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Sample records for producing marker-free transgenic

  1. [Produce of marker-free transgenic tobacco plants by FLP/frt recombination system].

    PubMed

    Shan, Xiao-Yi; Li, Bei; Zhang, Ju-Ren

    2006-09-01

    Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of the transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of the 2 microm plasmid of Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41% re-transgenic tobacco plants, which indicated that this systerm could make a great contribution to obtain the marker free transgenic plants. PMID:17037196

  2. 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. PMID:25371551

  3. 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

  4. Development of Marker-Free Transgenic Potato Tubers Enriched in Caffeoylquinic Acids and Flavonols.

    PubMed

    Li, Yang; Tang, Wenzhao; Chen, Jing; Jia, Ru; Ma, Lianjie; Wang, Shaoli; Wang, Jiao; Shen, Xiangling; Chu, Zhaohui; Zhu, Changxiang; Ding, Xinhua

    2016-04-13

    Potato (Solanum tuberosum L.) is a major crop worldwide that meets human economic and nutritional requirements. Potato has several advantages over other crops: easy to cultivate and store, cheap to consume, and rich in a variety of secondary metabolites. In this study, we generated three marker-free transgenic potato lines that expressed the Arabidopsis thaliana flavonol-specific transcriptional activator AtMYB12 driven by the tuber-specific promoter Patatin. Marker-free potato tubers displayed increased amounts of caffeoylquinic acids (CQAs) (3.35-fold increases on average) and flavonols (4.50-fold increase on average). Concentrations of these metabolites were associated with the enhanced expression of genes in the CQA and flavonol biosynthesis pathways. Accumulation of CQAs and flavonols resulted in 2-fold higher antioxidant capacity compared to wild-type potatoes. Tubers from these marker-free transgenic potatoes have therefore improved antioxidant properties. PMID:27019017

  5. Large-scale production of functional human lysozyme from marker-free transgenic cloned cows

    PubMed Central

    Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

    2016-01-01

    Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future. PMID:26961596

  6. Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

    PubMed

    Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

    2016-01-01

    Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future. PMID:26961596

  7. Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision.

    PubMed

    Woo, Hee-Jong; Qin, Yang; Park, Soo-Yun; Park, Soon Ki; Cho, Yong-Gu; Shin, Kong-Sik; Lim, Myung-Ho; Cho, Hyun-Suk

    2015-01-01

    Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice. PMID:26172549

  8. Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision

    PubMed Central

    Woo, Hee-Jong; Qin, Yang; Park, Soo-Yun; Park, Soon Ki; Cho, Yong-Gu; Shin, Kong-Sik; Lim, Myung-Ho; Cho, Hyun-Suk

    2015-01-01

    Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice. PMID:26172549

  9. Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants.

    PubMed

    Saelim, Laddawan; Phansiri, Salak; Suksangpanomrung, Malinee; Netrphan, Supatcharee; Narangajavana, Jarunya

    2009-03-01

    The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar "KU50" was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19-21% was observed. Among the total number of ESP explants, 32-38% regained normal extended shoot phenotype and 88-96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries. PMID:19093119

  10. Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

    PubMed Central

    Yang, Ching-Fu; Chen, Kuan-Chun; Cheng, Ying-Hui; Raja, Joseph A. J.; Huang, Ya-Ling; Chien, Wan-Chu; Yeh, Shyi-Dong

    2014-01-01

    Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. PMID:25030413

  11. Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System

    PubMed Central

    Kasai, Yuki; Harayama, Shigeaki

    2016-01-01

    The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after

  12. Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System.

    PubMed

    Kasai, Yuki; Harayama, Shigeaki

    2016-01-01

    The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after

  13. Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

    PubMed

    Chakraborti, Dipankar; Sarkar, Anindya; Mondal, Hossain A; Schuermann, David; Hohn, Barbara; Sarmah, Bidyut K; Das, Sampa

    2008-10-01

    A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view. PMID:18663453

  14. Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination

    PubMed Central

    2013-01-01

    Background Antibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype. Results Allium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding

  15. Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects.

    PubMed

    Manimaran, P; Ramkumar, G; Sakthivel, K; Sundaram, R M; Madhav, M S; Balachandran, S M

    2011-01-01

    Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program. PMID:21672619

  16. Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration.

