23) induces left ventricular hypertrophy (LVH). However, the opposite directional relationship, namely a potential effect of LVH on FGF23, remains uncertain. Here we evaluated the effects of LVH on FGF23 using cardiomyocyte-specific calcineurin A transgenic mice. At six weeks, these mice showed severe LVH, with elevated levels of serum intact FGF23. FGF23 levels were elevated in cardiomyocytes, but not osteocytes, of the transgenic animals. Moreover, transverse aortic constriction also upregulated myocardial FGF23 expression in wild type mice. The promoter region of the FGF23 gene contains two putative nuclear factors of activated T cells (NFAT)-binding sites, with NFAT1 activating the promoter in a proximal NFAT-binding site dependent manner. Neither serum, urinary, or fractional excretion values of calcium and phosphate nor serum levels of 1,25(OH) 2 vitamin D were different between wild type and transgenic mice. Moreover, the renal expression of FGF receptors and α-Klotho was comparable. However, plasma levels of antidiuretic hormone were significantly increased in the transgenic mice, and aquaporin-2 immunohistochemical staining was mainly positive in the apical membrane of the collecting duct, compared to a primarily cytoplasmic staining in wild type mice. Real-time PCR analyses of kidney CYP27B1 and CYP24A1 expression in wild type mice showed that exogenous antidiuretic hormone blocked FGF23's actions on these vitamin D activating or inactivating enzymes. Finally, the renal resistance of transgenic mice to FGF23 was partly overcome by tolvaptan. Thus, LVH in transgenic mice is associated with an increase in myocardial and serum intact FGF23, with the kidneys being protected against FGF23 excess by elevated antidiuretic hormone levels. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Ameloblasts require active RhoA to generate normal dental enamel.

PubMed

Xue, Hui; Li, Yong; Everett, Eric T; Ryan, Kathleen; Peng, Li; Porecha, Rakhee; Yan, Yan; Lucchese, Anna M; Kuehl, Melissa A; Pugach, Megan K; Bouchard, Jessica; Gibson, Carolyn W

2013-08-01

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences. Transgenic and wild-type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F-actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho-associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E-cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited. © 2013 Eur J Oral Sci.

Prevention of UVB Radiation-induced Epidermal Damage by Expression of Heat Shock Protein 70*

PubMed Central

Matsuda, Minoru; Hoshino, Tatsuya; Yamashita, Yasuhiro; Tanaka, Ken-ichiro; Maji, Daisuke; Sato, Keizo; Adachi, Hiroaki; Sobue, Gen; Ihn, Hironobu; Funasaka, Yoko; Mizushima, Tohru

2010-01-01

Irradiation with UV light, especially UVB, causes epidermal damage via the induction of apoptosis, inflammatory responses, and DNA damage. Various stressors, including UV light, induce heat shock proteins (HSPs) and the induction, particularly that of HSP70, provides cellular resistance to such stressors. The anti-inflammatory activity of HSP70, such as its inhibition of nuclear factor kappa B (NF-κB), was recently revealed. These in vitro results suggest that HSP70 protects against UVB-induced epidermal damage. Here we tested this idea by using transgenic mice expressing HSP70 and cultured keratinocytes. Irradiation of wild-type mice with UVB caused epidermal damage such as induction of apoptosis, which was suppressed in transgenic mice expressing HSP70. UVB-induced apoptosis in cultured keratinocytes was suppressed by overexpression of HSP70. Irradiation of wild-type mice with UVB decreased the cutaneous level of IκB-α (an inhibitor of NF-κB) and increased the infiltration of leukocytes and levels of pro-inflammatory cytokines and chemokines in the epidermis. These inflammatory responses were suppressed in transgenic mice expressing HSP70. In vitro, the overexpression of HSP70 suppressed the expression of pro-inflammatory cytokines and chemokines and increased the level of IκB-α in keratinocytes irradiated with UVB. UVB induced an increase in cutaneous levels of cyclobutane pyrimidine dimers and 8-hydroxy-2′-deoxyguanosine, both of which were suppressed in transgenic mice expressing HSP70. This study provides genetic evidence that HSP70 protects the epidermis from UVB-induced radiation damage. The findings here also suggest that the protective action of HSP70 is mediated by anti-apoptotic, anti-inflammatory, and anti-DNA damage effects. PMID:20018843

Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

PubMed Central

Huber, M C; Bosch, F X; Sippel, A E; Bonifer, C

1994-01-01

The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion. Images PMID:7937145

Competitive Expression of Endogenous Wheat CENH3 May Lead to Suppression of Alien ZmCENH3 in Transgenic Wheat × Maize Hybrids.

PubMed

Chen, Wei; Zhu, Qilin; Wang, Haiyan; Xiao, Jin; Xing, Liping; Chen, Peidu; Jin, Weiwei; Wang, Xiu-E

2015-11-20

Uniparental chromosome elimination in wheat × maize hybrid embryos is widely used in double haploid production of wheat. Several explanations have been proposed for this phenomenon, one of which is that the lack of cross-species CENH3 incorporation may act as a barrier to interspecies hybridization. However, it is unknown if this mechanism applies universally. To study the role of CENH3 in maize chromosome elimination of wheat × maize hybrid embryos, maize ZmCENH3 and wheat αTaCENH3-B driven by the constitutive CaMV35S promoter were transformed into wheat variety Yangmai 158. Five transgenic lines for ZmCENH3 and six transgenic lines for αTaCENH3-B were identified. RT-PCR analysis showed that the transgene could be transcribed at a low level in all ZmCENH3 transgenic lines, whereas transcription of endogenous wheat CENH3 was significantly up-regulated. Interestingly, the expression levels of both wheat CENH3 and ZmCENH3 in the ZmCENH3 transgenic wheat × maize hybrid embryos were higher than those in the non-transformed Yangmai 158 × maize hybrid embryos. This indicates that the alien ZmCENH3 in wheat may induce competitive expression of endogenous wheat CENH3, leading to suppression of ZmCENH3 over-expression. Eliminations of maize chromosomes in hybrid embryos of ZmCENH3 transgenic wheat × maize and Yangmai 158 × maize were compared by observations on micronuclei presence, by marker analysis using maize SSRs (simple sequence repeats), and by FISH (fluorescence in situ hybridization) using 45S rDNA as a probe. The results indicate that maize chromosome elimination events in the two crosses are not significantly different. Fusion protein ZmCENH3-YFP could not be detected in ZmCENH3 transgenic wheat by either Western blotting or immnunostaining, whereas accumulation and loading of the αTaCENH3-B-GFP fusion protein was normal in αTaCENH3-B transgenic lines. As ZmCENH3-YFP did not accumulate after AM114 treatment, we speculate that low levels of ZmCENH3 protein in transgenic wheat may be one of the factors that lead to failure of suppression of maize chromatin elimination in ZmCENH3 transgenic wheat × maize hybrids. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

PubMed

Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

2015-01-01

The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

PubMed Central

Zhou, Hongxia; Huang, Cao; Yang, Min; Landel, Carlisle P; Xia, Pedro Yuxing; Liu, Yong-Jian; Xia, Xu Gang

2009-01-01

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases. PMID:19214245

Expression of Folate Pathway Genes in the Cartilage of Hoxd4 and Hoxc8 Transgenic Mice

PubMed Central

Kruger, Claudia; Talmadge, Catherine; Kappen, Claudia

2014-01-01

BACKGROUND Hox transcription factors are well known for their role in skeletal patterning in vertebrates. They regulate gene expression during the development of cartilage, the precursor to mature bone. We previously reported that overexpression of the homeobox genes Hoxc8 and Hoxd4 results in severe cartilage defects, reduced proteoglycan content, accumulation of immature chondrocytes, and decreased maturation to hypertrophy. We have also shown that Hoxd4 transgenic mice whose diets were supplemented with folate had their skeletal development restored. Since folate is required for growth and differentiation of chondrocytes, we hypothesized that the beneficial effect of folate in Hoxd4 transgenic mice might indicate a local deficiency in folate utilization, possibly caused by deregulation of genes encoding folate transport proteins or folate metabolic enzymes. METHODS We assayed the prevalence of transcripts for 22 folate transport proteins and metabolizing enzymes, here collectively referred to as folate pathway genes. Quantitative real-time PCR was performed on cDNA samples derived from RNA isolated from primary chondrocytes of individual rib cartilages from Hoxd4 and Hoxc8 transgenic mice, respectively. RESULTS This study shows that the Hox transgenes produce overexpression of Hoxd4 and Hoxc8 in primary chondrocytes from perinatal transgenic mice. However, no differences were found in expression levels of the folate pathway genes in transgenic cells compared to littermate controls. CONCLUSIONS Our results provide evidence that folate pathway genes are only indirect targets of Hox transgene overexpression in our transgenic animals. These expression studies provide a baseline for future studies into the role of folate metabolism in chondrocyte differentiation. PMID:16586448

Transcriptional insulation of the human keratin 18 gene in transgenic mice.

PubMed Central

Neznanov, N; Thorey, I S; Ceceña, G; Oshima, R G

1993-01-01

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes. Images PMID:7681143

Analysis of fiber-type differences in reporter gene expression of β-gal transgenic muscle.

PubMed

Tai, Phillip W L; Smith, Catherine L; Angello, John C; Hauschka, Stephen D

2012-01-01

β-galactosidase (β-gal) is among the most frequently used markers for studying a wide variety of biological mechanisms, e.g., gene expression, cell migration, stem cell conversion to different cell types, and gene silencing. Many of these studies require the histochemical detection of relative β-gal levels in tissue cross-sections mounted onto glass slides and visualized by microscopy. This is particularly useful for the analysis of promoter activity in skeletal muscle tissue since the β-gal levels can vary dramatically between different anatomical muscles and myofiber types. The differences in promoter activity can be due to a myofiber's developmental history, innervation, response to normal or experimental physiological signals, and its disease state. It is thus important to identify the individual fiber types within muscle cross-sections and to correlate these with transgene expression signals. Here, we provide a detailed description of how to process and analyze muscle tissues to determine the fiber-type composition and β-gal transgene expression within cryosections.

Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk.

PubMed

Mikus, Tomás; Poplstein, Martin; Sedláková, Jirina; Landa, Vladimír; Jeníkova, Gabriela; Trefil, Pavel; Lidický, Jan; Malý, Petr

2004-10-01

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

The hrpZ Gene of Pseudomonas syringae pv. phaseolicola Enhances Resistance to Rhizomania Disease in Transgenic Nicotiana benthamiana and Sugar Beet

PubMed Central

Pavli, Ourania I.; Kelaidi, Georgia I.; Tampakaki, Anastasia P.; Skaracis, George N.

2011-01-01

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZPsph). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZPsph was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZPsph showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZPsph developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed. PMID:21394206

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

PubMed

Pavli, Ourania I; Kelaidi, Georgia I; Tampakaki, Anastasia P; Skaracis, George N

2011-03-04

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean

PubMed Central

Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

2015-01-01

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity. PMID:26635848

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

PubMed

Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

2015-01-01

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

PubMed Central

Kalinina, Olena; Zeller, Simon L.; Schmid, Bernhard

2011-01-01

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes. PMID:22132219

Petunia Floral Defensins with Unique Prodomains as Novel Candidates for Development of Fusarium Wilt Resistance in Transgenic Banana Plants

PubMed Central

Ghag, Siddhesh B.; Shekhawat, Upendra K. Singh; Ganapathi, Thumballi R.

2012-01-01

Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C- terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium–mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana. PMID:22745785

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

PubMed Central

Liu, Wenting; Liang, Zongsuo; Wang, Xinhua; Sibbald, Susan; Hunter, David; Tian, Lining

2013-01-01

Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species. PMID:23690914

Effects of mutant human Ki-ras{sup G12C} gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.

2008-08-15

Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras{sup G12C} allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 {mu}g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras{sup G12C} allele in the lung, and resulted in themore » development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 {mu}g/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 {mu}g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 {mu}g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras{sup G12C} expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models.« less

Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, W.M.; Dausman, J.; Beard, C.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protectionmore » analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.« less

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants.

PubMed

Mora, A; Earle, E D

2001-04-01

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED50 levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED50 level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED50 showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.

Memory Impairment in Transgenic Alzheimer Mice Requires Cellular Prion Protein

PubMed Central

Gimbel, David A.; Nygaard, Haakon B.; Coffey, Erin E.; Gunther, Erik C.; Laurén, Juha; Gimbel, Zachary A.; Strittmatter, Stephen M.

2012-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide are thought to play a key role in the pathophysiology of Alzheimer’s disease (AD). Recently, we reported that synthetic Aβ oligomers bind to cellular prion protein (PrPC) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Aβ peptide. We hypothesized that PrPC is essential for the ability of brain-derived Aβ to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1ΔE9 into Prnp−/− mice to examine the necessity of PrPC for AD-related phenotypes. Neither APP expression nor Aβ level is altered by PrPC absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrPC expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1ΔE9 transgenic mice. The AD transgenic mice with intact PrPC expression exhibit deficits in spatial learning and memory. Mice lacking PrPC, but containing Aβ plaque derived from APPswe/PSen1ΔE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrPC expression dissociates Aβ accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrPC. PMID:20445063

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests

PubMed Central

Yarasi, Bharathi; Sadumpati, Vijayakumar; Immanni, China Pasalu; Vudem, Dasavantha Reddy; Khareedu, Venkateswara Rao

2008-01-01

Background Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Results Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. Conclusion In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding. PMID:18854007

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests.

PubMed

Yarasi, Bharathi; Sadumpati, Vijayakumar; Immanni, China Pasalu; Vudem, Dasavantha Reddy; Khareedu, Venkateswara Rao

2008-10-14

Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which approximately 21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Expression of the B subunit of Escherichia coli heat-labile enterotoxin as a fusion protein in transgenic tomato.

PubMed

Walmsley, A M; Alvarez, M L; Jin, Y; Kirk, D D; Lee, S M; Pinkhasov, J; Rigano, M M; Arntzen, C J; Mason, H S

2003-06-01

Epitopes often require co-delivery with an adjuvant or targeting protein to enable recognition by the immune system. This paper reports the ability of transgenic tomato plants to express a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) and an immunocontraceptive epitope. The fusion protein was found to assemble into pentamers, as evidenced by its ability to bind to gangliosides, and had an average expression level of 37.8 microg g(-1) in freeze-dried transgenic tissues. Processing of selected transgenic fruit resulted in a 16-fold increase in concentration of the antigen with minimal loss in detectable antigen. The species-specific nature of this epitope was shown by the inability of antibodies raised against non-target species to detect the LTB fusion protein. The immunocontraceptive ability of this vaccine will be tested in future pilot mice studies.

Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

PubMed Central

Cerutti, H; Johnson, A M; Gillham, N W; Boynton, J E

1997-01-01

The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. PMID:9212467

Why is golden rice golden (yellow) instead of red?

PubMed

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-05-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

Why Is Golden Rice Golden (Yellow) Instead of Red?1[w

PubMed Central

Schaub, Patrick; Al-Babili, Salim; Drake, Rachel; Beyer, Peter

2005-01-01

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed. PMID:15821145

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

PubMed

Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

2015-08-24

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Tumorigenic potential of pituitary tumor transforming gene (PTTG) in vivo investigated using a transgenic mouse model, and effects of cross breeding with p53 (+/-) transgenic mice.

PubMed

Fong, Miranda Y; Farghaly, Hanan; Kakar, Sham S

2012-11-20

Pituitary tumor-transforming gene (PTTG) is an oncogene that is overexpressed in variety of tumors and exhibits characteristics of a transforming gene. Previous transgenic mouse models to access the tumorigenic potential in the pituitary and ovary have resulted in dysplasia without formation of visible tumors, possibly due to the insufficient expression of PTTG. PTTG expression level is critical for ovarian tumorigenesis in a xenograft model. Therefore, the tumorigenic function of PTTG in vivo remains unclear. We generated a transgenic mouse that overexpresses PTTG driven by the CMV promoter to determine whether PTTG functions as a transforming oncogene that is capable of initiating tumorigenesis. Transgenic animals were generated by microinjection of PTTG transgene into the male pronucleus of FVB 0.5 day old embryos. Expression levels of PTTG in tissues of transgenic animals were analyzed using an immunohistochemical analysis. H&E staining and immunohistostaining were performed to examine the type of tumor in transgenic and PTTG transgenic/p53+/- animals. PTTG transgenic offspring (TgPTTG) were monitored for tumor development at various ages. H&E analysis was performed to identify the presence of cancer and hyperplastic conditions verified with the proliferation marker PCNA and the microvessel marker CD31. Immunohistochemistry was performed to determine transgene expression, revealing localization to the epithelium of the fallopian tube, with more generalized expression in the liver, lung, kidney, and spleen. At eight months of age, 2 out of 15 TgPTTG developed ovarian cancer, 2 out of 15 developed benign tumors, 2 out of 15 developed cervical dysplasia, and 3 out of 15 developed adenomyosis of the uterus. At ten months of age, 2 out of 10 TgPTTG developed adenocarcinoma of the ovary, 1 out of 10 developed a papillary serous adenocarcinoma, and 2 out of 10 presented with atypia of ovarian epithelial cells. Tumorigenesis is a multi-step process, often requiring multiple oncogenes and/or inactivation of tumor suppressor genes. Therefore, to understand the contribution of p53 to PTTG induced tumorigenesis, we crossbred TgPTTG to p53+/- mice and maintained those 8 to 10 months. TgPTTG/p53+/- animals developed sarcomas faster than p53+/- alone as well as different tumor types in addition to cervical carcinomas in situ in 10 out of 17 females. We conclude that while PTTG is a functional transforming oncogene, it requires an additional partner to effectively promote tumorigenesis through the loss of p53 include or between function or modulation.

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease

PubMed Central

Beyene, Getu; Chauhan, Raj Deepika; Ilyas, Muhammad; Wagaba, Henry; Fauquet, Claude M.; Miano, Douglas; Alicai, Titus; Taylor, Nigel J.

2017-01-01

Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD. PMID:28149300

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.

Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybridmore » promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes.« less

On the use of the T-REx tetracycline-inducible gene expression system in vivo.

PubMed

Dobrovolsky, Vasily N; Heflich, Robert H

2007-10-15

Components of the commercially available T-REx system were used to create two types of transgenic mice. The first contained the tetracycline-repressor transgene under the control of the CMV promoter/enhancer; the second type contained a green fluorescent protein (GFP) reporter transgene under the control of the CMV promoter/enhancer with a tetracycline repressor operator sequence. Transgene expression was unpredictable in animals containing the individual transgenes. Animals with the reporter transgene expressed GFP in only some tissues (e.g., pancreas, kidney), and one line of reporter transgenic animals developed kidney disease, presumably due to expression of the transgene. The two types of transgenic animals were crossbred to produce double-transgenic animals with the object of regulating the expression of the reporter in vivo. When a similar double-transgenic system was constructed in cultured cells, the repressor protein suppressed the transcription of the reporter transgene. The presence of the repressor in double-transgenic animals had no effect on the expression of the reporter; double transgenic animals developed the same kidney disease that was seen in singly transgenic mice with the reporter. Our results indicate that transgenes under the control of the CMV promoter in the T-REx system express somewhat unpredictably and in only a limited number of tissues, making the use of this system for the development of in vivo models problematical. Copyright 2007 Wiley Periodicals, Inc.

A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

PubMed Central

Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

2014-01-01

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

Suppression of glucose-6-phosphate-isomerase induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of glucose-6-phosphate-isomerase.

PubMed

Hirota, Tomoya; Tsuboi, Hiroto; Iizuka-Koga, Mana; Takahashi, Hiroyuki; Asashima, Hiromitsu; Yokosawa, Masahiro; Kondo, Yuya; Ohta, Masaru; Wakasa, Yuhya; Matsumoto, Isao; Takaiwa, Fumio; Sumida, Takayuki

2017-05-01

To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI 325-339 ) in mice model of GPI-induced arthritis (GIA). We generated transgenic rice expressing T-cell epitope of hGPI 325-339 and APL12 contained in the seed endosperm. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined for IL-17 production in splenocytes and inguinal lymph node (iLN) cells, and analyzed the expression levels of functional molecules in splenocytes. Prophylactic treatment of GIA mice with APL12 transgenic (APL12-TG) rice seeds significantly reduced the severity of arthritis and titers of serum anti-GPI antibodies compared with non-transgenic (Non-TG) rice-treated mice. APL12-TG and hGPI 325-339 transgenic (hGPI 325-339 -TG) rice seeds improved the histopathological arthritis scores and decreased IL-17 production compared with non-TG rice-treated mice. APL12-TG rice-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4  +  CD25  +  Foxp3 +  cells in the spleen compared with non-TG rice- and hGPI 325-339 -TG rice-treated mice. APL12-TG rice seeds improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on Treg cells in spleen.

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids.

PubMed

Mi, Xiaoxiao; Liu, Xue; Yan, Haolu; Liang, Lina; Zhou, Xiangyan; Yang, Jiangwei; Si, Huaijun; Zhang, Ning

2017-01-01

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0±1.43 (ST2) and 36.6±0.99 (35S3) aphids per plant, which corresponds to 24.9-53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti.

PubMed

Kokoza, V; Ahmed, A; Cho, W L; Jasinskiene, N; James, A A; Raikhel, A

2000-08-01

Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20-22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes

PubMed Central

Zheng, Desong; Sun, Quanxi; Liu, Jiang; Li, Yaxiao; Hua, Jinping

2016-01-01

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis. PMID:27433934

Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene.

PubMed

Fu, Jianming; Ristic, Zoran

2010-06-01

We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.

PubMed

Kumar, S; Arul, L; Talwar, D

2010-01-01

We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

NASA Technical Reports Server (NTRS)

Love, J.; Scott, A. C.; Thompson, W. F.; Brown, C. S. (Principal Investigator)

2000-01-01

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

Confirmation of germ-line transmission in the red fluorescence protein (RFP) transgenic cloned male cat.

PubMed

Cho, Su-Jin; Lee, Young S; Lee, Jae-Ik; Bang, Jae-Il; Yang, Jing; Klassen, Henry; Kong, Il-Keun

2010-12-01

The production of transgenic animals is highly desirable for biotechnology and basic research. We investigated the reproductive ability of a red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) by natural mating with a domestic female cat. The RFP expression levels were examined in early embryogenesis, tissues from 45-day-old two fetuses, and four RFP TG cat offspring. The RFP gene was detected in tissue samples from one dead kitten, including several organs and the skin. Also, under a fluorescent light source, we were able to directly detect the RFP expression of in in vitro-produced blastocysts derived with sperm from the RFP TG cat. These results indicate that the RFP TG cat exhibits normal reproductive fertility, stable germ-line transmission of the RFP transgene, and characteristic RFP expression in its offspring. We isolated feline neural progenitor cells from a 45-day-old fetus derived from the natural mating of the RFP TG cat with a domestic female cat. Isolated brain and retinal progenitor cells were successfully passaged at least four times post isolation (day 23), and showed a high RFP expression level. This method of producing genetically modified cloned cats will be important for generating biomedical models of human diseases.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

PubMed Central

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

PubMed

Omar, Ahmad A; Murata, Mayara M; El-Shamy, Hesham A; Graham, James H; Grosser, Jude W

2018-04-01

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

PubMed

Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

2014-08-01

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. © 2014. Published by The Company of Biologists Ltd.

Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

PubMed

Murfett, J; McClure, B A

1998-06-01

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

Expression of a Heterologous S-Adenosylmethionine Decarboxylase cDNA in Plants Demonstrates That Changes in S-Adenosyl-l-Methionine Decarboxylase Activity Determine Levels of the Higher Polyamines Spermidine and Spermine1

PubMed Central

Thu-Hang, Pham; Bassie, Ludovic; Safwat, Gehan; Trung-Nghia, Pham; Christou, Paul; Capell, Teresa

2002-01-01

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering. PMID:12177487

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

PubMed

Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

2010-05-01

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum.

PubMed

Qiu, Jian; Sun, Shuquan; Luo, Shiqiao; Zhang, Jichuan; Xiao, Xianzhou; Zhang, Liqun; Wang, Feng; Liu, Shizhong

2014-04-01

This study developed a new purple coloured Taraxacum brevicorniculatum plant through genetic transformation using the Arabidopsis AtPAP1 gene, which overproduced anthocyanins in its vegetative tissues. Rubber-producing Taraxacum plants synthesise high-quality natural rubber (NR) in their roots and so are a promising alternative global source of this raw material. A major factor in its commercialization is the need for multipurpose exploitation of the whole plant. To add value to the aerial tissues, red/purple plants of the rubber-producing Taraxacum brevicorniculatum species were developed through heterologous expression of the production of anthocyanin pigment 1 (AtPAP1) transcription factor from Arabidopsis thaliana. The vegetative tissue of the transgenic plants showed an average of a 48-fold increase in total anthocyanin content over control levels, but with the exception of pigmentation, the transgenic plants were phenotypically comparable to controls and displayed similar growth vigor. Southern blot analysis confirmed that the AtPAP1 gene had been integrated into the genome of the high anthocyanin Taraxacum plants. The AtPAP1 expression levels were estimated by quantitative real-time PCR and were highly correlated with the levels of total anthocyanins in five independent transgenic lines. High levels of three cyanidin glycosides found in the purple plants were characterized by high performance liquid chromatography-mass spectrum analysis. The presence of NR was verified by NMR and infrared spectroscopy, and confirmed that NR biosynthesis had not been affected in the transgenic Taraxacum lines. In addition, other major phenylpropanoid products such as chlorogenic acid and quercetin glycosides were also enhanced in the transgenic Taraxacum. The red/purple transgenic Taraxacum lines described in this study would increase the future application of the species as a rubber-producing crop due to its additional health benefits.

Microarray expression profiling identifies genes with altered expression in HDL-deficient mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Callow, Matthew J.; Dudoit, Sandrine; Gong, Elaine L.

2000-05-05

Based on the assumption that severe alterations in the expression of genes known to be involved in HDL metabolism may affect the expression of other genes we screened an array of over 5000 mouse expressed sequence tags (ESTs) for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apo AI) knockout mice, Scavenger Receptor BI (SR-BI) transgenic mice and control mice were co-hybridized to microarrays. Two-sample t-statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared withmore » the control mice. In the SR-BI group we found 9 array elements representing at least 5 genes to be significantly altered on the basis of an adjusted p value of less than 0.05. In the apo AI knockout group 8 array elements representing 4 genes were altered compared with the control group (p < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments of apo AI knockout and SR-BI transgenic mice.« less

Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium.

PubMed

Zhang, Yuman; Lian, Lijuan; Liu, Qing; Xiao, Na; Fang, Rongxiang; Liu, Qinglin; Chen, Xiaoying

2013-04-01

Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5' and 3' RACE methods. CmNRRa expression levels in various tissues were monitored by real-time RT-PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium-mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real-time RT-PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa-RNAi transgenic plants flowered 40-64 days earlier, while CmNRRa-overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year-round, and may improve commercial chrysanthemum production in the flower industry. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwasaki, Yuko; Iwasaki, Hitoshi; Yatoh, Shigeru

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic {beta}-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, {beta}{epsilon}{tau}{alpha}2, MafA, andmore » IRS-2 were suppressed, partially explaining the loss and dysfunction of {beta}-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous {beta}-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts {beta}-cell mass and function.« less

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period.

PubMed

Borejsza-Wysocka, Ewa; Norelli, John L; Aldwinckle, Herb S; Malnoy, Mickael

2010-06-03

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years. Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.

Single-Event Transgene Product Levels Predict Levels in Genetically Modified Breeding Stacks.

PubMed

Gampala, Satyalinga Srinivas; Fast, Brandon J; Richey, Kimberly A; Gao, Zhifang; Hill, Ryan; Wulfkuhle, Bryant; Shan, Guomin; Bradfisch, Greg A; Herman, Rod A

2017-09-13

The concentration of transgene products (proteins and double-stranded RNA) in genetically modified (GM) crop tissues is measured to support food, feed, and environmental risk assessments. Measurement of transgene product concentrations in breeding stacks of previously assessed and approved GM events is required by many regulatory authorities to evaluate unexpected transgene interactions that might affect expression. Research was conducted to determine how well concentrations of transgene products in single GM events predict levels in breeding stacks composed of these events. The concentrations of transgene products were compared between GM maize, soybean, and cotton breeding stacks (MON-87427 × MON-89034 × DAS-Ø15Ø7-1 × MON-87411 × DAS-59122-7 × DAS-40278-9 corn, DAS-81419-2 × DAS-44406-6 soybean, and DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 × MON-88913-8 × DAS-81910-7 cotton) and their component single events (MON-87427, MON-89034, DAS-Ø15Ø7-1, MON-87411, DAS-59122-7, and DAS-40278-9 corn, DAS-81419-2, and DAS-44406-6 soybean, and DAS-21023-5, DAS-24236-5, SYN-IR102-7, MON-88913-8, and DAS-81910-7 cotton). Comparisons were made within a crop and transgene product across plant tissue types and were also made across transgene products in each breeding stack for grain/seed. Scatter plots were generated comparing expression in the stacks to their component events, and the percent of variability accounted for by the line of identity (y = x) was calculated (coefficient of identity, I 2 ). Results support transgene concentrations in single events predicting similar concentrations in breeding stacks containing the single events. Therefore, food, feed, and environmental risk assessments based on concentrations of transgene products in single GM events are generally applicable to breeding stacks composed of these events.

Silencing the HaHR3 Gene by Transgenic Plant-mediated RNAi to Disrupt Helicoverpa armigera Development

PubMed Central

Xiong, Yehui; Zeng, Hongmei; Zhang, Yuliang; Xu, Dawei; Qiu, Dewen

2013-01-01

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests. PMID:23630449

Optimization of Acidothermus Celluloyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-Translational Modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Hooker, Brian S.; Quesenberry, Ryan D.

2005-10-01

Biochemical characteristics of Acidothermus cellulolyticus endoglucanase (E1) and its physiological effects in transgenic tobacco (Nicotiana tabacum) has been studied previously. In an attempt to obtain a high level of production of intact E1 in transgenic plants, the E1 gene was expressed under the control of strong Mac promoter (a hybrid promoter of manopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region) or tomato Rubisco small subunit (RbcS-3C) promoter with different 5’ untranslated leader (UTL) sequence and targeted to different subcellular comartmentations with various transit peptides. The expression of E1 protein in transgenic tobacco plants was determined via E1more » activity, protein immunobloting, and RNA gel-blotting analyses. Effects of different transit peptides on E1 protein production and its stability were examined in transgenic tobacco plants carrying one of six transgene expression vectors with the same (Mac) promoter and transcription terminator (Tmas). Transgenic tobacco plants with apoplast transit peptide (Mm-apo) had the highest average E1 activity and protein accumulation , while E1 protein was more stable in transgenic plants with no transit peptide (Mm) than others. The E1 expression under tomato RbcS-3C promoter was higher than that under Mac promoter based on the average E1 activity, E1 protein accumulation, and RNA gel-blotting. The E1 expression was increased more than two fold when the 5’-UTL of alfalfa mosaic virus RNA4 gene replaced the UTL of RbcS-3C promoter, while the UTL of alfalfa mosaic virus RNA4 gene was less effective than the UTL of Mac promoter. The optimal combination of promoter, 5’-UTL, and subcellular compartmentation (transit peptide) for E1 protein production in transgenic tobacco plants are discussed.« less

Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress

PubMed Central

Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P.; Dobaria, Jentilal R.

2016-01-01

Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

PubMed Central

Rugh, C L; Wilde, H D; Stack, N M; Thompson, D M; Summers, A O; Meagher, R B

1996-01-01

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution. Images Fig. 2 Fig. 3 Fig. 4 PMID:8622910

Overexpression of Arabidopsis AnnAt8 Alleviates Abiotic Stress in Transgenic Arabidopsis and Tobacco

PubMed Central

Yadav, Deepanker; Ahmed, Israr; Shukla, Pawan; Boyidi, Prasanna; Kirti, Pulugurtha Bharadwaja

2016-01-01

Abiotic stress results in massive loss of crop productivity throughout the world. Because of our limited knowledge of the plant defense mechanisms, it is very difficult to exploit the plant genetic resources for manipulation of traits that could benefit multiple stress tolerance in plants. To achieve this, we need a deeper understanding of the plant gene regulatory mechanisms involved in stress responses. Understanding the roles of different members of plant gene families involved in different stress responses, would be a step in this direction. Arabidopsis, which served as a model system for the plant research, is also the most suitable system for the functional characterization of plant gene families. Annexin family in Arabidopsis also is one gene family which has not been fully explored. Eight annexin genes have been reported in the genome of Arabidopsis thaliana. Expression studies of different Arabidopsis annexins revealed their differential regulation under various abiotic stress conditions. AnnAt8 (At5g12380), a member of this family has been shown to exhibit ~433 and ~175 fold increase in transcript levels under NaCl and dehydration stress respectively. To characterize Annexin8 (AnnAt8) further, we have generated transgenic Arabidopsis and tobacco plants constitutively expressing AnnAt8, which were evaluated under different abiotic stress conditions. AnnAt8 overexpressing transgenic plants exhibited higher seed germination rates, better plant growth, and higher chlorophyll retention when compared to wild type plants under abiotic stress treatments. Under stress conditions transgenic plants showed comparatively higher levels of proline and lower levels of malondialdehyde compared to the wild-type plants. Real-Time PCR analyses revealed that the expression of several stress-regulated genes was altered in AnnAt8 over-expressing transgenic tobacco plants, and the enhanced tolerance exhibited by the transgenic plants can be correlated with altered expressions of these stress-regulated genes. Our findings suggest a role for AnnAt8 in enhancing abiotic stress tolerance at different stages of plant growth and development. PMID:27135239

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

PubMed Central

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

PubMed Central

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

PubMed

Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

2011-01-01

The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically. These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation. © 2011 John Wiley & Sons A/S.

Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Cheng; Graduate University of Chinese Academy of Sciences, Beijing; Jin, Xia

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in themore » pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.« less

Golden bananas in the field: elevated fruit pro-vitamin A from the expression of a single banana transgene.

PubMed

Paul, Jean-Yves; Khanna, Harjeet; Kleidon, Jennifer; Hoang, Phuong; Geijskes, Jason; Daniells, Jeff; Zaplin, Ella; Rosenberg, Yvonne; James, Anthony; Mlalazi, Bulukani; Deo, Pradeep; Arinaitwe, Geofrey; Namanya, Priver; Becker, Douglas; Tindamanyire, James; Tushemereirwe, Wilberforce; Harding, Robert; Dale, James

2017-04-01

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro-vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA-biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β-carotene equivalent (β-CE) in the fruit. Expression of a Fe'i banana-derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β-CE. Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop 'Golden Rice 2', also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1-aminocyclopropane-1-carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild-type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate-limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm.

PubMed

Ding, Liping; Chen, Yajuan; Wei, Xiaoli; Ni, Mi; Zhang, Jiewei; Wang, Hongzhi; Zhu, Zhen; Wei, Jianhua

2017-01-01

Transgenic poplar lines 'Shanxin' (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7-9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

PubMed Central

Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

1993-01-01

The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

PubMed

Lin, Heng-Ju; Lee, Shu-Hua; Wu, Jen-Leih; Duann, Yeh-Fang; Chen, Jyh-Yih

2013-12-01

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Evaluation of an FRDA-EGFP genomic reporter assay in transgenic mice.

PubMed

Sarsero, Joseph P; Holloway, Timothy P; Li, Lingli; McLenachan, Samuel; Fowler, Kerry J; Bertoncello, Ivan; Voullaire, Lucille; Gazeas, Sophie; Ioannou, Panos A

2005-04-01

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron-sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin-EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA-EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.

Memory impairment in transgenic Alzheimer mice requires cellular prion protein.

PubMed

Gimbel, David A; Nygaard, Haakon B; Coffey, Erin E; Gunther, Erik C; Laurén, Juha; Gimbel, Zachary A; Strittmatter, Stephen M

2010-05-05

Soluble oligomers of the amyloid-beta (Abeta) peptide are thought to play a key role in the pathophysiology of Alzheimer's disease (AD). Recently, we reported that synthetic Abeta oligomers bind to cellular prion protein (PrP(C)) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Abeta peptide. We hypothesized that PrP(C) is essential for the ability of brain-derived Abeta to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1DeltaE9 into Prnp-/- mice to examine the necessity of PrP(C) for AD-related phenotypes. Neither APP expression nor Abeta level is altered by PrP(C) absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrP(C) expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1DeltaE9 transgenic mice. The AD transgenic mice with intact PrP(C) expression exhibit deficits in spatial learning and memory. Mice lacking PrP(C), but containing Abeta plaque derived from APPswe/PSen1DeltaE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrP(C) expression dissociates Abeta accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrP(C).

Expression of VGRNb-PE immunotoxin in transplastomic lettuce (Lactuca sativa L.).

PubMed

Mirzaee, Malihe; Jalali-Javaran, Mokhtar; Moieni, Ahmad; Zeinali, Sirous; Behdani, Mahdi

2018-05-01

This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.

Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

PubMed Central

Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

1997-01-01

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

Methods of affecting nitrogen assimilation in plants

DOEpatents

Coruzzi, Gloria; Gutierrez, Rodrigo A.; Nero, Damion C.

2016-10-11

Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.

PubMed

Dacquin, Romain; Starbuck, Michael; Schinke, Thorsten; Karsenty, Gérard

2002-06-01

Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter. Copyright 2002 Wiley-Liss, Inc.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

PubMed

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

PubMed Central

Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

2017-01-01

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

AMT1;1 transgenic rice plants with enhanced NH4 + permeability show superior growth and higher yield under optimal and suboptimal NH4 + conditions

PubMed Central

Rothstein, Steven J.

2014-01-01

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4 +). The NH4 + uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4 + transport in rice plants. However, little is known about its involvement in NH4 + uptake in rice roots and subsequent effects on NH4 + assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4 + permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4 + content in the shoots and roots than the WT. Direct NH4 + fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4 + contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4 + levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions. PMID:24420570

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.)

PubMed Central

Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

2017-01-01

Coconut (Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae (Palmaceae). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1. Its transcriptional activities and interactions with the acetyl-CoA carboxylase (BCCP2) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis, high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase (P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined. PMID:28179911

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

PubMed

Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

2017-01-01

Coconut ( Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae ( Palmaceae ). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1 . Its transcriptional activities and interactions with the acetyl-CoA carboxylase ( BCCP2 ) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis , high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase ( P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

A novel platform for biologically active recombinant human interleukin-13 production.

PubMed

Wang, David J; Brandsma, Martin; Yin, Ziqin; Wang, Aiming; Jevnikar, Anthony M; Ma, Shengwu

2008-06-01

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Independent effects of apolipoprotein AV and apolipoprotein CIII on plasma triglyceride concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baroukh, Nadine N.; Bauge, Eric; Akiyama, Jennifer

2003-08-15

Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered Both the apolipoprotein A5 and C3 genes have repeatedly been shownmore » to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered triglycerides. To overcome these confounding factors and address their relationship, we generated independent lines of mice that either over-expressed (''double transgenic'') or completely lacked (''double knockout'') both apolipoprotein genes. We report that both ''double transgenic'' and ''double knockout'' mice display intermedia tetriglyceride concentrations compared to over-expression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the ''double transgenic'' mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV is a potent triglyceride modulator despite its low concentration. Together, these data indicate that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.« less

Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin

PubMed Central

Achouri, Younes; Hahn, Stephan; Many, Marie-Christine; Craps, Julie; Refetoff, Samuel; Liao, Xiao-Hui; Dumont, Jacques E.; Van Sande, Jacqueline; Corvilain, Bernard; Miot, Françoise; De Deken, Xavier

2016-01-01

Background: The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function. Methods: Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays. Results: Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected. Conclusions: This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype. PMID:27599561

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

PubMed

Glasser, Stephan W; Eszterhas, Susan K; Detmer, Emily A; Maxfield, Melissa D; Korfhagen, Thomas R

2005-04-01

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco.

PubMed

Osakabe, Yuriko; Osakabe, Keishi; Chiang, Vincent L

2009-01-01

We characterized promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clalpha, from Pinus taeda. Histochemical- and quantitative assays of GUS expression in the vascular tissue were performed using transgenic tobacco plants expressing promoter-GUS reporters. Deletion analysis of the Pta4Clalpha promoter showed that the region -524 to -252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLalpha was also detected in the xylem cells under bending stress. DNA-protein complexes were detected in the reactions of the Pta4CLalpha promoter fragments with the nuclear proteins of xylem of P. taeda. The AC elements in the Pta4CLalpha promoter appeared to have individual roles during xylem development that are activated in a coordinated manner in response to stress in transgenic tobacco.

Gene Transfer into Rat Brain Using Adenoviral Vectors

PubMed Central

Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

2010-01-01

Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines

PubMed Central

Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

2014-01-01

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

Lipidomic and metabolic changes in the P4-type ATPase ATP10D deficient C57BL/6J wild type mice upon rescue of ATP10D function.