    PubMed

    Gayen, Srimonta; Mandal, Chandi Charan; Samanta, Milan Kumar; Dey, Avishek; Sen, Soumitra Kumar

    2016-04-01

    Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW. PMID:26925624

  17. Production of marker-free transgenic Jatropha curcas expressing hybrid Bacillus thuringiensis δ-endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth (Archips micaceanus)

    PubMed Central

    2014-01-01

    Background The potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice. Results Here, we report the production of a marker-free transgenic line of J. curcas (L10) expressing Cry1Ab/1Ac using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L10 carries a single copy of marker-free T-DNA that contains the Cry1Ab/1Ac gene under the control of a maize phosphoenolpyruvate carboxylase gene promoter (P Pepc :Cry1Ab/1Ac:T Nos ). The P Pepc :Cry1Ab/1Ac:T Nos gene was highly expressed in leaves of L10 plants. Insecticidal bioassays using leaf explants of L10 resulted in 80-100% mortality of larvae of A. micaceanus at 4 days after infestation. Conclusion The results demonstrate that the hybrid Bt δ-endotoxin protein Cry1Ab/1Ac expressed in Jatropha curcas displays strong insecticidal activity to A. micaceanus. The marker-free transgenic J. curcas line L10 can be used for breeding of insect resistance to A. micaceanus. PMID:24808924

  18. How To Produce and Characterize Transgenic Plants.

    ERIC Educational Resources Information Center

    Savka, Michael A.; Wang, Shu-Yi; Wilson, Mark

    2002-01-01

    Explains the process of establishing transgenic plants which is a very important tool in plant biology and modern agriculture. Produces transgenic plants with the ability to synthesize opines. (Contains 17 references.) (YDS)

  19. A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.).

    PubMed

    Breitler, Jean-Christophe; Meynard, Donaldo; Van Boxtel, Jos; Royer, Monique; Bonnot, François; Cambillau, Laurence; Guiderdoni, Emmanuel

    2004-06-01

    A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars. PMID:15359604

  20. Retransformation of marker-free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and wasabi defensin genes.

    PubMed

    Khan, Raham Sher; Darwish, Nader Ahmed; Khattak, Bushra; Ntui, Valentine Otang; Kong, Kynet; Shimomae, Kazuki; Nakamura, Ikuo; Mii, Masahiro

    2014-09-01

    Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations. PMID:24802621

  1. [DEVELOPMENT OF MARKER-FREE TRANSFORMANTS BY SITE-SPECIFIC RECOMBINASES].

    PubMed

    Sekan, A S; Isaenkov, S V; Blume, Ya B

    2015-01-01

    To produce transgenic plants in current biotechnology selectable marker genes are used that lead to the selectivity.of transformants from non-transformed organisms. However, after the transgenic event has been occurred, the presence of these genes in transformed genome in general is uselless. Moreover, the continued presence of this kind of genes in transgenic plants with their further commercialization may raise certain public concern. Therefore, various techniques have been developed in recent years to obtain marker free transgenic plants. In the present review the main strategies for removal of selective marker DNA sequences that are used in genetic engineering are described. The most popular among them is site-specific recombination technology. The particular attention is paid to site-specific recombinase system Cre/loxP. The using of a new approach with site-specific recombinase system Cre/loxP under the control of 35S promoter to generate marker-free genetically modified plants is described. PMID:26841495

  2. Development of antibiotic marker-free creeping bentgrass resistance against herbicides.

    PubMed

    Lee, Ki-Won; Kim, Ki-Yong; Kim, Kyung-Hee; Lee, Byung-Hyun; Kim, Jin-Seog; Lee, Sang-Hoon

    2011-01-01

    Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species. PMID:21173055

  3. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  4. Transgenic silkworms produce recombinant human type III procollagen in cocoons.