PubMed

Sigruener, Alexander; Wolfrum, Christian; Boettcher, Alfred; Kopf, Thomas; Liebisch, Gerhard; Orsó, Evelyn; Schmitz, Gerd

2017-01-01

Sequence variants near the human gene for P4-type ATPase, class V, type 10D (ATP10D) were shown to significantly associate with circulating hexosylceramide d18:1/16:0 and d18:1/24:1 levels, obesity, insulin resistance, plasma high density lipoprotein (HDL), coronary stenotic index and intracranial atherosclerotic index. In mice Atp10d is associated with HDL modulation and C57BL/6 mice expressing a truncated, non-functional form of ATP10D easily develop obesity and insulin resistance on high-fat diet. We analyzed metabolic differences of ATP10D deficient C57BL/6J wild type and ATP10D transgenic C57BL/6J BAC129 mice. ATP10D transgenic mice gain 25% less weight on high-fat diet concomitant with a reduced increase in fat cell mass but independent of adipocyte size change. ATP10D transgenic mice also had 26% lower triacylglycerol levels with approximately 76% bound to very low density lipoprotein while in ATP10D deficient wild type mice 57% are bound to low density lipoprotein. Furthermore increased oxygen consumption and CO2 production, 38% lower glucose and 69% lower insulin levels and better insulin sensitivity were observed in ATP10D transgenic mice. Besides decreased hexosylceramide species levels were detected. Part of these effects may be due to reduced hepatic stearoyl-CoA desaturase 1 (SCD1) expression in ATP10D transgenic mice, which was reflected by altered fatty acid and lipid species patterns. There was a significant decrease in the hepatic 18:1 to 18:0 free fatty acid ratio in transgenic mice. The ratio of 16:1 to 16:0 was not significantly different. Interestingly both ratios were significantly reduced in plasma total fatty acids. In summary we found that ATP10D reduces high-fat diet induced obesity and improves insulin sensitivity. ATP10D transgenic mice showed altered hepatic expression of lipid-metabolism associated genes, including Scd1, along with changes in hepatic and plasma lipid species and plasma lipoprotein pattern.

Overexpression of Jatropha Gibberellin 2-oxidase 6 (JcGA2ox6) Induces Dwarfism and Smaller Leaves, Flowers and Fruits in Arabidopsis and Jatropha

PubMed Central

Hu, Ying-Xiong; Tao, Yan-Bin; Xu, Zeng-Fu

2017-01-01

Gibberellins (GAs) are plant hormones that play fundamental roles in plant growth and development. Gibberellin 2-oxidase (GA2ox) plays a direct role in determining the levels of bioactive GAs by catalyzing bioactive GAs or their immediate precursors to inactive forms. In this study, a GA2ox gene, designated JcGA2ox6, was isolated from Jatropha curcas. JcGA2ox6 is expressed in all tissues of adult Jatropha, with the highest expression level in male flowers and the lowest expression level in young leaves. Overexpression of JcGA2ox6 in Arabidopsis resulted in a typical dwarf phenotype, along with late flowering, smaller leaves and flowers, shorter siliques and smaller seeds. Similarly, when JcGA2ox6 was overexpressed in Jatropha, the transgenic plants exhibited a dwarf phenotype with dark-green leaves and smaller inflorescences, flowers, fruits and seeds. However, the flowering time of Jatropha was not affected by overexpression of JcGA2ox6, unlike that in the transgenic Arabidopsis. Moreover, the number of flowers per inflorescence, the weight of 10 seeds and the seed oil content were significantly decreased in transgenic Jatropha. The results indicated that overexpression of JcGA2ox6 had a great impact on the vegetative and reproductive growth of transgenic Jatropha. Furthermore, we found that the dwarf phenotype of transgenic Jatropha was caused by a decrease in endogenous bioactive GA4, which was correlated with the degree of dwarfism. PMID:29312375

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds.

PubMed

Liu, Wen Xian; Liu, Hua Liang; Qu, Le Qing

2013-09-01

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.

Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1β gene product

PubMed Central

Björkdahl, O; Åkerblad, P; Gjörloff-wingren, A; Leanderson, T; Dohlsten, M

1999-01-01

To evaluate the biological effects of over-expression of interleukin-1β (IL-1β) on the immune system we have generated transgenic mice, expressing the IL-1β gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Eμ). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B-cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L− T cells and local secretion of IL-2 and IL-4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL-1β in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL-1β in controlling lymphoid microarchitecture and, when over-expressed, breaking the threshold in T-helper–B-cell interaction. PMID:10233687

Stable expression of mtlD gene imparts multiple stress tolerance in finger millet.

PubMed

Hema, Ramanna; Vemanna, Ramu S; Sreeramulu, Shivakumar; Reddy, Chandrasekhara P; Senthil-Kumar, Muthappa; Udayakumar, Makarla

2014-01-01

Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.

A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

PubMed

Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

2014-04-01

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

PubMed

de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis)

PubMed Central

de Oliveira, Raquel S.; Oliveira-Neto, Osmundo B.; Moura, Hudson F. N.; de Macedo, Leonardo L. P.; Arraes, Fabrício B. M.; Lucena, Wagner A.; Lourenço-Tessutti, Isabela T.; de Deus Barbosa, Aulus A.; da Silva, Maria C. M.; Grossi-de-Sa, Maria F.

2016-01-01

Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests. PMID:26925081

Polyamines attenuate ethylene-mediated defense responses to abrogate resistance to Botrytis cinerea in tomato

USDA-ARS?s Scientific Manuscript database

Transgenic tomato (Solanum lycopersicum) lines over-expressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild type plants and were examined for responses to the...

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus.

PubMed

Vigne, Emmanuelle; Komar, Véronique; Fuchs, Marc

2004-04-01

One of the major environmental safety issues over transgenic crops containing virus-derived genes relates to the outcome of recombination events between viral transgene transcripts and RNAs from indigenous virus populations. We addressed this issue by assessing the emergence of viable Grapevine fanleaf virus (GFLV) recombinants in transgenic grapevines expressing the GFLV coat protein (CP) gene. Test plants consisted of nontransgenic scions grafted onto transgenic and nontransgenic rootstocks that were exposed over 3 years to nematode-mediated GFLV infection in two distinct vineyard sites. The CP gene of challenging GFLV isolates was amplified from scions by IC-RT-PCR, and characterized by RFLP and nucleotide sequencing using strain F13 as reference since it provided the CP transgene. Analysis of EcoRI and StyI RFLP banding patterns from 347 challenging GFLV isolates and sequence data from 85 variants revealed no characteristics similar to strain F13 and no difference in the molecular variability among isolates from 190 transgenic and 157 nontransgenic plants, or from plants within (253 individuals) or outside (94 individuals) of the two sites. Interestingly, five GFLV recombinants were identified in three nontransgenic plants located outside of the two field settings. This survey indicates that transgenic grapevines did not assist the emergence of viable GFLV recombinants to detectable levels nor did they affect the molecular diversity of indigenous GFLV populations during the trial period. This is the first report on safety assessment of recombination with a transgenic crop expressing a CP gene under field conditions of heavy disease pressure but low, if any, selection pressure against recombinant viruses.

Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

PubMed

Deng, Shoulong; Li, Guiguan; Zhang, Jinlong; Zhang, Xiaosheng; Cui, Maosheng; Guo, Yong; Liu, Guoshi; Li, Guangpeng; Feng, Jianzhong; Lian, Zhengxing

2013-07-01

An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance. Copyright © 2013 Elsevier Inc. All rights reserved.

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

PubMed Central

2010-01-01

Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

Generating high temperature tolerant transgenic plants: Achievements and challenges.

PubMed

Grover, Anil; Mittal, Dheeraj; Negi, Manisha; Lavania, Dhruv

2013-05-01

Production of plants tolerant to high temperature stress is of immense significance in the light of global warming and climate change. Plant cells respond to high temperature stress by re-programming their genetic machinery for survival and reproduction. High temperature tolerance in transgenic plants has largely been achieved either by over-expressing heat shock protein genes or by altering levels of heat shock factors that regulate expression of heat shock and non-heat shock genes. Apart from heat shock factors, over-expression of other trans-acting factors like DREB2A, bZIP28 and WRKY proteins has proven useful in imparting high temperature tolerance. Besides these, elevating the genetic levels of proteins involved in osmotic adjustment, reactive oxygen species removal, saturation of membrane-associated lipids, photosynthetic reactions, production of polyamines and protein biosynthesis process have yielded positive results in equipping transgenic plants with high temperature tolerance. Cyclic nucleotide gated calcium channel proteins that regulate calcium influxes across the cell membrane have recently been shown to be the key players in induction of high temperature tolerance. The involvement of calmodulins and kinases in activation of heat shock factors has been implicated as an important event in governing high temperature tolerance. Unfilled gaps limiting the production of high temperature tolerant transgenic plants for field level cultivation are discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

PubMed

Ewulonu, U K; Snyder, L; Silver, L M; Schimenti, J C

1996-03-01

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Hepatitis B surface antigen gene expression is regulated by sex steroids and glucocorticoids in transgenic mice.

PubMed Central

Farza, H; Salmon, A M; Hadchouel, M; Moreau, J L; Babinet, C; Tiollais, P; Pourcel, C

1987-01-01

We have investigated the basis for liver-specific and sex-linked expression of hepatitis B surface antigen (HBsAg) gene in transgenic mice by monitoring the level of liver HBsAg mRNA and serum HBsAg at different stages of development and in response to sex-hormone regulation. Transcription of the HBsAg gene starts at day 15 of development, together with that of the albumin gene, and reaches a comparable level at birth. HBsAg mRNA level and HBsAg production are parallel in males and females during prenatal development and until the first month of life, but HBsAg gene expression increases 5-10 times in males at puberty. After castration, the level of expression decreases dramatically in both males and females and is subsequently increased by injection of testosterone or estradiol. Glucocorticoids also regulated positively expression of the HBsAg gene. Our results suggest that sex hormones play a role in hepatitis B virus gene expression during natural infection and could explain the difference in incidence of chronic carriers between men and women. Images PMID:3469661

An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

PubMed

Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

2004-03-01

Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.

An N‐terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

PubMed Central

KIS, MIHALY; BURBRIDGE, EMMA; BROCK, IAN W.; HEGGIE, LAURA; DIX, PHILIP J.; KAVANAGH, TONY A.

2004-01-01

• Background and Aims Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N‐terminal and C‐terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. • Methods Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N‐terminal or the C‐terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV‐35S) or the tobacco RUBISCO‐SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium‐mediated transformation. To study the effects of the N‐ and C‐terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. • Key Results Transgenic tobacco plants can exhibit a ten‐fold increase in peroxidase activity compared with wild‐type tobacco levels, and the majority of this activity is located in the symplast. The N‐terminal extension is essential for the production of high levels of recombinant protein, while the C‐terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. • Conclusions There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N‐terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development. PMID:14749254

Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper.

PubMed

Seo, Hyo-Hyoun; Park, Sangkyu; Park, Soomin; Oh, Byung-Jun; Back, Kyoungwhan; Han, Oksoo; Kim, Jeong-Il; Kim, Young Soon

2014-01-01

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL-1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

PubMed

Li, Yan; Lian, Di; Deng, Shoulong; Zhang, Xiaosheng; Zhang, Jinlong; Li, Wenting; Bai, Hai; Wang, Zhixian; Wu, Hongping; Fu, Juncai; Han, Hongbing; Feng, Jianzhong; Liu, Guoshi; Lian, Ling; Lian, Zhengxing

2016-01-01

Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth. In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference. Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

PubMed Central

Seo, Hyo-Hyoun; Park, Sangkyu; Park, Soomin; Oh, Byung-Jun; Back, Kyoungwhan; Han, Oksoo; Kim, Jeong-Il; Kim, Young Soon

2014-01-01

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL−1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease. PMID:24848280

Isogenic transgenic homozygous fish induced by artificial parthenogenesis.

PubMed

Nam, Y K; Cho, Y S; Kim, D S

2000-12-01

As a model system for vertebrate transgenesis, fish have many attractive advantages, especially with respect to the characteristics of eggs, allowing us to produce isogenic, transgenic, homozygous vertebrates by combining with chromosome-set manipulation. Here, we describe the large-scale production of isogenic transgenic homozygous animals using our experimental organism, the mud loach Misgurnus mizolepis, by the simple process of artificial parthenogenesis in a single generation. These isogenic fish have retained transgenic homozygous status in a stable manner during the subsequent 5 years, and exhibited increased levels of transgene expression. Furthermore, their isogenic nature was confirmed by cloned transgenic homozygous offspring produced via another step of parthenogenic reproduction of the isogenic homozygous transgenic fish. These results demonstrate that a combination of transgenesis and artificial parthenogenesis will make the rapid utilization of genetically pure homozygous transgenic system in vertebrate transgenesis possible.

Transgenic Wheat Expressing a Barley UDP-Glucosyltransferase Detoxifies Deoxynivalenol and Provides High Levels of Resistance to Fusarium graminearum.

PubMed

Li, Xin; Shin, Sanghyun; Heinen, Shane; Dill-Macky, Ruth; Berthiller, Franz; Nersesian, Natalya; Clemente, Thomas; McCormick, Susan; Muehlbauer, Gary J

2015-11-01

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

PubMed Central

Drummond, Heather A.; Gousette, Monette U.; Storm, Megan V.; Abraham, Nader G.; Csongradi, Eva

2012-01-01

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139±3 versus 153±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments,. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption. PMID:22323644

Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice

NASA Technical Reports Server (NTRS)

Chakravarthy, M. V.; Fiorotto, M. L.; Schwartz, R. J.; Booth, F. W.

2001-01-01

Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

PubMed Central

Mattei, Elisabetta; Corbi, Nicoletta; Di Certo, Maria Grazia; Strimpakos, Georgios; Severini, Cinzia; Onori, Annalisa; Desantis, Agata; Libri, Valentina; Buontempo, Serena; Floridi, Aristide; Fanciulli, Maurizio; Baban, Dilair; Davies, Kay E.; Passananti, Claudio

2007-01-01

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter “A”. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics. PMID:17712422

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal gene transfer.

PubMed

Jin, Chuan; Fotaki, Grammatiki; Ramachandran, Mohanraj; Nilsson, Berith; Essand, Magnus; Yu, Di

2016-07-01

Chimeric antigen receptor (CAR) T-cell therapy is a new successful treatment for refractory B-cell leukemia. Successful therapeutic outcome depends on long-term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV-S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19(+) target cell recognition as LV-engineered T cells and are as effective in controlling tumor growth in vivo We propose that NILV-S/MAR vectors are superior to current options as they enable long-term transgene expression without the risk of insertional mutagenesis and genotoxicity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Population dynamics of Sesamia inferens on transgenic rice expressing Cry1Ac and CpTI in southern China.

PubMed

Han, Lanzhi; Liu, Peilei; Wu, Kongming; Peng, Yufa; Wang, Feng

2008-10-01

Genetically modified insect-resistant rice lines containing the cry1Ac gene from Bacillus thuringiensis (Bt) or the CpTI (cowpea trypsin inhibitor) gene developed for the management of lepidopterous pests are highly resistant to the major target pests, Chilo suppressalis (Walker), Cnaphalocrocis medinalis (Guenée), and Scirpophaga incertulas (Walker), in the main rice-growing areas of China. However, the effects of these transgenic lines on Sesamia inferens (Walker), an important lepidopterous rice pest, are currently unknown. Because different insect species have varying susceptibility to Bt insecticidal proteins that may affect population dynamics, research into the effects of these transgenic rice lines on the population dynamics of S. inferens was conducted in Fuzhou, southern China, in 2005 and 2006. The results of laboratory, field cage, and field plot experiments show that S. inferens has comparatively high susceptibility to the transgenic line during the early growing season, with significant differences observed in larval density and infestation levels between transgenic and control lines. Because of a decrease in Cry1Ac levels in the plant as it ages, the transgenic line provided only a low potential for population suppression late in the growing season. There is a correlation between the changing expression of Cry1Ac and the impact of transgenic rice on the population dynamics of S. inferens during the season. These results indicate that S. inferens may become a major pest in fields of prospective commercially released transgenic rice, and more attention should be paid to developing an effective alternative management strategy.

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

PubMed

Chien, Yu; Cheng, Wei-Cheng; Wu, Menq-Rong; Jiang, Si-Tse; Shen, Che-Kun James; Chung, Bon-chu

2013-10-01

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

PubMed

Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

2015-01-01

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

Field-grown miR156 transgenic switchgrass reproduction, yield, global gene expression analysis, and bioconfinement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Chelsea R.; Millwood, Reginald J.; Tang, Yuhong

Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum). However, regulatory issues arising from gene flow may prevent commercialization of engineered switchgrass in the eastern United States where the species is native. And, depending on its expression level, microRNA156 (miR156) can reduce, delay, or eliminate flowering, which may serve to decrease transgene flow. Here, in this unique field study of transgenic switchgrass that was permitted to flower, two low (T14 and T35) and two medium (T27 and T37) miR156-overexpressing 'Alamo' lines with the transgene under the control of the constitutivemore » maize (Zea mays) ubiquitin 1 promoter, along with nontransgenic control plants, were grown in eastern Tennessee over two seasons.« less

Field-grown miR156 transgenic switchgrass reproduction, yield, global gene expression analysis, and bioconfinement

DOE PAGES

Johnson, Chelsea R.; Millwood, Reginald J.; Tang, Yuhong; ...

2017-11-30

Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum). However, regulatory issues arising from gene flow may prevent commercialization of engineered switchgrass in the eastern United States where the species is native. And, depending on its expression level, microRNA156 (miR156) can reduce, delay, or eliminate flowering, which may serve to decrease transgene flow. Here, in this unique field study of transgenic switchgrass that was permitted to flower, two low (T14 and T35) and two medium (T27 and T37) miR156-overexpressing 'Alamo' lines with the transgene under the control of the constitutivemore » maize (Zea mays) ubiquitin 1 promoter, along with nontransgenic control plants, were grown in eastern Tennessee over two seasons.« less

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants.

PubMed

Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

2016-01-01

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

PubMed Central

Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

2016-01-01

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants. PMID:27073898

Rosuvastatin does not affect human apolipoprotein A-I expression in genetically modified mice: a clue to the disputed effect of statins on HDL.

PubMed

Marchesi, Marta; Parolini, Cinzia; Caligari, Silvia; Gilio, Donatella; Manzini, Stefano; Busnelli, Marco; Cinquanta, Paola; Camera, Marina; Brambilla, Marta; Sirtori, Cesare R; Chiesa, Giulia

2011-11-01

Besides a significant reduction of low-density lipoprotein (LDL) cholesterol, statins moderately increase high-density lipoprotein (HDL) levels. In vitro studies have indicated that this effect may be the result of an increased expression of apolipoprotein (apo)A-I, the main protein component of HDL. The aim of the present study was to investigate in vivo the effect of rosuvastatin on apoA-I expression and secretion in a transgenic mouse model for human apoA-I. Human apoA-I transgenic mice were treated for 28 days with 5, 10 or 20 mg·kg(-1) ·day(-1) of rosuvastatin, the most effective statin in raising HDL levels. Possible changes of apoA-I expression by treatment were investigated by quantitative real-time RT-PCR on RNA extracted from mouse livers. The human apoA-I secretion rate was determined in primary hepatocytes isolated from transgenic mice from each group after treatment. Rosuvastatin treatment with 5 and 10 mg·kg(-1) ·day(-1) did not affect apoA-I plasma levels, whereas a significant decrease was observed in mice treated with 20 mg·kg(-1) ·day(-1) of rosuvastatin (-16%, P < 0.01). Neither relative hepatic mRNA concentrations of apoA-I nor apoA-I secretion rates from primary hepatocytes were influenced by rosuvastatin treatment at each tested dose. In human apoA-I transgenic mice, rosuvastatin treatment does not increase either apoA-I transcription and hepatic secretion, or apoA-I plasma levels. These results support the hypothesis that other mechanisms may account for the observed HDL increase induced by statin therapy in humans. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Patterns of expression of position-dependent integrated transgenes in mouse embryo.

PubMed Central

Bonnerot, C; Grimber, G; Briand, P; Nicolas, J F

1990-01-01

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution. Images PMID:1696727

Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

PubMed Central

Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

2013-01-01

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration.

PubMed

Mor, Danielle E; Tsika, Elpida; Mazzulli, Joseph R; Gould, Neal S; Kim, Hanna; Daniels, Malcolm J; Doshi, Shachee; Gupta, Preetika; Grossman, Jennifer L; Tan, Victor X; Kalb, Robert G; Caldwell, Kim A; Caldwell, Guy A; Wolfe, John H; Ischiropoulos, Harry

2017-11-01

Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.