    PubMed

    Tomita, Masahiro; Munetsuna, Hiroto; Sato, Tsutomu; Adachi, Takahiro; Hino, Rika; Hayashi, Masahiro; Shimizu, Katsuhiko; Nakamura, Namiko; Tamura, Toshiki; Yoshizato, Katsutoshi

    2003-01-01

    We describe the generation of transgenic silkworms that produce cocoons containing recombinant human collagen. A fusion cDNA was constructed encoding a protein that incorporated a human type III procollagen mini-chain with C-propeptide deleted, a fibroin light chain (L-chain), and an enhanced green fluorescent protein (EGFP). This cDNA was ligated downstream of the fibroin L-chain promoter and inserted into a piggyBac vector. Silkworm eggs were injected with the vectors, producing worms displaying EGFP fluorescence in their silk glands. The cocoons emitted EGFP fluorescence, indicating that the promoter and fibroin L-chain cDNAs directed the synthesized products to be secreted into cocoons. The presence of fusion proteins in cocoons was demonstrated by immunoblotting, collagenase-sensitivity tests, and amino acid sequencing. The fusion proteins from cocoons were purified to a single electrophoretic band. This study demonstrates the viability of transgenic silkworms as a tool for producing useful proteins in bulk. PMID:12483223

  5. Environmental risk assessments for transgenic crops producing output trait enzymes

    PubMed Central

    Tuttle, Ann; Shore, Scott; Stone, Terry

    2009-01-01

    The environmental risks from cultivating crops producing output trait enzymes can be rigorously assessed by testing conservative risk hypotheses of no harm to endpoints such as the abundance of wildlife, crop yield and the rate of degradation of crop residues in soil. These hypotheses can be tested with data from many sources, including evaluations of the agronomic performance and nutritional quality of the crop made during product development, and information from the scientific literature on the mode-of-action, taxonomic distribution and environmental fate of the enzyme. Few, if any, specific ecotoxicology or environmental fate studies are needed. The effective use of existing data means that regulatory decision-making, to which an environmental risk assessment provides essential information, is not unnecessarily complicated by evaluation of large amounts of new data that provide negligible improvement in the characterization of risk, and that may delay environmental benefits offered by transgenic crops containing output trait enzymes. PMID:19924556

  6. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

    PubMed

    Joyce, Priya; Hermann, Scott; O'Connell, Anthony; Dinh, Quang; Shumbe, Leonard; Lakshmanan, Prakash

    2014-05-01

    Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques. PMID:24330327

  7. Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population.

    PubMed

    Zapiola, María L; Mallory-Smith, Carol A

    2012-10-01

    Gene flow is the most frequently expressed public concern related to the deregulation of transgenic events (Snow 2002; Ellstrand 2003). However, assessing the potential for transgene escape is complex because it depends on the opportunities for unintended gene flow, and establishment and persistence of the transgene in the environment (Warwick et al. 2008). Creeping bentgrass (Agrostis stolonifera L.), a turfgrass species widely used on golf courses, has been genetically engineered to be resistant to glyphosate, a nonselective herbicide. Outcrossing species, such as creeping bentgrass (CB), which have several compatible species, have greater chances for gene escape and spontaneous hybridization (i.e. natural, unassisted sexual reproduction between taxa in the field), which challenges transgene containment. Several authors have emphasized the need for evidence of spontaneous hybridization to infer the potential for gene flow (Armstrong et al. 2005). Here we report that a transgenic intergeneric hybrid has been produced as result of spontaneous hybridization of a feral-regulated transgenic pollen receptor (CB) and a nontransgenic pollen donor (rabbitfoot grass, RF, Polypogon monspeliensis (L.) Desf.). We identified an off-type transgenic seedling and confirmed it to be CB × RF intergeneric hybrid. This first report of a transgenic intergeneric hybrid produced in situ with a regulated transgenic event demonstrates the importance of considering all possible avenues for transgene spread at the landscape level before planting a regulated transgenic crop in the field. Spontaneous hybridization adds a level of complexity to transgene monitoring, containment, mitigation and remediation programmes. PMID:22625177

  8. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    PubMed

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition. PMID:27565868

  9. PERSISTENCE IN SOIL OF TRANSGENIC PLANT PRODUCED BACILLUS THURINGIENSIS VAR. KURSTAKI O-ENDOTOXIN1

    EPA Science Inventory

    Transgenic plants that produce pesticidal proteins will release these proteins into the soil when these plants are incorporated into the soil by tillage or as leaf litter. Little is known about the fate and persistence of transgenic plant pesticidal products in the soil. We used ...

  10. Transgenic pigs produce functional human factor VIII in milk.

    PubMed

    Paleyanda, R K; Velander, W H; Lee, T K; Scandella, D H; Gwazdauskas, F C; Knight, J W; Hoyer, L W; Drohan, W N; Lubon, H

    1997-10-01

    Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma. PMID:9335047

  11. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

    PubMed Central

    Tada, Minoru; Tatematsu, Ken-Ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. PMID:26261057

  12. High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

    PubMed Central

    Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

    2014-01-01

    Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms. PMID:25162624

  13. A Standardized Lepidopteran Bioassay to Investigate the Bioactivity of Insecticidal Proteins Produced in Transgenic Crops.

    PubMed

    Graser, Gerson; Walters, Frederick S

    2016-01-01

    Insecticidal bioassays are the only reliable method to investigate the biological activity of an insecticidal protein and therefore provide an essential toolkit for the characterization and potency determination of these proteins. Here we present a standardized method for a lepidopteran larval bioassay, which is optimized to specifically estimate activity of insecticidal proteins produced in transgenic plants. The treatment can be either applied to the surface of the artificial diet, or blended into the diet. PMID:26614295

  14. Overexpression of human loricrin in transgenic mice produces a normal phenotype.

    PubMed Central

    Yoneda, K; Steinert, P M

    1993-01-01

    The cornified cell envelope (CE) of terminally differentiating stratified squamous epithelial cells is a complex multiprotein assembly about 15 nm thick of which loricrin is a major component. We have produced transgenic mice bearing the human loricrin transgene in order to study the role of loricrin in CE assembly, structure, and function. By analyses of RNA and protein, we show that the human transgene is expressed in mouse epithelial tissues in an appropriate developmental manner but at an overall level about twice that of endogenous mouse loricrin. Thus the 20-kbp construct used contains all necessary regulatory elements. By immunogold electron microscopy, all of the expressed protein is incorporated into the CE. That no alternations were noted indicates that overproduction of the loricrin component of the CE does not affect the flexible structure or function of the epithelial tissues. Furthermore, these data imply that loricrin may be the last protein to be deposited onto, and thus lines, the intracellular surface of the CE, where it may be accessible to interact with the subjacent keratin intermediate-filament network. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8248167

  15. Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.

    PubMed

    Peréz Aguirreburualde, María Sol; Gómez, María Cristina; Ostachuk, Agustín; Wolman, Federico; Albanesi, Guillermo; Pecora, Andrea; Odeon, Anselmo; Ardila, Fernando; Escribano, José M; Dus Santos, María José; Wigdorovitz, Andrés

    2013-02-15

    Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 μg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 μg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection. PMID:23291101

  16. Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

    PubMed

    Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

    2006-01-01

    The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops. PMID:16382182

  17. Marker-free cell discrimination by holographic optical tweezers

    NASA Astrophysics Data System (ADS)

    Schaal, F.; Warber, M.; Zwick, S.; van der Kuip, H.; Haist, T.; Osten, W.

    2009-06-01

    We introduce a method for marker-free cell discrimination based on optical tweezers. Cancerous, non-cancerous, and drug-treated cells could be distinguished by measuring the trapping forces using holographic optical tweezers. We present trapping force measurements on different cell lines: normal pre-B lymphocyte cells (BaF3; "normal cells"), their Bcr-Abl transformed counterparts (BaF3-p185; "cancer cells") as a model for chronic myeloid leukaemia (CML) and Imatinib treated BaF3-p185 cells. The results are compared with reference measurements obtained by a commercial flow cytometry system.

  18. Large-scale production and evaluation of marker-free indica rice IR64 expressing phytoferritin genes.

    PubMed

    Oliva, Norman; Chadha-Mohanty, Prabhjit; Poletti, Susanna; Abrigo, Editha; Atienza, Genelou; Torrizo, Lina; Garcia, Ruby; Dueñas, Conrado; Poncio, Mar Aristeo; Balindong, Jeanette; Manzanilla, Marina; Montecillo, Florencia; Zaidem, Maricris; Barry, Gerard; Hervé, Philippe; Shou, Huxia; Slamet-Loedin, Inez H