A Novel G-Protein-Coupled Receptors Gene from Upland Cotton Enhances Salt Stress Tolerance in Transgenic Arabidopsis.

PubMed

Lu, Pu; Magwanga, Richard Odongo; Lu, Hejun; Kirungu, Joy Nyangasi; Wei, Yangyang; Dong, Qi; Wang, Xingxing; Cai, Xiaoyan; Zhou, Zhongli; Wang, Kunbo; Liu, Fang

2018-04-12

Plants have developed a number of survival strategies which are significant for enhancing their adaptation to various biotic and abiotic stress factors. At the transcriptome level, G-protein-coupled receptors (GPCRs) are of great significance, enabling the plants to detect a wide range of endogenous and exogenous signals which are employed by the plants in regulating various responses in development and adaptation. In this research work, we carried out genome-wide analysis of target of Myb1 ( TOM1 ), a member of the GPCR gene family. The functional role of TOM1 in salt stress tolerance was studied using a transgenic Arabidopsis plants over-expressing the gene. By the use of the functional domain PF06454, we obtained 16 TOM genes members in Gossypium hirsutum , 9 in Gossypium arboreum , and 11 in Gossypium raimondii . The genes had varying physiochemical properties, and it is significant to note that all the grand average of hydropathy (GRAVY) values were less than one, indicating that all are hydrophobic in nature. In all the genes analysed here, both the exonic and intronic regions were found. The expression level of Gh_A07G0747 (GhTOM) was significantly high in the transgenic lines as compared to the wild type; a similar trend in expression was observed in all the salt-related genes tested in this study. The study in epidermal cells confirmed the localization of the protein coded by the gene TOM1 in the plasma membrane. Analysis of anti-oxidant enzymes showed higher concentrations of antioxidants in transgenic lines and relatively lower levels of oxidant substances such as H₂O₂. The low malondialdehyde (MDA) level in transgenic lines indicated that the transgenic lines had relatively low level of oxidative damage compared to the wild types. The results obtained indicate that Gh_A07G0747 (GhTOM) can be a putative target gene for enhancing salt stress tolerance in plants and could be exploited in the future for the development of salt stress-tolerant cotton cultivars.

A Novel G-Protein-Coupled Receptors Gene from Upland Cotton Enhances Salt Stress Tolerance in Transgenic Arabidopsis

PubMed Central

Lu, Hejun; Kirungu, Joy Nyangasi; Wei, Yangyang; Dong, Qi; Wang, Xingxing; Cai, Xiaoyan; Zhou, Zhongli; Wang, Kunbo; Liu, Fang

2018-01-01

Plants have developed a number of survival strategies which are significant for enhancing their adaptation to various biotic and abiotic stress factors. At the transcriptome level, G-protein-coupled receptors (GPCRs) are of great significance, enabling the plants to detect a wide range of endogenous and exogenous signals which are employed by the plants in regulating various responses in development and adaptation. In this research work, we carried out genome-wide analysis of target of Myb1 (TOM1), a member of the GPCR gene family. The functional role of TOM1 in salt stress tolerance was studied using a transgenic Arabidopsis plants over-expressing the gene. By the use of the functional domain PF06454, we obtained 16 TOM genes members in Gossypium hirsutum, 9 in Gossypium arboreum, and 11 in Gossypium raimondii. The genes had varying physiochemical properties, and it is significant to note that all the grand average of hydropathy (GRAVY) values were less than one, indicating that all are hydrophobic in nature. In all the genes analysed here, both the exonic and intronic regions were found. The expression level of Gh_A07G0747 (GhTOM) was significantly high in the transgenic lines as compared to the wild type; a similar trend in expression was observed in all the salt-related genes tested in this study. The study in epidermal cells confirmed the localization of the protein coded by the gene TOM1 in the plasma membrane. Analysis of anti-oxidant enzymes showed higher concentrations of antioxidants in transgenic lines and relatively lower levels of oxidant substances such as H2O2. The low malondialdehyde (MDA) level in transgenic lines indicated that the transgenic lines had relatively low level of oxidative damage compared to the wild types. The results obtained indicate that Gh_A07G0747 (GhTOM) can be a putative target gene for enhancing salt stress tolerance in plants and could be exploited in the future for the development of salt stress-tolerant cotton cultivars. PMID:29649144

Characterization of a new, inducible transgenic mouse model with GFP expression in melanocytes and their precursors.

PubMed

Joshi, Sandeep S; Tandukar, Bishal; Castaneda, Maira; Jiang, Shunlin; Diwakar, Ganesh; Hertzano, Ronna P; Hornyak, Thomas J

2018-01-01

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties. Published by Elsevier B.V.

Deletion of the human beta-globin LCR 5'HS4 or 5'HS1 differentially affects beta-like globin gene expression in beta-YAC transgenic mice.

PubMed

Fedosyuk, Halyna; Peterson, Kenneth R

2007-01-01

A 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC) was modified by homologous recombination to delete 2.9 kb of cross-species conserved sequence similarity encompassing the LCR 5' hypersensitive site (HS) 4 (Delta5'HS4 beta-YAC). In three transgenic mouse lines, completion of the gamma- to beta-globin switch during definitive erythropoiesis was delayed relative to wild-type beta-YAC mice. In addition, quantitative per-copy human beta-like globin mRNA levels were similar to wild-type beta-YAC transgenic lines, although beta-globin gene expression was slightly decreased in the day 12 fetal liver of Delta5'HS4 beta-YAC mice. A 0.8 kb 5'HS1 fragment was similarly deleted in the YAC. Three Delta5'HS1 beta-YAC transgenic lines were established. epsilon-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type beta-YAC mice, but gamma-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, gamma-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the beta-like globin genes were unaffected by deletion of 5'HS1. Decreased expression of the epsilon- and gamma-globin genes is the first phenotype ascribed to a 5'HS1 mutation in the human beta-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.

Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis.

PubMed

Li, Zhao; Zhou, Miaoping; Zhang, Zengyan; Ren, Lijuan; Du, Lipu; Zhang, Boqiao; Xu, Huijun; Xin, Zhiyong

2011-03-01

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1-RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.

Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

PubMed

Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-03-01

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. © 2016 Federation of European Biochemical Societies.

Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

PubMed

Wakasa, Yuhya; Takaiwa, Fumio

2016-01-01

Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid, drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate accumulation.

PubMed

Ma, Lichao; Wang, Yanrong; Liu, Wenxian; Liu, Zhipeng

2014-11-01

GDP-mannose 3', 5'-epimerase (GME) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (ascorbic acid) biosynthetic pathway in higher plants. In this study, a novel cDNA fragment (MsGME) encoding a GME protein was isolated and characterised from alfalfa (Medicago sativa). An expression analysis confirmed that MsGME expression was induced by salinity, PEG and acidity stresses. MsGME overexpression in Arabidopsis enhanced tolerance of the transgenic plants to salt, drought and acid. Real-time PCR analysis revealed that the transcript levels of GDP-D-mannose pyrophosphorylase (GMP), L-galactose-phosphate 1-P phosphatase (GP) and GDP-L-galactose phosphorylase (GGP) were increased in transgenic Arabidopsis (T3 generation). Moreover, the ascorbate content was increased in transgenic Arabidopsis. Our results suggest that MsGME can effectively enhance tolerance of transgenic Arabidopsis to acid, drought and salt by increasing ascorbate accumulation.

Classical swine fever virus replicated poorly in cells from MxA transgenic pigs.

PubMed

Zhao, Yicheng; Wang, Tiedong; Yao, Li; Liu, Bo; Teng, Chunbo; Ouyang, Hongsheng

2016-08-17

In addition to their value as livestock, pigs are susceptible to classical swine fever virus (CSFV) and can serve as reservoirs for CSFV, allowing it to develop into an epizootic. CSFV, a pestivirus of the Flaviviridae family, has a single-stranded RNA genome. Recent research has indicated that the human MxA protein inhibits the life cycles of certain RNA viruses, such as members of the Bunyaviridae family, the Flaviviridae family and others. To produce pigs with antiviral protection against CSFV, transgenic pigs expressing human MxA were generated by nuclear transplantation. Cells from three MxA transgenic piglets were used to investigate in vitro antiviral activity of MxA aganist CSFV, and the results of in vitro indirect immunofluorescence assays, virus titration and real-time PCR indicated that the MxA transgenic pig has an antiviral capacity against CSFV. Transgene with human MxA on pigs is feasible. High levels of MxA expression do inhibit CSFV in vitro at early time points post-infection at 60-96dpi.

Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

PubMed

Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

2016-03-01

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

Dose-dependent effects of higher methionine levels on the transcriptome and metabolome of transgenic Arabidopsis seeds.

PubMed

Cohen, Hagai; Amir, Rachel

2017-05-01

Higher methionine levels in transgenic Arabidopsis seeds trigger the accumulation of stress-related transcripts and primary metabolites. These responses depend on the levels of methionine within seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. To reveal the regulatory role of the Arabidopsis thaliana CYSTATHIONINE γ-SYNTHASE (AtCGS), methionine main regulatory enzyme, in the synthesis of methionine in seeds, we generated transgenic RNAi seeds with targeted repression of AtCGS during late developmental stages of seeds. Unexpectedly, these seeds accumulated 2.5-fold more methionine than wild-type seeds. To study the nature of these seeds, transcriptomic and primary metabolite profiling were employed using Affymetrix ATH1 microarray and gas chromatography-mass spectrometry analyses, respectively. The results were compared to transgenic Arabidopsis seeds expressing a feedback-insensitive form of AtCGS (named SSE-AtD-CGS) that were previously showed to accumulate up to sixfold more soluble methionine than wild-type seeds. Statistical assessments showed that the nature of transcriptomic and metabolic changes that occurred in RNAi::AtCGS seeds were relatively similar, but to lesser extents, to those previously reported for SSE-AtD-CGS seeds, and linked to the induction of global transcriptomic and metabolic responses associated with stronger desiccation stress. As transgenic seeds obtained by both manipulations exhibited higher, but different methionine levels, the data strongly suggest that these changes depend on the absolute amounts of methionine within seeds and much less to the expression level of AtCGS.

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration.

PubMed

Kaźmierczak, Beata; Kuźma-Kozakiewicz, Magdalena; Usarek, Ewa; Barańczyk-Kuźma, Anna

2011-01-01

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

Expression and biological effects of high levels of serum IgE in epsilon heavy chain transgenic mice.

PubMed

Adamczewski, M; Köhler, G; Lamers, M C

1991-03-01

We have generated and examined transgenic mice carrying a rearranged immunoglobulin transgene coding for the heavy chain of an IgE antibody. These mice produce the secreted form of the recombinant epsilon heavy chain. Serum IgE levels were increased at least 100-fold over control values. Transgenic epsilon mRNA was detected in spleen and thymus, not in liver and heart. Transgenic epsilon production in vitro was slightly up-regulated by T cells, but not affected by interleukin 4 in vitro or Nippostrongylus infestation in vivo. The B cell and T cell compartments and antigen-specific IgE, IgG1 and IgM responses as well as the increase in endogenous IgE after Nippostrongylus infestation in transgenic mice were normal. These data indicate that the presence of high levels of transgenic IgE did not induce class-specific suppressive mechanisms. Transgenic IgE bound to Fc epsilon receptor type I and Fc epsilon receptor type II and mediated histamine release from mast cells in vitro and an allergic skin reaction in vivo. It inhibited an ovalbumin-specific skin reaction in ovalbumin-immunized transgenic mice only during the initial phases of the immune response. This result has a bearing on the feasibility of immune therapy of allergic diseases with substances that block binding of IgE to its receptors.

Ectopically expressing MdPIP1;3, an aquaporin gene, increased fruit size and enhanced drought tolerance of transgenic tomatoes.

PubMed

Wang, Lin; Li, Qing-Tian; Lei, Qiong; Feng, Chao; Zheng, Xiaodong; Zhou, Fangfang; Li, Lingzi; Liu, Xuan; Wang, Zhi; Kong, Jin

2017-12-19

Water deficit severely reduces apple growth and production, is detrimental to fruit quality and size. This problem is exacerbated as global warming is implicated in producing more severe drought stress. Thus water-efficiency has becomes the major target for apple breeding. A desired apple tree can absorb and transport water efficiently, which not only confers improved drought tolerance, but also guarantees fruit size for higher income returns. Aquaporins, as water channels, control water transportation across membranes and can regulate water flow by changing their amount and activity. The exploration of molecular mechanism of water efficiency and the gene wealth will pave a way for molecular breeding of drought tolerant apple tree. In the current study, we screened out a drought inducible aquaporin gene MdPIP1;3, which specifically enhanced its expression during fruit expansion in 'Fuji' apple (Malus domestica Borkh. cv. Red Fuji). It localized on plasma membranes and belonged to PIP1 subfamily. The tolerance to drought stress enhanced in transgenic tomato plants ectopically expressing MdPIP1;3, showing that the rate of losing water in isolated transgenic leaves was slower than wild type, and stomata of transgenic plants closed sensitively to respond to drought compared with wild type. Besides, length and diameter of transgenic tomato fruits increased faster than wild type, and in final, fruit sizes and fresh weights of transgenic tomatoes were bigger than wild type. Specially, in cell levels, fruit cell size from transgenic tomatoes was larger than wild type, showing that cell number per mm 2 in transgenic fruits was less than wild type. Altogether, ectopically expressing MdPIP1;3 enhanced drought tolerance of transgenic tomatoes partially via reduced water loss controlled by stomata closure in leaves. In addition, the transgenic tomato fruits are larger and heavier with larger cells via more efficient water transportation across membranes. Our research will contribute to apple production, by engineering apples with big fruits via efficient water transportation when well watered and enhanced drought tolerance in transgenic apples under water deficit.

Hsf-1 affects podocyte markers NPHS1, NPHS2 and WT1 in a transgenic mouse model of TTRVal30Met-related amyloidosis.

PubMed

Petrakis, Ioannis; Mavroeidi, Vasiliki; Stylianou, Kostas; Andronikidi, Eva; Lioudaki, Eirini; Perakis, Kostas; Stratigis, Spyridon; Vardaki, Eleftheria; Zafeiri, Maria; Giannakakis, Kostantinos; Plaitakis, Andreas; Amoiridis, George; Saraiva, Maria Joao; Daphnis, Eugene

2013-09-01

Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes. Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition. Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness. Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression.

Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis

USDA-ARS?s Scientific Manuscript database

Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expressi...

Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

PubMed Central

Tata, Purushothama Rao; Pardo-Saganta, Ana; Prabhu, Mythili; Vinarsky, Vladimir; Law, Brandon M.; Fontaine, Benjamin A.; Tager, Andrew M.

2013-01-01

Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter–driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell–specific transgene expression using a cytokeratin 5 (also known as keratin 5)–driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype. PMID:23848320

Development of Orally Bioavailable Therapeutics by the Chloroplast Expression System to Counter Muscle Degeneration, Weakness, and Fibrosis in DMD

DTIC Science & Technology

2016-11-01

validation (Daniell) (months 1-16): 1a. Plant transgenic tobacco seeds and grow (months 1-4, typical growing period; repeated 2 times). 1b. Harvest...GFP. Adult mice were orally fed with leaf materials from transgenic tobacco plants, with the amount adjusted to GFP expression levels, for three... Breeding Cmdx and C57 mice for colony maintenance and expansion (Barton) (months 1-30) 3a. Purchase mice (month 1). 3b. Breed mice for older age

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO2 levels.

PubMed

Tamoi, Masahiro; Hiramatsu, Yoshie; Nedachi, Shigeki; Otori, Kumi; Tanabe, Noriaki; Maruta, Takanori; Shigeoka, Shigeru

2011-05-01

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO(2) levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO(2) levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO(2) levels.

Impact of age and vector construct on striatal and nigral transgene expression

PubMed Central

Polinski, Nicole K; Manfredsson, Fredric P; Benskey, Matthew J; Fischer, D Luke; Kemp, Christopher J; Steece-Collier, Kathy; Sandoval, Ivette M; Paumier, Katrina L; Sortwell, Caryl E

2016-01-01

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD. PMID:27933309

Co-expression of P173S Mutant Rice EPSPS and igrA Genes Results in Higher Glyphosate Tolerance in Transgenic Rice

PubMed Central

Fartyal, Dhirendra; Agarwal, Aakrati; James, Donald; Borphukan, Bhabesh; Ram, Babu; Sheri, Vijay; Yadav, Renu; Manna, Mrinalini; Varakumar, Panditi; Achary, V. Mohan M.; Reddy, Malireddy K.

2018-01-01

Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice EPSPS glyphosate tolerant mutant gene (OsmEPSPS) alone and in combination with the glyphosate detoxifying encoding igrA gene, recently characterized from Pseudomonas. The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with OsmEPSPS and igrA genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the OsmEPSPS transgenic plants. This study highlights the advantage of igrA led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant OsmEPSPS gene, which offered a better option to tackle in vivo glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants. PMID:29487608

Co-expression of P173S Mutant Rice EPSPS and igrA Genes Results in Higher Glyphosate Tolerance in Transgenic Rice.

PubMed

Fartyal, Dhirendra; Agarwal, Aakrati; James, Donald; Borphukan, Bhabesh; Ram, Babu; Sheri, Vijay; Yadav, Renu; Manna, Mrinalini; Varakumar, Panditi; Achary, V Mohan M; Reddy, Malireddy K

2018-01-01

Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice EPSPS glyphosate tolerant mutant gene ( OsmEPSPS ) alone and in combination with the glyphosate detoxifying encoding igrA gene, recently characterized from Pseudomonas . The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with OsmEPSPS and igrA genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the OsmEPSPS transgenic plants. This study highlights the advantage of igrA led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant OsmEPSPS gene, which offered a better option to tackle in vivo glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants.

Co-transforming bar and CsALDH Genes Enhanced Resistance to Herbicide and Drought and Salt Stress in Transgenic Alfalfa (Medicago sativa L.)

PubMed Central

Duan, Zhen; Zhang, Daiyu; Zhang, Jianquan; Di, Hongyan; Wu, Fan; Hu, Xiaowen; Meng, Xuanchen; Luo, Kai; Zhang, Jiyu; Wang, Yanrong

2015-01-01

Drought and high salinity are two major abiotic factors that restrict the productivity of alfalfa. By application of the Agrobacterium-mediated transformation method, an oxidative responsive gene, CsALDH12A1, from the desert grass Cleistogenes songorica together with the bar gene associated with herbicide resistance, were co-transformed into alfalfa (Medicago sativa L.). From the all 90 transformants, 16 were positive as screened by spraying 1 mL L-1 10% Basta solution and molecularly diagnosis using PCR. Real-time PCR analysis indicated that drought and salt stress induced high CsALDH expression in the leaves of the transgenic plants. The CsALDH expression levels under drought (15 d) and salt stress (200 mM NaCl) were 6.11 and 6.87 times higher than in the control plants, respectively. In comparison to the WT plants, no abnormal phenotypes were observed among the transgenic plants, which showed significant enhancement of tolerance to 15 d of drought and 10 d of salinity treatment. Evaluation of the physiological and biochemical indices during drought and salt stress of the transgenic plants revealed relatively lower Na+ content and higher K+ content in the leaves relative to the WT plants, a reduction of toxic on effects and maintenance of osmotic adjustment. In addition, the transgenic plants could maintain a higher relative water content level, higher shoot biomass, fewer changes in the photosystem, decreased membrane injury, and a lower level of osmotic stress. These results indicate that the co-expression of the introduced bar and CsALDH genes enhanced the herbicide, drought and salt tolerance of alfalfa and therefore can potentially be used as a novel genetic resource for the future breeding programs to develop new cultivars. PMID:26734025

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

PubMed Central

Kota, Madhuri; Daniell, Henry; Varma, Sam; Garczynski, Stephen F.; Gould, Fred; Moar, William J.

1999-01-01

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field. PMID:10051556

Stable Expression of mtlD Gene Imparts Multiple Stress Tolerance in Finger Millet

PubMed Central

Hema, Ramanna; Vemanna, Ramu S.; Sreeramulu, Shivakumar; Reddy, Chandrasekhara P.; Senthil-Kumar, Muthappa; Udayakumar, Makarla

2014-01-01

Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet. PMID:24922513

Amyloidosis in transgenic mice expressing murine amyloidogenic apolipoprotein A-II (Apoa2c).

PubMed

Ge, Fengxia; Yao, Junjie; Fu, Xiaoying; Guo, Zhanjun; Yan, Jingmin; Zhang, Beiru; Zhang, Huanyu; Tomozawa, Hiroshi; Miyazaki, Junichi; Sawashita, Jinko; Mori, Masayuki; Higuchi, Keiichi

2007-07-01

In mice, apolipoprotein A-II (apoA-II) self-associates to form amyloid fibrils (AApoAII) in an age-associated manner. We postulated that the two most important factors in apoA-II amyloidosis are the Apoa2(c) allele, which codes for the amyloidogenic protein APOA2C (Gln5, Ala38) and transmission of amyloid fibrils. To characterize further the contribution of the Apoa2(c) allele to amyloidogenesis and improve detection of amyloidogenic materials, we established transgenic mice that overexpress APOA2C protein under the cytomegalovirus (CMV) immediate early gene (CMV-IE) enhancer/chicken beta promoter. Compared to transgene negative (Tg(-/-)) mice that express apoA-II protein mainly in the liver, mice homozygous (Tg(+/+)) and heterozygous (Tg(+/-)) for the transgene express a high level of apoA-II protein in many tissues. They also have higher plasma concentrations of apoA-II, higher ratios of ApoA-II/apolipoprotein A-I (ApoA-I) and higher concentrations of high-density lipoprotein (HDL) cholesterol. Following injection of AApoAII fibrils into Tg(+/+) mice, amyloid deposition was observed in the testis, liver, kidney, heart, lungs, spleen, tongue, stomach and intestine but not in the brain. In Tg(+/+) mice, but not in Tg(-/-) mice, amyloid deposition was induced by injection of less than 10(-8) mug AApoAII fibrils. Furthermore, deposition in Tg(+/+) mice occurred more rapidly and to a greater extent than in Tg(-/-) mice. These studies indicate that increased levels of APOA2C protein lead to earlier and greater amyloid deposition and enhanced sensitivity to the transmission of amyloid fibrils in transgenic mice. This transgenic mouse model should prove valuable for studies of amyloidosis.

Metabolic changes in transgenic maize mature seeds over-expressing the Aspergillus niger phyA2.

PubMed

Rao, Jun; Yang, Litao; Guo, Jinchao; Quan, Sheng; Chen, Guihua; Zhao, Xiangxiang; Zhang, Dabing; Shi, Jianxin

2016-02-01

Non-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2. Genetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC-MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

Increased phytochrome B alleviates density effects on tuber yield of field potato crops.

PubMed

Boccalandro, Hernán E; Ploschuk, Edmundo L; Yanovsky, Marcelo J; Sánchez, Rodolfo A; Gatz, Christiane; Casal, Jorge J

2003-12-01

The possibility that reduced photomorphogenic responses could increase field crop yield has been suggested often, but experimental support is still lacking. Here, we report that ectopic expression of the Arabidopsis PHYB (phytochrome B) gene, a photoreceptor involved in detecting red to far-red light ratio associated with plant density, can increase tuber yield in field-grown transgenic potato (Solanum tuberosum) crops. Surprisingly, this effect was larger at very high densities, despite the intense reduction in the red to far-red light ratios and the concomitant narrowed differences in active phytochrome B levels between wild type and transgenics at these densities. Increased PHYB expression not only altered the ability of plants to respond to light signals, but they also modified the light environment itself. This combination resulted in larger effects of enhanced PHYB expression on tuber number and crop photosynthesis at high planting densities. The PHYB transgenics showed higher maximum photosynthesis in leaves of all strata of the canopy, and this effect was largely due to increased leaf stomatal conductance. We propose that enhanced PHYB expression could be used in breeding programs to shift optimum planting densities to higher levels.

Hydrogel Macroporosity and the Prolongation of Transgene Expression and the Enhancement of Angiogenesis

PubMed Central

Shepard, Jaclyn A.; Virani, Farrukh R.; Goodman, Ashley G.; Gossett, Timothy D.; Shin, Seungjin; Shea, Lonnie D.

2012-01-01

The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37°C. The macropores were interconnected, as evidenced by homogeneous cell seeding in vitro and complete cell infiltration in vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications. PMID:22800542

Promoting scopolamine biosynthesis in transgenic Atropa belladonna plants with pmt and h6h overexpression under field conditions.

PubMed

Xia, Ke; Liu, Xiaoqiang; Zhang, Qiaozhuo; Qiang, Wei; Guo, Jianjun; Lan, Xiaozhong; Chen, Min; Liao, Zhihua

2016-09-01

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94-5.13 mg g(-1)) in field conditions, can provide more valuable plant materials for scopolamine production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

PubMed Central

Bidlack, Felicitas B.; Xia, Yan; Pugach, Megan K.

2017-01-01

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel. PMID:29201008

Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo.

PubMed

Bidlack, Felicitas B; Xia, Yan; Pugach, Megan K

2017-01-01

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes ( Tg ) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180 Tg , CTRNC Tg and LRAP Tg mice to generate M180 Tg and CTRNC Tg double transgene and M180 Tg , CTRNC Tg , LRAP Tg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene ( p < 0.05). The presence of the LRAP Tg did not improve the phenotype of M180 Tg /CTRNC Tg /KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth

PubMed Central

Kim, Hyun Uk; Lee, Kyeong-Ryeol; Jung, Su-Jin; Shin, Hyun A; Go, Young Sam; Suh, Mi-Chung; Kim, Jong Bum

2017-01-01

Summary The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves do not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a 3-fold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol, were reduced more in senescence-induced LEC2 than endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Senescence-induced LEC2 upregulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expression of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues. PMID:25790072

Senescence-inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth.

PubMed

Kim, Hyun Uk; Lee, Kyeong-Ryeol; Jung, Su-Jin; Shin, Hyun A; Go, Young Sam; Suh, Mi-Chung; Kim, Jong Bum

2015-12-01

The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves does not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a threefold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol were reduced more in senescence-induced LEC2 than in endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Senescence-induced LEC2 up-regulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expressions of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

PubMed

Wert, S E; Glasser, S W; Korfhagen, T R; Whitsett, J A

1993-04-01

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.

E-Peptides Control Bioavailability of IGF-1

PubMed Central

Piszczek, Agnieszka; Perlas, Emarald; Winn, Nadine; Nastasi, Tommaso; Rosenthal, Nadia

2012-01-01

Insulin-like growth factor 1 (IGF-1) is a potent cytoprotective growth factor that has attracted considerable attention as a promising therapeutic agent. Transgenic over-expression of IGF-1 propeptides facilitates protection and repair in a broad range of tissues, although transgenic mice over-expressing IGF-1 propeptides display little or no increase in IGF-1 serum levels, even with high levels of transgene expression. IGF-1 propeptides are encoded by multiple alternatively spliced transcripts including C-terminal extension (E) peptides, which are highly positively charged. In the present study, we use decellularized mouse tissue to show that the E-peptides facilitate in vitro binding of murine IGF-1 to the extracellular matrix (ECM) with varying affinities. This property is independent of IGF-1, since proteins consisting of the E-peptides fused to relaxin, a related member of the insulin superfamily, bound equally avidly to decellularized ECM. Thus, the E-peptides control IGF-1 bioavailability by preventing systemic circulation, offering a potentially powerful way to tether IGF-1 and other therapeutic proteins to the site of synthesis and/or administration. PMID:23251442

The physiological response of Populus tremula x alba leaves to the down-regulation of PIP1 aquaporin gene expression under no water stress

PubMed Central

Secchi, Francesca; Zwieniecki, Maciej A.

2013-01-01

In order to study the role of PIP1 aquaporins in leaf water and CO2 transport, several lines of PIP1-deficient transgenic Populus tremula x alba were generated using a reverse genetic approach. These transgenic lines displayed no visible developmental or morphological phenotypes when grown under conditions of no water stress. Major photosynthetic parameters were also not affected by PIP1 down regulation. However, low levels of PIP1 expression resulted in greater leaf hydraulic resistance (an increase of 27%), which effectively implicated PIP1 role in water transport. Additionally, the expression level of PIP1 genes in the various transgenic lines was correlated with reductions in mesophyll conductance to CO2 (gm), suggesting that in poplar, these aquaporins influenced membrane permeability to CO2. Overall, although analysis showed that PIP1 genes contributed to the mass transfer of water and CO2 in poplar leaves, their down-regulation did not dramatically impair the physiological needs of this fast growing tree when cultivated under conditions of no stress. PMID:24379822

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

PubMed

Boosalis, Michael S; Sangerman, Jose I; White, Gary L; Wolf, Roman F; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H; Li, Biaoru; Pace, Betty S; Nouraie, Mehdi; Faller, Douglas V; Perrine, Susan P

2015-01-01

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate.

PubMed

Arwert, Esther N; Lal, Rohit; Quist, Sven; Rosewell, Ian; van Rooijen, Nico; Watt, Fiona M

2010-11-16

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

Production of phytotoxic cationic α-helical antimicrobial peptides in plant cells using inducible promoters.

PubMed

Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

2014-01-01

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes.

Production of Phytotoxic Cationic α-Helical Antimicrobial Peptides in Plant Cells Using Inducible Promoters

PubMed Central

Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

2014-01-01

Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes. PMID:25387106

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin.

PubMed

Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2009-06-30

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

PubMed Central

Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2009-01-01

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida–mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies. PMID:19549857

Molecular cloning and functional analysis of the drought tolerance gene MsHSP70 from alfalfa (Medicago sativa L.).

PubMed

Li, Zhenyi; Long, Ruicai; Zhang, Tiejun; Wang, Zhen; Zhang, Fan; Yang, Qingchuan; Kang, Junmei; Sun, Yan

2017-03-01

Heat shock proteins (HSPs) are a ubiquitously expressed class of protective proteins that play a key role in plant response to stressful conditions. This study aimed to characterize and investigate the function of an HSP gene in alfalfa (Medicago sativa). MsHSP70, which contains a 2028-bp open reading frame, was identified through homology cloning. MsHSP70 shares high sequence identity (94.47%) with HSP70 from Medicago truncatula. Expression analysis of MsHSP70 in alfalfa organs revealed a relatively higher expression level in aerial organs such as flowers, stems and leaves than in roots. MsHSP70 was induced by heat shock, abscisic acid (ABA) and hydrogen peroxide. Transgenic Arabidopsis seedlings overexpressing MsHSP70 were hyposensitive to polyethylene glycol (PEG) and ABA treatments, suggesting that exogenous expression of MsHSP70 enhanced Arabidopsis tolerance to these stresses. Examination of physiological indexes related to drought and ABA stress demonstrated that in comparison with non-transgenic plants, T3 transgenic Arabidopsis plants had an increased proline content, higher superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content. Furthermore, higher relative water content (RWC) was detected in transgenic plants compared with non-transgenic plants under drought stress. These findings clearly indicate that molecular manipulation of MsHSP70 in plants can have substantial effects on stress tolerance.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

PubMed Central

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A.; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P.

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches. PMID:29423380

Mice Orally Immunized with a Transgenic Plant Expressing the Glycoprotein of Crimean-Congo Hemorrhagic Fever Virus ▿

PubMed Central

Ghiasi, S. M.; Salmanian, A. H.; Chinikar, S.; Zakeri, S.

2011-01-01

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories. PMID:22012978

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24.

PubMed

Huang, Xuan; Wang, Jian; Du, Zhen; Zhang, Chen; Li, Lan; Xu, Ziqin

2013-10-01

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T1 lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T1 plants. Resistance to stripe rust in transgenic T2 and T3 XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27-36 %) was recorded for transgenic T2 and T3 XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat.

Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus.

PubMed

Ghiasi, S M; Salmanian, A H; Chinikar, S; Zakeri, S

2011-12-01

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley.

PubMed

Chauhan, Harsh; Boni, Rainer; Bucher, Rahel; Kuhn, Benjamin; Buchmann, Gabriele; Sucher, Justine; Selter, Liselotte L; Hensel, Goetz; Kumlehn, Jochen; Bigler, Laurent; Glauser, Gaëtan; Wicker, Thomas; Krattinger, Simon G; Keller, Beat

2015-10-01

The wheat gene Lr34 encodes an ABCG-type transporter which provides durable resistance against multiple pathogens. Lr34 is functional as a transgene in barley, but its mode of action has remained largely unknown both in wheat and barley. Here we studied gene expression in uninfected barley lines transgenic for Lr34. Genes from multiple defense pathways contributing to basal and inducible disease resistance were constitutively active in seedlings and mature leaves. In addition, the hormones jasmonic acid and salicylic acid were induced to high levels, and increased levels of lignin as well as hordatines were observed. These results demonstrate a strong, constitutive re-programming of metabolism by Lr34. The resistant Lr34 allele (Lr34res) encodes a protein that differs by two amino acid polymorphisms from the susceptible Lr34sus allele. The deletion of a single phenylalanine residue in Lr34sus was sufficient to induce the characteristic Lr34-based responses. Combination of Lr34res and Lr34sus in the same plant resulted in a reduction of Lr34res expression by 8- to 20-fold when the low-expressing Lr34res line BG8 was used as a parent. Crosses with the high-expressing Lr34res line BG9 resulted in an increase of Lr34sus expression by 13- to 16-fold in progenies that inherited both alleles. These results indicate an interaction of the two Lr34 alleles on the transcriptional level. Reduction of Lr34res expression in BG8 crosses reduced the negative pleiotropic effects of Lr34res on barley growth and vigor without compromising disease resistance, suggesting that transgenic combination of Lr34res and Lr34sus can result in agronomically useful resistance. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Transcriptional-metabolic networks in beta-carotene-enriched potato tubers: the long and winding road to the Golden phenotype.

PubMed

Diretto, Gianfranco; Al-Babili, Salim; Tavazza, Raffaela; Scossa, Federico; Papacchioli, Velia; Migliore, Melania; Beyer, Peter; Giuliano, Giovanni

2010-10-01

Vitamin A deficiency is a public health problem in a large number of countries. Biofortification of major staple crops (wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and potato [Solanum tuberosum]) with β-carotene has the potential to alleviate this nutritional problem. Previously, we engineered transgenic "Golden" potato tubers overexpressing three bacterial genes for β-carotene synthesis (CrtB, CrtI, and CrtY, encoding phytoene synthase, phytoene desaturase, and lycopene β-cyclase, respectively) and accumulating the highest amount of β-carotene in the four aforementioned crops. Here, we report the systematic quantitation of carotenoid metabolites and transcripts in 24 lines carrying six different transgene combinations under the control of the 35S and Patatin (Pat) promoters. Low levels of B-I expression are sufficient for interfering with leaf carotenogenesis, but not for β-carotene accumulation in tubers and calli, which requires high expression levels of all three genes under the control of the Pat promoter. Tubers expressing the B-I transgenes show large perturbations in the transcription of endogenous carotenoid genes, with only minor changes in carotenoid content, while the opposite phenotype (low levels of transcriptional perturbation and high carotenoid levels) is observed in Golden (Y-B-I) tubers. We used hierarchical clustering and pairwise correlation analysis, together with a new method for network correlation analysis, developed for this purpose, to assess the perturbations in transcript and metabolite levels in transgenic leaves and tubers. Through a "guilt-by-profiling" approach, we identified several endogenous genes for carotenoid biosynthesis likely to play a key regulatory role in Golden tubers, which are candidates for manipulations aimed at the further optimization of tuber carotenoid content.

Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

PubMed Central

Ramalho, José S; Anders, Ross; Jaissle, Gesine B; Seeliger, Mathias W; Huxley, Clare; Seabra, Miguel C

2002-01-01

Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases. PMID:12401133

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

PubMed

Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

2013-05-01

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

Arabidopsis gene expression patterns are altered during spaceflight

NASA Astrophysics Data System (ADS)

Paul, Anna-Lisa; Popp, Michael P.; Gurley, William B.; Guy, Charles; Norwood, Kelly L.; Ferl, Robert J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613-621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced - but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment opportunities.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

PubMed

Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

2016-04-01

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

PubMed

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae.

PubMed

Mahmoud, Sulley Ben; Ramos, John E; Shatters, Robert G; Hall, David G; Lapointe, Stephen L; Niedz, Randall P; Rougé, Pierre; Cave, Ronald D; Borovsky, Dov

2017-03-01

Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (10 9 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×10 6 cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus. Published by Elsevier Inc.

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

PubMed

Ryan, G R; Dai, X M; Dominguez, M G; Tong, W; Chuan, F; Chisholm, O; Russell, R G; Pollard, J W; Stanley, E R

2001-07-01

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Infertility in transgenic mice overexpressing the bovine growth hormone gene: luteal failure secondary to prolactin deficiency.

PubMed

Cecim, M; Kerr, J; Bartke, A

1995-05-01

Overexpression of growth hormone (GH) in transgenic mice is associated with various degrees of impairment of female reproductive functions. Transgenic PEPCK.bGH mice express high GH levels, and only around 20% of the females will carry gestation to Day 7. The objective of the present study was to investigate luteal function in PEPCK.bGH mice during early pregnancy, when CL are fully dependent on the pituitary. Plasma progesterone levels measured on Days 2 or 7 postcoitum (p.c.) were lower in transgenic than in normal females. In transgenic females with a previous history of infertility, daily injections of 1 mg progesterone starting on Day 2 p.c. significantly increased the proportion of animals pregnant on Day 7. When ovaries from transgenic mice were transplanted into ovariectomized normal littermates, the recipients exhibited normal vaginal cycles and responded to mating by vaginal cytology changes consistent with pseudopregnancy. In contrast, ovariectomized transgenic females bearing transplants of ovaries from normal mice had slightly prolonged estrous cycles and failed to become pseudopregnant after mating. Plasma progesterone levels on Days 2 and 7 p.c. in normal females with transgenic ovaries were not different from plasma progesterone levels measured in normal females into which normal ovaries had been transplanted. Twice-daily injections of 100 micrograms of prolactin (PRL) in saline or in polyvinylpyrrolidone starting on the evening of Day 2 p.c. were able to rescue luteal function. The proportion of PRL-injected transgenic animals that were pregnant on Day 7 was significantly higher than that of saline-injected transgenic controls and resembled the pregnancy rate of normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.

PubMed

Zhou, Fei; Badillo-Corona, Jesus A; Karcher, Daniel; Gonzalez-Rabade, Nuria; Piepenburg, Katrin; Borchers, A-M Inka; Maloney, Alan P; Kavanagh, Tony A; Gray, John C; Bock, Ralph

2008-12-01

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

PubMed Central

Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

2013-01-01

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

Gene transfer strategies in animal transgenesis.

PubMed

Montoliu, Lluís

2002-01-01

Position effects in animal transgenesis have prevented the reproducible success and limited the initial expectations of this technique in many biotechnological projects. Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression domain. An expression domain is thought to contain all regulatory elements that are needed to specifically control the expression of a given gene in time and space. The lack of profound knowledge on the chromatin structure of expression domains of biotechnological interest, such as mammary gland-specific genes, explains why most standard expression vectors have failed to drive high-level, position-independent, and copy-number-dependent expression of transgenes in a reproducible manner. In contrast, the application of artificial chromosome-type constructs to animal transgenesis usually ensures optimal expression levels. YACs, BACs, and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant key regulatory sequences, previously unknown, that are characteristic for each expression domain. These elements contribute to insulating the artificial chromosome-type constructs from chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes.

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

PubMed

Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

2011-02-01

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

PubMed

Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

2017-09-15

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

PubMed Central

ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

2009-01-01

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune responses against lentiviral vectors was effective in causing a decrease in transgene expression only if the immune response was directed against the transgene. A systemic immune response against vector components alone did not cause brain inflammation, possibly because vector-derived epitopes were not being presented in the CNS. PMID:15960605

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

PubMed

Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Selective reconstitution of liver cholesterol biosynthesis promotes lung maturation but does not prevent neonatal lethality in Dhcr7 null mice.

PubMed

Yu, Hongwei; Li, Man; Tint, G Stephen; Chen, Jianliang; Xu, Guorong; Patel, Shailendra B

2007-04-04

Targeted disruption of the murine 3beta-hydroxysterol-Delta7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver. We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice. The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity.

Transgenic rats with green, red, and blue fluorescence: powerful tools for bioimaging, cell trafficking, and differentiation

NASA Astrophysics Data System (ADS)

Murakami, Takashi; Kobayashi, Eiji

2005-04-01

The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats" system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

PubMed

Liu, Desheng; Makaroff, Christopher A

2015-03-05

Eco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth. To further investigate the function(s) of AtCTF7, a tagged version of AtCTF7 and several AtCTF7 deletion constructs were created and transformed into wild type or ctf7 +/- plants. Transgenic plants expressing 35S:NTAP:AtCTF7∆299-345 (AtCTF7∆B) displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes exhibited chromosome fragmentation and uneven chromosome segregation. Mutant ovules contained abnormal megasporocyte-like cells during pre-meiosis, megaspores experienced elongated meiosis and megagametogenesis, and defective megaspores/embryo sacs were produced at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and dwarfism that were inherited in a non-Mendelian fashion. Transcripts for epigenetically regulated transposable elements (TEs) were elevated in transgenic plants. Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B. High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

Gene expression profiling: cell cycle deregulation and aneuploidy do not cause breast cancer formation in WAP-SVT/t transgenic animals.

PubMed

Klein, Andreas; Guhl, Eva; Zollinger, Raphael; Tzeng, Yin-Jeh; Wessel, Ralf; Hummel, Michael; Graessmann, Monika; Graessmann, Adolf

2005-05-01

Microarray studies revealed that as a first hit the SV40 T/t antigen causes deregulation of 462 genes in mammary gland cells (ME cells) of WAP-SVT/t transgenic animals. The majority of deregulated genes are cell proliferation specific and Rb-E2F dependent, causing ME cell proliferation and gland hyperplasia but not breast cancer formation. In the breast tumor cells a further 207 genes are differentially expressed, most of them belonging to the cell communication category. In tissue culture breast tumor cells frequently switch off WAP-SVT/t transgene expression and regain the morphology and growth characteristics of normal ME cells, although the tumor-revertant cells are aneuploid and only 114 genes regain the expression level of normal ME cells. The profile of retransformants shows that only 38 deregulated genes are tumor-specific, and that none of them is considered to be a typical breast cancer gene.

Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.

PubMed

Hook, Vivian Y H; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-08-21

Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.

Chromatin insulation by a transcriptional activator

PubMed Central

Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

2003-01-01

In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

PubMed

Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2018-05-31

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka

PubMed Central

Froschauer, Alexander; Kube, Lisa; Kegler, Alexandra; Rieger, Christiane; Gutzeit, Herwig O.

2015-01-01

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level. PMID:26148066

Metabolic engineering of tomato fruit organic acid content guided by biochemical analysis of an introgression line.

PubMed

Morgan, Megan J; Osorio, Sonia; Gehl, Bernadette; Baxter, Charles J; Kruger, Nicholas J; Ratcliffe, R George; Fernie, Alisdair R; Sweetlove, Lee J

2013-01-01

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.

Metabolic Engineering of Tomato Fruit Organic Acid Content Guided by Biochemical Analysis of an Introgression Line1[W][OA

PubMed Central

Morgan, Megan J.; Osorio, Sonia; Gehl, Bernadette; Baxter, Charles J.; Kruger, Nicholas J.; Ratcliffe, R. George; Fernie, Alisdair R.; Sweetlove, Lee J.

2013-01-01

Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity. PMID:23166354

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

PubMed

Chew, Bee Lynn; Fisk, Ian D; Fray, Rupert; Tucker, Gregory A; Bodi, Zsuzsanna; Ferguson, Alison; Xia, Wei; Seymour, Graham B

2017-01-01

This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

[TSA improve transgenic porcine cloned embryo development and transgene expression].

PubMed

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines

PubMed Central

Takeda, Kozue; Iida, Machiko; Kumasaka, Mayuko; Matsumoto, Yoshinari; Kato, Masashi

2010-01-01

Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma. PMID:20422010

c-RET molecule in malignant melanoma from oncogenic RET-carrying transgenic mice and human cell lines.

PubMed

Ohshima, Yuichiro; Yajima, Ichiro; Takeda, Kozue; Iida, Machiko; Kumasaka, Mayuko; Matsumoto, Yoshinari; Kato, Masashi

2010-04-21

Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma.

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

PubMed Central

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses.

PubMed

Montero-Barrientos, Marta; Hermosa, Rosa; Cardoza, Rosa E; Gutiérrez, Santiago; Nicolás, Carlos; Monte, Enrique

2010-05-15

The ability of some Trichoderma strains, a biological control agent, to overcome extreme environmental conditions has previously been reported and related to heat-shock proteins (HSPs). These proteins are induced environmentally and are involved in important processes, acting as molecular chaperones in all organisms. In a previous study, we demonstrated, by overexpression, that the Trichoderma harzianum hsp70 gene conferred tolerance to heat and other abiotic stresses to this fungus. In this work, we investigate the function of the T. harzianum T34 hsp70 gene in Arabidopsis thaliana. We analyze transgenic plant responses under adverse environmental conditions and the expression levels of a set of seven stress genes, using quantitative RT-PCR. As expected, transgenic plants expressing the T. harzianum hsp70 gene exhibited enhanced tolerance to heat stress. In addition, they did not show growth inhibition and, after heat pre-treatment, transgenic seedlings were more tolerant to osmotic, salt and oxidative stresses with respect to the wild-type behavior. Transgenic lines also had increased transcript levels of the Na(+)/H(+) exchanger 1 (SOS1) and ascorbate peroxidase 1 (APX1) genes, involved in salt and oxidative stress responses, respectively. However, the heat-shock factor (HSF) and four HSP genes tested were down-regulated in 35S:hsp70 plants. Overall, our results indicate that hsp70 confers tolerance to heat and other abiotic stresses and that the fungal HSP70 protein acts as a negative regulator of the HSF transcriptional activity in Arabidopsis. (c) 2009 Elsevier GmbH. All rights reserved.

Mouse Mammary Tumor Virus c-rel Transgenic Mice Develop Mammary Tumors

PubMed Central

Romieu-Mourez, Raphaëlle; Kim, Dong W.; Min Shin, Sang; Demicco, Elizabeth G.; Landesman-Bollag, Esther; Seldin, David C.; Cardiff, Robert D.; Sonenshein, Gail E.

2003-01-01

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-κB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-κB. Analysis of the composition of NF-κB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-κB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-κB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis. PMID:12897145

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea.

PubMed

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L; Mukherjee, Sunil Kumar; Sahoo, Lingaraj

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea

PubMed Central

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L.; Mukherjee, Sunil Kumar

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea. PMID:29077738

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

PubMed

Merentie, Mari; Rissanen, Riina; Lottonen-Raikaslehto, Line; Huusko, Jenni; Gurzeler, Erika; Turunen, Mikko P; Holappa, Lari; Mäkinen, Petri; Ylä-Herttuala, Seppo

2018-01-01

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

A Universal Role for Inositol 1,4,5-Trisphosphate-Mediated Signaling in Plant Gravitropism1[W

PubMed Central

Perera, Imara Y.; Hung, Chiu-Yueh; Brady, Shari; Muday, Gloria K.; Boss, Wendy F.

2006-01-01

Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants. PMID:16384898

Cytokinin signaling regulates cambial development in poplar

PubMed Central

Nieminen, Kaisa; Immanen, Juha; Laxell, Marjukka; Kauppinen, Leila; Tarkowski, Petr; Dolezal, Karel; Tähtiharju, Sari; Elo, Annakaisa; Decourteix, Mélanie; Ljung, Karin; Bhalerao, Rishikesh; Keinonen, Kaija; Albert, Victor A.; Helariutta, Ykä

2008-01-01

Although a substantial proportion of plant biomass originates from the activity of vascular cambium, the molecular basis of radial plant growth is still largely unknown. To address whether cytokinins are required for cambial activity, we studied cytokinin signaling across the cambial zones of 2 tree species, poplar (Populus trichocarpa) and birch (Betula pendula). We observed an expression peak for genes encoding cytokinin receptors in the dividing cambial cells. We reduced cytokinin levels endogenously by engineering transgenic poplar trees (P. tremula × tremuloides) to express a cytokinin catabolic gene, Arabidopsis CYTOKININ OXIDASE 2, under the promoter of a birch CYTOKININ RECEPTOR 1 gene. Transgenic trees showed reduced concentration of a biologically active cytokinin, correlating with impaired cytokinin responsiveness. In these trees, both apical and radial growth was compromised. However, radial growth was more affected, as illustrated by a thinner stem diameter than in WT at same height. To dissect radial from apical growth inhibition, we performed a reciprocal grafting experiment. WT scion outgrew the diameter of transgenic stock, implicating cytokinin activity as a direct determinant of radial growth. The reduced radial growth correlated with a reduced number of cambial cell layers. Moreover, expression of a cytokinin primary response gene was dramatically reduced in the thin-stemmed transgenic trees. Thus, a reduced level of cytokinin signaling is the primary basis for the impaired cambial growth observed. Together, our results show that cytokinins are major hormonal regulators required for cambial development. PMID:19064928

Tumorigenic properties of iron regulatory protein 2 (IRP2) mediated by its specific 73-amino acids insert.

PubMed

Maffettone, Carmen; Chen, Guohua; Drozdov, Ignat; Ouzounis, Christos; Pantopoulos, Kostas

2010-04-13

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2(Delta73) (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2(Delta73) failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2(Delta73)-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology.

Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

PubMed Central

Maffettone, Carmen; Chen, Guohua; Drozdov, Ignat; Ouzounis, Christos; Pantopoulos, Kostas

2010-01-01

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2Δ73 (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2Δ73 failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2Δ73-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology. PMID:20405006

Transient, Inducible, Placenta-Specific Gene Expression in Mice

PubMed Central

Fan, Xiujun; Petitt, Matthew; Gamboa, Matthew; Huang, Mei; Dhal, Sabita; Druzin, Maurice L.; Wu, Joseph C.

2012-01-01

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues. PMID:23011919

GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury.

PubMed

Duann, Pu; Lianos, Elias A

2009-09-01

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury

PubMed Central

Duann, Pu; Lianos, Elias A.

2009-01-01

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-β1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury. PMID:19587144

Nuclear-encoded mitochondrial complex I gene expression is restored to normal levels by inhibition of unedited ATP9 transgene expression in Arabidopsis thaliana.

PubMed

Busi, María V; Gómez-Casati, Diego F; Perales, Mariano; Araya, Alejandro; Zabaleta, Eduardo

2006-01-01

Mitochondria play an important role during sporogenesis in plants. The steady state levels of the nuclear-encoded mitochondrial complex I (nCI), PSST, TYKY and NADHBP transcripts increase in flowers of male-sterile plants with impairment of mitochondrial function generated by the expression of the unedited version of ATP9 (u-ATP9). This suggests a nuclear control of nCI genes in response to the mitochondrial flaw. To evaluate this hypothesis, transgenic plants carrying the GUS reporter gene, under the control of the PSST, TYKY and NADHBP promoters, were constructed. We present evidence that suppression by antisense strategy of the expression of u-ATP9 restores the normal levels of three nCI transcripts, indicating that the increase in PSST, TYKY and NADHBP in plants with a mitochondrial flaw occurs at the transcriptional level. The data presented here support the hypothesis that a mitochondrial dysfunction triggers a retrograde signaling which induce some nuclear-encoded mitochondrial genes. Moreover, these results demonstrate that this is a valuable experimental model for studying nucleus-mitochondria cross-talk events.

Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts.

PubMed

Mishra, Pragya; Singh, Shweta; Rathinam, Maniraj; Nandiganti, Muralimohan; Ram Kumar, Nikhil; Thangaraj, Arulprakash; Thimmegowda, Vinutha; Krishnan, Veda; Mishra, Vagish; Jain, Neha; Rai, Vandna; Pattanayak, Debasis; Sreevathsa, Rohini

2017-02-22

Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

PubMed

Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

2016-03-01

Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

Muscle-specific transgenic expression of porcine myostatin propeptide enhances muscle growth in mice.

PubMed

Wang, Kaiyun; Li, Zicong; Li, Yang; Zeng, Jinyong; He, Chang; Yang, Jinzeng; Liu, Dewu; Wu, Zhenfang

2013-10-01

Myostatin is a well-known negative regulator of skeletal muscle growth. Inhibition of myostatin activity results in increased muscle mass. Myostatin propeptide, as a myostatin antagonist, could be applied to promote meat production in livestock such as pigs. In this study, we generated a transgenic mouse model expressing porcine myostatin propeptide under the control of muscle-specific regulatory elements. The mean body weight of transgenic mice from a line expressing the highest level of porcine myostatin propeptide was increased by 5.4 % (P = 0.023) and 3.2 % (P = 0.031) in males and females, respectively, at 8 weeks of age. Weight of carcass, fore limb and hind limb was respectively increased by 6.0 % (P = 0.038), 9.0 % (P = 0.014), 8.7 % (P = 0.036) in transgenic male mice, compared to wild-type male controls at the age of 9 weeks. Similarly, carcass, fore limb and hind limb of transgenic female mice was 11.4 % (P = 0.002), 14.5 % (P = 0.006) and 14.5 % (P = 0.03) respectively heavier than that of wild-type female mice. The mean cross-section area of muscle fiber was increased by 17 % (P = 0.002) in transgenic mice, in comparison with wild-type controls. These results demonstrated that porcine myostatin propeptide is effective in enhancement of muscle growth. The present study provided useful information for future study on generation of transgenic pigs overexpressing porcine myostatin propeptide for improvement of muscle mass.

A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.

PubMed

Chen, Eryong; Wang, Xiaoqian; Gong, Qian; Butt, Hamama Islam; Chen, Yanli; Zhang, Chaojun; Yang, Zuoren; Wu, Zhixia; Ge, Xiaoyang; Zhang, Xianlong; Li, Fuguang; Zhang, Xueyan

2017-09-05

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding. Copyright © 2017. Published by Elsevier B.V.

Persistence of transgene expression influences CD8+ T-cell expansion and maintenance following immunization with recombinant adenovirus.

PubMed

Finn, Jonathan D; Bassett, Jennifer; Millar, James B; Grinshtein, Natalie; Yang, Teng Chih; Parsons, Robin; Evelegh, Carole; Wan, Yonghong; Parks, Robin J; Bramson, Jonathan L

2009-12-01

Previous studies determined that the CD8(+) T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8(+) T-cell maintenance following immunization with a recombinant adenovirus.

Assaying for pollen drift from transgenic Rainbow to nontransgenic Kapoho papaya under commercial and experimental field conditions in Hawaii

USDA-ARS?s Scientific Manuscript database

In 1992 papaya ringpsot virus (PRSV) was discovered in Puna district of Hawaii Island where 95% of the state of Hawaii’s papaya was being grown. By 1998 production in Puna had decreased 50% from 1998 levels. A PRSV-resistant transgenic papaya ‘Rainbow’ expressing the coat protein gene of PRSV was ...

Potential Effects of Oilseed Rape Expressing Oryzacystatin-1 (OC-1) and of Purified Insecticidal Proteins on Larvae of the Solitary Bee Osmia bicornis

PubMed Central

Konrad, Roger; Ferry, Natalie; Gatehouse, Angharad M. R.; Babendreier, Dirk

2008-01-01

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis ( = O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products. PMID:18628826

Expression of the Beet necrotic yellow vein virus p25 protein induces hormonal changes and a root branching phenotype in Arabidopsis thaliana.

PubMed

Peltier, Claire; Schmidlin, Laure; Klein, Elodie; Taconnat, Ludivine; Prinsen, Els; Erhardt, Mathieu; Heintz, Dimitri; Weyens, Guy; Lefebvre, Marc; Renou, Jean-Pierre; Gilmer, David

2011-06-01

The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.

Pathogen Phytosensing: Plants to Report Plant Pathogens.

PubMed

Mazarei, Mitra; Teplova, Irina; Hajimorad, M Reza; Stewart, C Neal

2008-04-14

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or 'phytosensors', by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable reporter gene could provide biological evidence to define the functional differences between pathogens, and provide new technology and applications for transgenic plants as phytosensors.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

PubMed

Yu, Dawei; Zhang, Shoufeng; Du, Weihua; Zhang, Jinxia; Fan, Zongxing; Hao, Haisheng; Liu, Yan; Zhao, Xueming; Qin, Tong; Zhu, Huabin

2014-01-01

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L.

PubMed

El-Esawi, Mohamed A; Elkelish, Amr; Elansary, Hosam O; Ali, Hayssam M; Elshikh, Mohamed; Witczak, Jacques; Ahmad, Margaret

2017-01-01

Lactuca serriola L. is a herbaceous species, used for human nutrition and medicinal purposes. The high antioxidant capacity of L. serriola indicates the possibility of enhancing its edible and health potential by increasing the flavonoid and phenolic contents. The present study aimed at enhancing the production of phenolics and flavonoids by hairy root cultures in Lactuca serriola transformed with Agrobacterium rhizogenes strain AR15834 harbouring the rolB gene. The genetic transformation of rolB in transformed roots was validated, and rolB expression level was evaluated using real-time qPCR analysis. Expression levels of flavonoid biosynthesis genes (CHI, PAL, FLS, and CHS) were assessed in the hairy and nontransformed roots. Results showed higher expression levels in the transgenic roots than in the nontransformed ones ( p < 0.01). Transgenic hairy roots exhibited a 54.8-96.7% increase in the total phenolic content, 38.1-76.2% increase in the total flavonoid content, and 56.7-96.7% increase in the total reducing power when compared with the nontransgenic roots ( p < 0.01). DPPH results also revealed that the transgenic hairy roots exhibited a 31.6-50% increase in antioxidant potential, when compared to normal roots. This study addressed the enhancement of secondary metabolite biosynthesis by hairy root induction in L. serriola .

Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds1

PubMed Central

Chen, Guoping; Hackett, Rachel; Walker, David; Taylor, Andy; Lin, Zhefeng; Grierson, Donald

2004-01-01

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed. PMID:15347800

Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

PubMed Central

Vincentz, M; Caboche, M

1991-01-01

A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level. Images PMID:2022181

Ectopic Expression of GsPPCK3 and SCMRP in Medicago sativa Enhances Plant Alkaline Stress Tolerance and Methionine Content

PubMed Central

Zhao, Yang; Zhao, Chaoyue; DuanMu, Huizi; Yu, Yang; Ji, Wei; Zhu, Yanming

2014-01-01

So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions. However, the biological functions of soybean PPCKs, especially in alkali stress response, are not yet well known. In previous studies, we constructed a Glycine soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2 and GsPPCK3) as potential alkali stress responsive genes. In this study, we confirmed the induced expression of GsPPCK3 under alkali stress and investigated its tissue expression specificity by using quantitative real-time PCR analysis. Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3 overexpression improved plant alkali tolerance, as evidenced by lower levels of relative ion leakage and MDA content and higher levels of chlorophyll content and root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP genes into alfalfa, and demonstrated the increased alkali tolerance of GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP co-overexpression promoted the PEPC activity, net photosynthetic rate and citric acid content of transgenic alfalfa under alkali stress. Moreover, we also observed the up-regulated expression of PEPC, CS (citrate synthase), H+-ATPase and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher methionine content than wild type alfalfa. Taken together, results presented in this study supported the positive role of GsPPCK3 in plant response to alkali stress, and provided an effective way to simultaneously improve plant alkaline tolerance and methionine content, at least in legume crops. PMID:24586886

Gene Transfer to Chicks Using Lentiviral Vectors Administered via the Embryonic Chorioallantoic Membrane

PubMed Central

Hen, Gideon; Yosefi, Sara; Shinder, Dmitry; Or, Adi; Mygdal, Sivan; Condiotti, Reba; Galun, Eithan; Bor, Amir; Sela-Donenfeld, Dalit; Friedman-Einat, Miriam

2012-01-01

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides. PMID:22606269

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

PubMed

Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

2017-08-22

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

Transgene Expression and Repression in Transgenic Rats Bearing the Phosphoenolpyruvate Carboxykinase-Simian Virus 40 T Antigen or the Phosphoenolpyruvate Carboxykinase-Transforming Growth Factor-α Constructs

PubMed Central

Haas, Michael J.; Dragan, Yvonne P.; Hikita, Hiroshi; Shimel, Randee; Takimoto, Koichi; Heath, Susan; Vaughan, Jennifer; Pitot, Henry C.

1999-01-01

Transgenic Sprague-Dawley rats expressing either human transforming growth factor-α (TGFα) or simian virus 40 large and small T antigen (TAg), each under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, were developed as an approach to the study of the promotion of hepatocarcinogenesis in the presence of a transgene regulatable by diet and/or hormones. Five lines of PEPCK-TGFα transgenic rats were established, each genetic line containing from one to several copies of the transgene per haploid genome. Two PEPCK-TAg transgenic founder rats were obtained, each with multiple copies of the transgene. Expression of the transgene was undetectable in the TGFα transgenic rats and could not be induced when the animals were placed on a high-protein, low-carbohydrate diet. The transgene was found to be highly methylated in all of these lines. No pathological alterations in the liver and intestine were observed at any time (up to 2 years) during the lives of these rats. One line of transgenic rats expressing the PEPCK-TAg transgene developed pancreatic islet cell hyperplasias and carcinomas, with few normal islets evident in the pancreas. This transgene is integrated as a hypomethylated tandem array of 10 to 12 copies on chromosome 8q11. Expression of large T antigen is highest in pancreatic neoplasms, but is also detectable in the normal brain, kidney, and liver. Mortality is most rapid in males, starting at 5 months of age and reaching 100% by 8 months. Morphologically, islet cell differentiation in the tumors ranges from poor to well differentiated, with regions of necrosis and fibrosis. Spontaneous metastasis of TAg-positive tumor cells to regional lymph nodes was observed. These studies indicate the importance of DNA methylation in the repression of specific transgenes in the rat. However, the expression of the PEPCK-TAg induces neoplastic transformation in islet cells, probably late in neuroendocrine cell differentiation. T antigen expression during neoplastic development may result in a pervasive change in the islet cell growth properties with selection of a transformed phenotype as a possible requirement for cell viability. PMID:10393850

Age-dependent changes in nitric oxide synthase activity and protein expression in striata of mice transgenic for the Huntington's disease mutation.

PubMed

Pérez-Severiano, Francisca; Escalante, Bruno; Vergara, Paula; Ríos, Camilo; Segovia, José

2002-09-27

Huntington's disease (HD) is an autosomal hereditary neurodegenerative disorder caused by an abnormal expansion of the CAG repeats that code for a polyglutamine tract in a novel protein called huntingtin (htt). Both patients and experimental animals exhibit oxidative damage in specific areas of the brain, particularly the striatum. Nitric oxide (NO) is involved in many different physiological processes, and under pathological conditions it may promote oxidative damage through the formation of the highly reactive metabolite peroxynitrite; however, it may also play a role protecting cells from oxidative damage. We previously showed a correlation between the progression of the neurological phenotype and striatal oxidative damage in a line of transgenic mice, R6/1, which expresses a human mutated htt exon 1 with 116 CAG repeats. The purpose of the present work was to explore the participation of NO in the progressive oxidative damage that occurs in the striata of R6/1 mice. We analyzed the role of NO by measuring the activity of nitric oxide synthase (NOS) in the striata of transgenic and control mice at different ages. There was no difference in NOS activity between transgenic and wild-type mice at 11 weeks of age. In contrast, 19-week-old transgenic mice showed a significant increase in NOS activity, compared with same age controls. By 35 weeks of age, there was a decrease in NOS activity in transgenic mice when compared with wild-type controls. NOS protein expression was also determined in 11-, 19- and 35-week-old transgenic mice and wild-type littermates. Our results show increased neuronal NOS expression in 19-week-old transgenic mice, followed by a decreased level in 35-week-old mice, compared with controls, a phenomenon that parallels the changes in NOS enzyme activity. The present results suggest that NO is involved in the process leading to striatal oxidative damage and that it is associated with the onset of the progressive neurological phenotype in mice transgenic for the HD mutation.

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

PubMed

Matsui, Takeshi; Takita, Eiji; Sato, Toshio; Aizawa, Michie; Ki, Misa; Kadoyama, Yumiko; Hirano, Kenji; Kinjo, Satoko; Asao, Hiroshi; Kawamoto, Keiko; Kariya, Haruko; Makino, Sou-Ichi; Hamabata, Takashi; Sawada, Kazutoshi; Kato, Ko

2011-08-01

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

Effect of various classes of pesticides on expression of stress genes in transgenic C. elegans model of Parkinson's disease.

PubMed

Jadiya, Pooja; Mir, Snober S; Nazir, Aamir

2012-12-01

Neurodegenerative diseases are known to be associated with genetic and environmental factors. The multifactorial Parkinson's disease (PD) is triggered and/or further worsened by exposure to certain pesticides. Existing literature suggests a link between pesticide exposure and increased incidence of PD. We carried out the present study to look into the stress gene expression pattern of transgenic Caenorhabditis elegans (C. elegans) model of PD after exposure to pesticides from different classes. Expression level of sod-1, sod-2, sod-3, hsp-70, hsp-60, and hsp-16.2 stress responsive genes was determined using qPCR. Our findings demonstrate that the expression of stress related genes does not follow a generalized pattern to different toxicants; rather each pesticide class has a specific expression signature.

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

PubMed

Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

2016-11-01

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A

PubMed Central

Gururani, Mayank Anand; Venkatesh, Jelli; Ganesan, Markkandan; Strasser, Reto Jörg; Han, Yunjeong; Kim, Jeong-Il; Lee, Hyo-Yeon; Song, Pill-Soon

2015-01-01

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions. PMID:26010864

Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants.

PubMed

Kotakis, Christos; Vrettos, Nicholas; Kotsis, Dimitrios; Tsagris, Mina; Kotzabasis, Kiriakos; Kalantidis, Kriton

2010-10-12

Expression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors. Here we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery. Light intensity is one of the previously unknown factors that affect transgene stability at the post-transcriptional level. Our findings demonstrate an example of how environmental conditions could affect RNA silencing.

Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease.

PubMed

Ignacimuthu, S; Ceasar, S Antony

2012-03-01

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

Selection of differently temporally regulated African swine fever virus promoters with variable expression activities and their application for transient and recombinant virus mediated gene expression.

PubMed

Portugal, Raquel S; Bauer, Anja; Keil, Guenther M

2017-08-01

African swine fever virus threatens pig production worldwide due to the lack of vaccines, for which generation of both deletion and insertion mutants is considered. For development of the latter, operational ASFV promoters of different temporal regulation and strengths are desirable. We therefore compared the capacities of putative promoter sequences from p72, CD2v, p30, viral DNA polymerase and U104L genes to mediate expression of luciferase from transfected plasmids after activation in trans, or p30-, DNA polymerase- and U104L promoters in cis, using respective ASFV recombinants. We identified sequences with promoter activities upstream the viral ORFs, and showed that they differ in both their expression intensity regulating properties and in their temporal regulation. In summary, p30 and DNA polymerase promoters are recommended for high level early regulated transgene expression. For late expression, the p72, CD2v and U104L promoter are suitable. The latter however, only if low level transgene expression is aimed. Copyright © 2017 Elsevier Inc. All rights reserved.

Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

PubMed Central

Zha, Wenjun; Peng, Xinxin; Chen, Rongzhi; Du, Bo; Zhu, Lili; He, Guangcun

2011-01-01

Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants. PMID:21655219

Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

PubMed Central

2012-01-01

Background RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers. Results Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. Conclusions AC2 RSS in transgenic tobacco plants interferes with the silencing machinery. It causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. The changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level. PMID:23130567

Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed

PubMed Central

2011-01-01

Background High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling. Results Phenotypic analysis of one protein that was upregulated during salt-induced stress, cyclophilin 2 (OsCYP2), indicated that OsCYP2 transgenic rice seedlings had better tolerance to salt stress than did wild-type seedlings. Interestingly, wild-type seedlings exhibited a marked reduction in maximal photochemical efficiency under salt stress, whereas no such change was observed for OsCYP2-transgenic seedlings. OsCYP2-transgenic seedlings had lower levels of lipid peroxidation products and higher activities of antioxidant enzymes than wild-type seedlings. Spatiotemporal expression analysis of OsCYP2 showed that it could be induced by salt stress in both Shanyou 10 and Liangyoupeijiu seedlings, but Shanyou 10 seedlings showed higher OsCYP2 expression levels. Moreover, circadian rhythm expression of OsCYP2 in Shanyou 10 seedlings occurred earlier than in Liangyoupeijiu seedlings. Treatment with PEG, heat, or ABA induced OsCYP2 expression in Shanyou 10 seedlings but inhibited its expression in Liangyoupeijiu seedlings. Cold stress inhibited OsCYP2 expression in Shanyou 10 and Liangyoupeijiu seedlings. In addition, OsCYP2 was strongly expressed in shoots but rarely in roots in two rice hybrid varieties. Conclusions Together, these data suggest that OsCYP2 may act as a key regulator that controls ROS level by modulating activities of antioxidant enzymes at translation level. OsCYP2 expression is not only induced by salt stress, but also regulated by circadian rhythm. Moreover, OsCYP2 is also likely to act as a key component that is involved in signal pathways of other types of stresses-PEG, heat, cold, or ABA. PMID:21324151

Molecular Imaging of Vasa Vasorum Neovascularization via DEspR-targeted Contrast-enhanced Ultrasound Micro-imaging in Transgenic Atherosclerosis Rat Model

PubMed Central

Decano, Julius L.; Moran, Anne Marie; Ruiz-Opazo, Nelson; Herrera, Victoria L. M.

2011-01-01

Purpose Given that carotid vasa vasorum neovascularization is associated with increased risk for stroke and cardiac events, the present in vivo study was designed to investigate molecular imaging of carotid artery vasa vasorum neovascularization via target-specific contrast-enhanced ultrasound (CEU) micro-imaging. Procedures Molecular imaging was performed in male transgenic rats with carotid artery disease and non-transgenic controls using dual endothelin1/VEGFsp receptor (DEspR)-targeted microbubbles (MBD) and the Vevo770 micro-imaging system and CEU imaging software. Results DEspR-targeted CEU-positive imaging exhibited significantly higher contrast intensity signal (CIS)-levels and pre-/post-destruction CIS-differences in seven of 13 transgenic rats, in contrast to significantly lower CIS-levels and differences in control isotype-targeted microbubble (MBC)-CEU imaging (n =8) and in MBD CEU-imaging of five non-transgenic control rats (P<0.0001). Ex vivo immunofluorescence analysis demonstrated binding of MBD to DEspR-positive endothelial cells; and association of DEspR-targeted increased contrast intensity signals with DEspR expression in vasa vasorum neovessel and intimal lesions. In vitro analysis demonstrated dose-dependent binding of MBD to DEspR-positive human endothelial cells with increasing %cells bound and number of MBD per cell, in contrast to MBC or non-labeled microbubbles (P<0.0001). Conclusion In vivo DEspR-targeted molecular imaging detected increased DEspR-expression in carotid artery lesions and in expanded vasa vasorum neovessels in transgenic rats with carotid artery disease. Future studies are needed to determine predictive value for stroke or heart disease in this transgenic atherosclerosis rat model and translational applications. PMID:20972637

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo

PubMed Central

Iqbal, Asif J.; McNeill, Eileen; Kapellos, Theodore S.; Regan-Komito, Daniel; Norman, Sophie; Burd, Sarah; Smart, Nicola; Machemer, Daniel E. W.; Stylianou, Elena; McShane, Helen; Channon, Keith M.; Chawla, Ajay

2014-01-01

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115+ monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow–derived CD68-GFP monocytes to that of CX3CR1GFP monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1GFP monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. PMID:25030063

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

PubMed

Iqbal, Asif J; McNeill, Eileen; Kapellos, Theodore S; Regan-Komito, Daniel; Norman, Sophie; Burd, Sarah; Smart, Nicola; Machemer, Daniel E W; Stylianou, Elena; McShane, Helen; Channon, Keith M; Chawla, Ajay; Greaves, David R

2014-10-09

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation. © 2014 by The American Society of Hematology.

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

PubMed

Ghanbari Safari, Maryam; Baesi, Kazem; Hosseinkhani, Saman

2017-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Pancreatic Transduction by Helper-Dependent Adenoviral Vectors via Intraductal Delivery

PubMed Central

Morró, Meritxell; Teichenne, Joan; Jimenez, Veronica; Kratzer, Ramona; Marletta, Serena; Maggioni, Luca; Mallol, Cristina; Ruberte, Jesus; Kochanek, Stefan; Bosch, Fatima

2014-01-01

Abstract Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue. PMID:25046147

The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model.

PubMed

Ravelonandro, M; Scorza, R; Callahan, A; Levy, L; Jacquet, C; Monsion, M; Damsteegt, V

2000-11-01

Sharka or plum pox, caused by Plum pox virus (PPV: genus Potyvirus; Family Potyviridae), is the most serious disease of Prunus. Most cultivated Prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties. Success in developing virus-resistant herbaceous crops through genetic engineering led us to investigate this approach for resistance to PPV. Our programme aims to develop a biotechnological approach to PPV control that is effective and shown to be environmentally safe. The programme began with the cloning of the PPV coat protein (CP) gene and the development of a transformation system for plum (Prunus domestica). The CP construct was first tested in Nicotiana benthamiana in which it proved effective in producing transgenic plants with varying levels of CP expression. Some of these plants, particularly low PPV CP expressers, were resistant to PPV, or recovered from initial infection. Based on these results plum was transformed using the Agrobacterium tumefaciens system and both low and high PPV CP-expressing transgenic plum lines were obtained. These were inoculated with PPV by bud grafts in the greenhouse. Line C-5 proved to be highly resistant. It contained multiple copies of the insert, produced low levels of PPV CP mRNA, no detectable CP and the insert appeared to be methylated. These characteristics all suggest that the resistance of the C-5 clone is based on post-transcriptional gene silencing (PTGS). Field tests of C-5 and other transgenic lines in Poland, Romania and Spain have demonstrated that such trees when inoculated by bud-grafts allow a low level of PPV multiplication, from which they rapidly recover. C-5 plants exposed to natural infection for 3 years did not become infected, whereas control trees were infected in the first year. Hybrid plums having the C-5 PPV CP insert inherited from C-5 are virus-resistant, demonstrating the usefulness of C-5 as a parent in developing new PPV-resistant plum varieties. Research is in progress on the biorisks of PPV CP transgenic plants. Gene constructs that either produce no CP or CP that cannot be transmitted by aphids have been developed, tested in N. benthamiana and transferred to plum. Studies have begun on the potential for synergistic interactions between the PPV CP gene and the other common viruses of Prunus spp. In the future we will be participating in investigating the toxicity or/and the allergenicity of transgenic fruit products and, more importantly, transgenic lines will be developed that express transgenes only in vegetative parts of the plant and not in the fruit.

Overexpression of Mafb in Podocytes Protects against Diabetic Nephropathy

PubMed Central

Yoh, Keigyou; Ojima, Masami; Okamura, Midori; Nakamura, Megumi; Hamada, Michito; Shimohata, Homare; Moriguchi, Takashi; Yamagata, Kunihiro; Takahashi, Satoru

2014-01-01

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target. PMID:24722438

Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress.

PubMed

Rodrigues, Simone M; Andrade, Maxuel O; Gomes, Ana Paula Soares; Damatta, Fabio M; Baracat-Pereira, Maria C; Fontes, Elizabeth P B

2006-01-01

Despite extensive studies in eukaryotic aldehyde dehydrogenases, functional information about the ALDH7 antiquitin-like proteins is lacking. A soybean antiquitin homologue gene, designated GmTP55, has been isolated which encodes a dehydrogenase motif-containing 55 kDa protein induced by dehydration and salt stress. GmTP55 is closely related to the stress-induced plant antiquitin-like proteins that belong to the ALDH7 family. Transgenic tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants constitutively expressing GmTP55 have been obtained in order to examine the physiological role of this enzyme under a variety of stress conditions. Ectopic expression of GmTP55 in both Arabidopsis and tobacco conferred tolerance to salinity during germination and to water deficit during plant growth. Under salt stress, the germination efficiency of both transgenic tobacco and Arabidopsis seeds was significantly higher than that of their control counterparts. Likewise, under progressive drought, the transgenic tobacco lines apparently kept the shoot turgidity to a normal level, which contrasted with the leaf wilt phenotype of control plants. The transgenic plants also exhibited an enhanced tolerance to H(2)O(2)- and paraquat-induced oxidative stress. Both GmTP55-expressing Arabidopsis and tobacco seeds germinated efficiently in medium supplemented with H(2)O(2), whereas the germination of control seeds was drastically impaired. Similarly, transgenic tobacco leaf discs treated with paraquat displayed a significant reduction in the necrotic lesions as compared with control leaves. These transgenic lines also exhibited a lower concentration of lipid peroxidation-derived reactive aldehydes under oxidative stress. These results suggest that antiquitin may be involved in adaptive responses mediated by a physiologically relevant detoxification pathway in plants.

Wheat Chloroplast Targeted sHSP26 Promoter Confers Heat and Abiotic Stress Inducible Expression in Transgenic Arabidopsis Plants

PubMed Central

Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

2013-01-01

The small heat shock proteins (sHSPs) have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs) in the promoter of sHSP26 was performed. Moreover, the importance of 5′ untranslated region (UTR) has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress. PMID:23349883

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

NASA Astrophysics Data System (ADS)

Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

2000-05-01

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

Interferon-α Silencing by Small Interference RNA Increases Adenovirus Transduction and Transgene Expression in Huh7 Cells.

PubMed

Sobrevilla-Navarro, Ana Alondra; Sandoval-Rodríguez, Ana; García-Bañuelos, Jesús Javier; Armendariz-Borunda, Juan; Salazar-Montes, Adriana María

2018-04-01

Adenoviruses are the most common vectors used in clinical trials of gene therapy. In 2017, 21.2% of clinical trials used rAds as vectors. Systemic administration of rAds results in high tropism in the liver. Interferon types α and β are the major antiviral cytokines which orchestrate the host's immune response against rAd, limiting therapeutic gene expression and preventing subsequent vector administration. siRNA is small double-strand RNAs that temporally inhibit the expression of a specific gene. The aim is to evaluate the effect of IFN-α blocking by a specific siRNA on Ad-GFP transduction and on transgene expression in Huh7 cells in culture. Huh7 cells were cultured in DMEM and transfected with 70 nM of siRNA-IFN-α. Six hours later, the cells were exposed to 1 × 10 9  vp/ml of rAd-GFP for 24 h. Expression of IFN-α, TNF-α and the PKR gene was determined by RT-qPCR. Percentage of transduction was analyzed by flow cytometry and by qPCR. GFP expression was determined by western blot. 70 nM of siRNA-IFN-α inhibited 96% of IFN-α and 65% of TNF-α gene expression compared to an irrelevant siRNA. Percentage of transduction and transgene expression increased in these cells compared to an irrelevant siRNA. Inhibition of IFN-α expression by siRNA-IFN-α enabled a higher level of transduction and transgene expression GFP, highlighting the role of IFN-α in the elimination of adenovirus in transduced cells and thus suggesting that its inhibition could be an important strategy for gene therapy in clinical trials using adenovirus as a vector directed to liver diseases.

Effects of Transgenic Cry1Ac + CpTI Cotton on Non-Target Mealybug Pest Ferrisia virgata and Its Predator Cryptolaemus montrouzieri

PubMed Central

Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

2014-01-01

Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata. PMID:24751821

Effects of transgenic Cry1Ac + CpTI cotton on non-target mealybug pest Ferrisia virgata and its predator Cryptolaemus montrouzieri.

PubMed

Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

2014-01-01

Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata.

Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

PubMed Central

2011-01-01

Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs. PMID:22078230

Calcium-activated potassium channels as potential early markers of human cervical cancer

PubMed Central

Ramírez, Ana; Vera, Eunice; Gamboa-Domínguez, Armando; Lambert, Paul; Gariglio, Patricio; Camacho, Javier

2018-01-01

Cervical cancer is a major cause of cancer-associated mortality in women in developing countries. Thus, novel early markers are required. Ion channels have gained great interest as tumor markers, including cervical cancer. The calcium-activated potassium channel KCNMA1 (subunit α-1 from subfamily M) has been associated with different malignancies, including tumors such as breast and ovarian cancer that are influenced by hormones. The KCNMA1 channel blocker iberiotoxin decreases the proliferation of HeLa cervical cancer cells. Nevertheless, KCNMA1 channel expression during cervical carcinogenesis remains elusive. Therefore, KCNMA1 expression was studied in cervical cancer development. FVB transgenic mice expressing the E7-oncogene of high-risk human papilloma virus, and non-transgenic mice were treated with estradiol-releasing pellets during 3 or 6 months to induce cervical lesions. Twenty-four human cervical biopsies from non-cancerous, low- or high-grade intraepithelial lesions, or cervical cancer were also studied. mRNA and protein expression was assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. Cervical dysplasia and carcinoma were observed only in the transgenic mice treated with estradiol for 3 and 6 months, respectively. Estradiol treatment increased KCNMA1 mRNA and protein expression in all groups; however, the highest levels were observed in the transgenic mice with carcinoma. KCNMA1 protein expression in the squamous cells of the transformation zone was observed only in the transgenic mice with cervical dysplasia or cancer. Human biopsies from non-cancerous cervix did not display KCNMA1 protein expression; in contrast, the majority of the tissues with cervical lesions (16/18) displayed KCNMA1 protein expression. The lowest channel immunostaining intensity was observed in biopsies from low-grade dysplasia and the strongest in the carcinoma tissues. These results suggest KCNMA1 channels as potential early cervical cancer markers. PMID:29725443

Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

PubMed

Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

2015-04-22

Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

PubMed Central

Boosalis, Michael S.; Sangerman, Jose I.; White, Gary L.; Wolf, Roman F.; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H.; Li, Biaoru; Pace, Betty S.; Nouraie, Mehdi; Faller, Douglas V.; Perrine, Susan P.

2015-01-01

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies. PMID:26713848

Immunological thresholds in neurological gene therapy: highly efficient elimination of transduced cells might be related to the specific formation of immunological synapses between T cells and virus-infected brain cells

PubMed Central

Barcia, Carlos; Gerdes, Christian; Xiong, Wei-Dong; Thomas, Clare E.; Liu, Chunyan; Kroeger, Kurt M.; Castro, Maria G.; Lowenstein, Pedro R.

2007-01-01

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6–18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1 × 107 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1 × 107–1 × 10³ iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8+ T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1 × 107 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression. PMID:18084640

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

PubMed

Jornot, L; Morris, M A; Petersen, H; Moix, I; Rochat, T

2002-01-01

It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases. Copyright 2001 John Wiley & Sons, Ltd.

Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance.

PubMed

Tak, Himanshu; Negi, Sanjana; Ganapathi, T R

2017-03-01

Banana is an important fruit crop and its yield is hampered by multiple abiotic stress conditions encountered during its growth. The NAC (NAM, ATAF, and CUC) transcription factors are involved in plant response to biotic and abiotic stresses. In the present study, we studied the induction of banana NAC042 transcription factor in drought and high salinity conditions and its overexpression in transgenic banana to improve drought and salinity tolerance. MusaNAC042 expression was positively associated with stress conditions like salinity and drought and it encoded a nuclear localized protein. Transgenic lines of banana cultivar Rasthali overexpressing MusaNAC042 were generated by Agrobacterium-mediated transformation of banana embryogenic cells and T-DNA insertion was confirmed by PCR and Southern blot analysis. Our results using leaf disc assay indicated that transgenic banana lines were able to tolerate drought and high salinity stress better than the control plants and retained higher level of total chlorophyll and lower level of MDA content (malondialdehyde). Transgenic lines analyzed for salinity (250 mM NaCl) and drought (Soil gravimetric water content 0.15) tolerance showed higher proline content, better Fv/Fm ratio, and lower levels of MDA content than control suggesting that MusaNAC042 may be involved in responses to higher salinity and drought stresses in banana. Expression of several abiotic stress-related genes like those coding for CBF/DREB, LEA, and WRKY factors was altered in transgenic lines indicating that MusaNAC042 is an efficient modulator of abiotic stress response in banana.

Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

PubMed

Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

2011-06-15

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

A Field Trial of TCE Phytoremediation by Genetically Modified Poplars Expressing Cytochrome P450 2E1.

PubMed

Legault, Emily K; James, C Andrew; Stewart, Keith; Muiznieks, Indulis; Doty, Sharon L; Strand, Stuart E

2017-06-06

A controlled field study was performed to evaluate the effectiveness of transgenic poplars for phytoremediation. Three hydraulically contained test beds were planted with 12 transgenic poplars, 12 wild type (WT) poplars, or left unplanted, and dosed with equivalent concentrations of trichloroethylene (TCE). Removal of TCE was enhanced in the transgenic tree bed, but not to the extent of the enhanced removal observed in laboratory studies. Total chlorinated ethene removal was 87% in the CYP2E1 bed, 85% in the WT bed, and 34% in the unplanted bed in 2012. Evapotranspiration of TCE from transgenic leaves was reduced by 80% and diffusion of TCE from transgenic stems was reduced by 90% compared to WT. Cis-dichloroethene and vinyl chloride levels were reduced in the transgenic tree bed. Chloride ion accumulated in the planted beds corresponding to the TCE loss, suggesting that contaminant dehalogenation was the primary loss fate.

Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

PubMed

Duan, X; Li, X; Xue, Q; Abo-el-Saad, M; Xu, D; Wu, R

1996-04-01

We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice

PubMed Central

Kaufmann, Martin; Lee, Seong Min; Pike, J. Wesley

2015-01-01

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca++, PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis. PMID:26441239

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene.

PubMed

Wang, Guo-Kun; Zhang, Meng; Gong, Jiang-Feng; Guo, Qi-Fang; Feng, Ya-Nan; Wang, Wei

2012-12-01

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

Functional repression of PtSND2 represses growth and development by disturbing auxin biosynthesis, transport and signaling in transgenic poplar.

PubMed

Wang, Haihai; Tang, Renjie; Wang, Cuiting; Qi, Qi; Gai, Ying; Jiang, Xiangning; Zhang, Hongxia

2015-01-01

Using chimeric repressor silencing technology, we previously reported that functional repression of PtSND2 severely arrested wood formation in transgenic poplar (Populus). Here, we provide further evidence that auxin biosynthesis, transport and signaling were disturbed in these transgenic plants, leading to pleiotropic defects in their growth patterns, including inhibited leaf enlargement and vascular tissue development in the leaf central vein, suppressed cambial growth and fiber elongation in the stem, and arrested growth in the root system. Two transgenic lines, which displayed the most remarkable phenotypic deviation from the wild-type, were selected for detailed studies. In both transgenic lines, expression of genes for auxin biosynthesis, transport and signaling was down-regulated, and indole-3-acetic acid distribution was severely disturbed in the apical buds, leaves, stems and roots of field-grown transgenic plants. Transient transcription dual-luciferase assays of ProPtTYDC2::LUC, ProPttLAX2::LUC and ProPoptrIAA20.2::LUC in poplar protoplasts revealed that expression of auxin-related genes might be regulated by PtSND2 at the transcriptional level. All these results indicate that functional repression of PtSND2 altered auxin biosynthesis, transport and signaling, and thereby disturbed the normal growth and development of transgenic plants. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Breeding of transgenic mice expressing human tau isoform with P301L mutation and identification of homozygous transgenic mice].

PubMed

Wang, Yan-yan; Chen, Ru-zhui; Zhu, Xiao-nani; Liu, Jing; Li, Zhi-hui; Liu, Xiu-juan; Li, Zhi-hui; Na, Xin; Liang, Shan-shan; Qiu, Guo-guang; Zhang, Wei; Wang, Hai; Wang, Xue-lan

2012-05-01

To establish homozygous transgenic mouse strain expressing human tau isoform with P301L mutation. Five transgenic mice expressing human tau isoform with P301L mutation were obtained by microinjection into male nuclei. Homozygote and hemizygote were identified by PCR and real-time fluorescent quantitative PCR. Ninety five homozygous transgenic mice were selected, and the results indicated that homozygous transgenic mice were superior to hemizygote in simulating the changes of biological characteristics. Exogenous gene tau is able to stably transmit to next generation and the combination of SYBR Green real-time fluorescent quantitative PCR with the traditional mating is a fast, reliable and economical way to screen homozygous and hemizygous transgenic mice.

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

PubMed Central

Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

2011-01-01

Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells. PMID:21931783

ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton.

PubMed

Wang, Chunling; Lu, Guoqing; Hao, Yuqiong; Guo, Huiming; Guo, Yan; Zhao, Jun; Cheng, Hongmei

2017-09-01

ABP9 , encoding a bZIP transcription factor from maize, enhances tolerance to multiple stresses and may participate in the ABA signaling pathway in transgenic cotton by altering physiological and biochemical processes and stress-related gene expression. Abiotic stresses, such as soil salinity and drought, negatively affect growth, development, and yield in cotton. Gene ABP9, which encodes a bZIP transcription factor, binds to the abscisic acid (ABA)-responsive-element (ABRE2) motif of the maize catalase1 gene. Its expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses, but little is known about its role in cotton. In the present study, the ABP9 gene was introduced into upland cotton (Gossypium hirsutum L.) cultivar R15 by Agrobacterium tumefaciens-mediated transformation, and 12 independent transgenic cotton lines were obtained. Cotton plants over-expressing ABP9 have enhanced tolerance to salt and osmotic stress. Under stress, they developed better root systems in a greenhouse and higher germination, reduced stomatal aperture, and stomatal density in a growth chamber. Under drought conditions, survival rate and relative water content (RWC) of transgenic cotton were higher than those of R15 plants. Under salt and osmotic stresses, chlorophyll, proline, and soluble sugar contents significantly increased in transgenic cotton leaves and the malondialdehyde (MDA) content was lower than in R15. Overexpression of ABP9 also enhanced oxidative stress tolerance, reduced cellular levels of reactive oxygen species (ROS) through increased activities of antioxidative enzymes, and alleviated oxidative damage to cell. Interestingly, ABP9 over-expressing cotton was more sensitive to exogenous ABA than R15 at seed germination, root growth, stomatal aperture, and stomatal density. Moreover, ABP9 overexpression upregulated significantly the transcription levels of stress-related genes such as GhDBP2, GhNCED2, GhZFP1, GhERF1, GhHB1, and GhSAP1 under salt treatment. Conjointly, these results showed that overexpression of ABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton. The stress-tolerant transgenic lines provide valuable resources for cotton breeding.

Genetic Transformation and Analysis of Rice OsAPx2 Gene in Medicago sativa

PubMed Central

Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

2012-01-01

The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H2O2)concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H2O2 content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H2O2, sodium sulfite (Na2SO3), and sodium bicarbonate (NaHCO3). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H2O2 and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained. PMID:22848448

Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa.

PubMed

Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

2012-01-01

The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H(2)O(2))concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H(2)O(2) content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H(2)O(2), sodium sulfite (Na(2)SO(3)), and sodium bicarbonate (NaHCO(3)). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H(2)O(2) and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained.

Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress1

PubMed Central

Youssefian, Shohab; Nakamura, Michimi; Orudgev, Emin; Kondo, Noriaki

2001-01-01

O-Acetylserine(thiol) lyase (OASTL), a key enzyme of plant sulfur metabolism, catalyzes the formation of Cys from sulfide and O-acetylserine. The biosynthesis of Cys is regarded as the exclusive function of sulfur reduction in plants, and a key limiting step in the production of glutathione (GSH), a thiol implicated in various cellular functions, including sulfur transport, gene expression, scavenging of reactive oxygen species (ROS), and resistance to biotic and abiotic stresses. To examine whether an increased capacity for cysteine (Cys) biosynthesis alters cellular responses to such stresses, we studied the differential changes in thiol levels and ROS scavenging of transgenic tobacco (Nicotiana tabacum) plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, to SO2 and to the ROS generator, methyl viologen. Intracellular Cys and GSH contents were generally higher in cys1 transgenics than in controls under normal growth conditions, but became especially elevated in transgenic plants after SO2 exposure. An examination of differences in the ROS scavenging system of the transgenic plants also demonstrated the specific accumulation of Cu/Zn superoxide dismutase transcripts, known to be induced by Cys or GSH, and elevated cellular superoxide dismutase activities. The transgenic plants accordingly showed dramatic reductions in the extent of both foliar and photooxidative damage in response to acute SO2, as well as reduced levels of chlorosis and membrane damage following methyl viologen treatment. Overall, our results imply that OASTL plays a pivotal role in the synthesis of Cys and GSH that are required for regulation of plant responses to oxidative stress. PMID:11457951

Constitutive Expression of Mammalian Nitric Oxide Synthase in Tobacco Plants Triggers Disease Resistance to Pathogens

PubMed Central

Chun, Hyun Jin; Park, Hyeong Cheol; Koo, Sung Cheol; Lee, Ju Huck; Park, Chan Young; Choi, Man Soo; Kang, Chang Ho; Baek, Dongwon; Cheong, Yong Hwa; Yun, Dae-Jin; Chung, Woo Sik; Cho, Moo Je; Kim, Min Chul

2012-01-01

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H2O2, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance. PMID:23124383

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

PubMed Central

Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

1993-01-01

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

PubMed Central

Ligaba-Osena, Ayalew; Jones, Jenna; Donkor, Emmanuel; Chandrayan, Sanjeev; Pole, Farris; Wu, Chang-Hao; Vieille, Claire; Adams, Michael W. W.; Hankoua, Bertrand B.

2018-01-01

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability. PMID:29541080

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase.

PubMed

Ligaba-Osena, Ayalew; Jones, Jenna; Donkor, Emmanuel; Chandrayan, Sanjeev; Pole, Farris; Wu, Chang-Hao; Vieille, Claire; Adams, Michael W W; Hankoua, Bertrand B

2018-01-01

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava ( Manihot esculenta ), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus , together with the gene encoding a modified ADP-glucose pyrophosphorylase ( glgC ) from Escherichia coli , were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

PubMed

Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

2015-08-03

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars.

PubMed

Sahebi, Mahbod; Hanafi, Mohamed M; Rafii, M Y; Azizi, Parisa; Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars

PubMed Central

Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties. PMID:28191468

Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy

NASA Technical Reports Server (NTRS)

Criswell, D. S.; Booth, F. W.; DeMayo, F.; Schwartz, R. J.; Gordon, S. E.; Fiorotto, M. L.

1998-01-01

This study examined the association between local insulin-like growth factor I (IGF-I) overexpression and atrophy in skeletal muscle. We hypothesized that endogenous skeletal muscle IGF-I mRNA expression would decrease with hindlimb unloading (HU) in mice, and that transgenic mice overexpressing human IGF-I (hIGF-I) specifically in skeletal muscle would exhibit less atrophy after HU. Male transgenic mice and nontransgenic mice from the parent strain (FVB) were divided into four groups (n = 10/group): 1) transgenic, weight-bearing (IGF-I/WB); 2) transgenic, hindlimb unloaded (IGF-I/HU); 3) nontransgenic, weight-bearing (FVB/WB); and 4) nontransgenic, hindlimb unloaded (FVB/HU). HU groups were hindlimb unloaded for 14 days. Body mass was reduced (P < 0.05) after HU in both IGF-I (-9%) and FVB mice (-13%). Contrary to our hypothesis, we found that the relative abundance of mRNA for the endogenous rodent IGF-I (rIGF-I) was unaltered by HU in the gastrocnemius (GAST) muscle of wild-type FVB mice. High-level expression of hIGF-I peptide and mRNA was confirmed in the GAST and tibialis anterior (TA) muscles of the transgenic mice. Nevertheless, masses of the GAST and TA muscles were reduced (P < 0.05) in both FVB/HU and IGF-I/HU groups compared with FVB/WB and IGF-I/WB groups, respectively, and the percent atrophy in mass of these muscles did not differ between FVB and IGF-I mice. Therefore, skeletal muscle atrophy may not be associated with a reduction of endogenous rIGF-I mRNA level in 14-day HU mice. We conclude that high local expression of hIGF-I mRNA and peptide in skeletal muscle alone cannot attenuate unloading-induced atrophy of fast-twitch muscle in mice.

Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

PubMed

Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

2015-05-12

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis PlantsW⃞

PubMed Central

Aharoni, Asaph; Giri, Ashok P.; Deuerlein, Stephan; Griepink, Frans; de Kogel, Willem-Jan; Verstappen, Francel W. A.; Verhoeven, Harrie A.; Jongsma, Maarten A.; Schwab, Wilfried; Bouwmeester, Harro J.

2003-01-01

Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants. PMID:14630967

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

PubMed

Cheng, Chih-Lun; Huang, Shin-Jie; Wu, Chih-Lu; Gong, Hong-Yi; Ken, Chuian-Fu; Hu, Shao-Yang; Wu, Jen-Leih

2015-11-17

Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1β, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Expression of Colocasia esculenta tuber agglutinin in Indian mustard provides resistance against Lipaphis erysimi and the expressed protein is non-allergenic.

PubMed

Das, Ayan; Ghosh, Prithwi; Das, Sampa

2018-06-01

Transgenic Brassica juncea plants expressing Colocasia esculenta tuber agglutinin (CEA) shows the non-allergenic nature of the expressed protein leading to enhanced mortality and reduced fecundity of mustard aphid-Lipaphis erysimi. Lipaphis erysimi (common name: mustard aphid) is the most devastating sucking insect pest of Indian mustard (Brassica juncea L.). Colocasia esculenta tuber agglutinin (CEA), a GNA (Galanthus nivalis agglutinin)-related lectin has previously been reported by the present group to be effective against a wide array of hemipteran insects in artificial diet-based bioassays. In the present study, efficacy of CEA in controlling L. erysimi has been established through the development of transgenic B. juncea expressing this novel lectin. Southern hybridization of the transgenic plants confirmed stable integration of cea gene. Expression of CEA in T 0 , T 1 and T 2 transgenic plants was confirmed through western blot analysis. Level of expression of CEA in the T 2 transgenic B. juncea ranged from 0.2 to 0.47% of the total soluble protein. In the in planta insect bioassays, the CEA expressing B. juncea lines exhibited enhanced insect mortality of 70-81.67%, whereas fecundity of L. erysimi was reduced by 49.35-62.11% compared to the control plants. Biosafety assessment of the transgenic B. juncea protein containing CEA was carried out by weight of evidence approach following the recommendations by FAO/WHO (Evaluation of the allergenicity of genetically modified foods: report of a joint FAO/WHO expert consultation, 22-25 Jan, Rome, http://www.fao.org/docrep/007/y0820e/y0820e00.HTM , 2001), Codex (Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome; Codex, Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome, 2003) and ICMR (Indian Council of Medical Research, guidelines for safety assessment of food derived from genetically engineered plants, http://www.icmr.nic.in/guide/Guidelines%20for%20Genetically%20Engineered%20Plants.pdf , 2008). Bioinformatics analysis, pepsin digestibility, thermal stability assay, immuno-screening and allergenicity assessment in BALB/c mice model demonstrated that the expressed CEA protein from transgenic B. juncea does not incite any allergenic response. The present study establishes CEA as an efficient insecticidal and non-allergenic protein to be utilized for controlling mustard aphid and similar hemipteran insects through the development of genetically modified plants.

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

PubMed Central

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

PubMed

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong; Qu, Shaohong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. © 2015 American Society of Plant Biologists. All Rights Reserved.

Chrysanthemum WRKY gene DgWRKY5 enhances tolerance to salt stress in transgenic chrysanthemum.

PubMed

Liang, Qian-Yu; Wu, Yin-Huan; Wang, Ke; Bai, Zhen-Yu; Liu, Qing-Lin; Pan, Yuan-Zhi; Zhang, Lei; Jiang, Bei-Bei

2017-07-06

WRKY transcription factors play important roles in plant growth development, resistance and substance metabolism regulation. However, the exact function of the response to salt stress in plants with specific WRKY transcription factors remains unclear. In this research, we isolated a new WRKY transcription factor DgWRKY5 from chrysanthemum. DgWRKY5 contains two WRKY domains of WKKYGQK and two C 2 H 2 zinc fingers. The expression of DgWRKY5 in chrysanthemum was up-regulated under various treatments. Meanwhile, we observed higher expression levels in the leaves contrasted with other tissues. Under salt stress, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzymes in transgenic chrysanthemum were significantly higher than those in WT, whereas the accumulation of H 2 O 2 , O 2 - and malondialdehyde (MDA) was reduced in transgenic chrysanthemum. Several parameters including root length, root length, fresh weight, chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress. Moreover, the expression of stress-related genes DgAPX, DgCAT, DgNCED3A, DgNCED3B, DgCuZnSOD, DgP5CS, DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT. These results suggested that DgWRKY5 could function as a positive regulator of salt stress in chrysanthemum.

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

PubMed

Ali, Md Emran; Ishii, Yuko; Taniguchi, Jyun-Ichi; Waliullah, Sumyya; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

2018-05-01

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Large-scale production of tannase using the yeast Arxula adeninivorans.

PubMed

Böer, Erik; Breuer, Friederike Sophie; Weniger, Michael; Denter, Sylvia; Piontek, Michael; Kunze, Gotthard

2011-10-01

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L(-1) when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L(-1). The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L(-1). Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.

GENETIC CATHEPSIN B DEFICIENCY REDUCES β-AMYLOID IN TRANSGENIC MICE EXPRESSING HUMAN WILD-TYPE AMYLOID PRECURSOR PROTEIN

PubMed Central

Hook, Vivian Y. H.; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-01-01

Neurotoxic β-amyloid (Aβ) peptides participate in Alzheimer’s disease (AD); therefore, reduction of Aβ generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Aβ may identify targets for reducing Aβ. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decrease of Aβ40 and Aβ42 by 67% in brain, and decreases levels of the C-terminal β-secretase fragment (CTFβ) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ. The difference in reduction of Aβ in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in AD. PMID:19501042

Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

PubMed

McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

2012-01-01

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.