    2014-01-01

    Biofortification of rice (Oryza sativa L.) using a transgenic approach to increase the amount of iron in the grain is proposed as a low-cost, reliable, and sustainable solution to help developing countries combat anemia. In this study, we generated and evaluated a large number of rice or soybean ferritin over-accumulators in rice mega-variety IR64, including marker-free events, by introducing soybean or rice ferritin genes into the endosperm for product development. Accumulation of the protein was confirmed by ELISA, in situ immunological detection, and Western blotting. As much as a 37- and 19-fold increase in the expression of ferritin gene in single and co-transformed plants, respectively, and a 3.4-fold increase in Fe content in the grain over the IR64 wild type was achieved using this approach. Agronomic characteristics of a total of 1,860 progenies from 58 IR64 single independent transgenic events and 768 progenies from 27 marker-free transgenic events were evaluated and most trait characteristics did not show a penalty. Grain quality evaluation of high-Fe IR64 transgenic events showed quality similar to that of the wild-type IR64. To understand the effect of transgenes on iron homeostasis, transcript analysis was conducted on a subset of genes involved in iron uptake and loading. Gene expression of the exogenous ferritin gene in grain correlates with protein accumulation and iron concentration. The expression of NAS2 and NAS3 metal transporters increased during the grain milky stage. PMID:24482599

  19. Transgenic cattle produced by nuclear transfer of fetal fibroblasts carrying Ipr1 gene at a specific locus.

    PubMed

    Wang, Yong Sheng; He, Xiaoning; Du, Yue; Su, Jianmin; Gao, Mingqing; Ma, Yefei; Hua, Song; Quan, Fusheng; Liu, Jun; Zhang, Yong

    2015-09-01

    This study aimed to assess the effects of the intracellular pathogen resistance 1 (Ipr1) transgene on preventing infection of Mycobacterium bovis in cattle. A specific expression vector for the Ipr1 gene was constructed and inserted in the genome between surfactant protein A and methionine adenosyltransferase I of bovine fetal fibroblasts. After SCNT, cleavage (86.9% vs. 87.4%, P > 0.05) and blastocyst developmental rates (34.6% vs. 33.5%, P > 0.05) were similar between transgenic and nontransgenic bovine fetal fibroblasts. Four surviving and one dead Ipr1-transgenic female cattle were produced by transfer of the SCNT blastocysts. Polymerase chain reaction and Southern blot analyses confirmed that the Ipr1 transgene of the cattle was located at the expected site. Inserting Ipr1 gene did not affect the expression of the surrounding genes. Main death modality of M bovis-infected peripheral blood mononuclear cells (PBMCs) derived from Ipr1-transgenic cattle was apoptosis, whereas that of PBMCs from control cattle was necrosis. In addition, the number of colony-forming units in PBMCs of Ipr1-transgenic cattle was significantly lower than that of the control cattle (P < 0.05). The finding that expression of Ipr1 transgene in PBMCs significantly increased anti-M bovis activity suggested breeding anti-M bovis cattle population by the transgenic SCNT technique could be a feasible strategy. PMID:25998271

  20. Migratory beekeeping practices contribute insignificantly to transgenic pollen flow among fields of alfalfa produced for seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased use of genetically engineered crops in agriculture has raised concerns over pollinator-mediated gene flow between transgenic and conventional agricultural varieties. This study evaluated whether contracted migratory beekeeping practices influence transgenic pollen flow among spatially iso...

  1. Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer.

    PubMed

    Webster, Nicole L; Forni, Monica; Bacci, Maria Laura; Giovannoni, Roberto; Razzini, Riccardo; Fantinati, Paolo; Zannoni, Augusta; Fusetti, Lisa; Dalprà, Leda; Bianco, Maria Rosaria; Papa, Michele; Seren, Eraldo; Sandrin, Mauro S; Mc Kenzie, Ian F C; Lavitrano, Marialuisa

    2005-09-01

    Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins. PMID:15906394

  2. Pituitary Changes in Prop1 Transgenic Mice: Hormone Producing Tumors and Signet-ring Type Gonadotropes

    PubMed Central

    Egashira, Noboru; Minematsu, Takeo; Miyai, Syunsuke; Takekoshi, Susumu; Camper, Sally A.; Osamura, Robert Y.

    2008-01-01

    Prophet of Pit-1 (Prop1) is an early transcription factor that delays the appearance of gonadotropin in the developing pituitaries. Prop1 transgenic (Tg) mice have been shown to generate pituitary tumors that either produce TSH or are non-hormone producing. In our series of Prop1 Tg mice, only 5 out of 9 female mice produced pituitary adenomas, and the adenomas were only GH, PRL, GH and PRL, PRL and gonadotropin or TSH producing. The pituitary cells that surrounded these adenomas showed hyperplasia of the corresponding hormone producing cells; i.e. the GH cells were increased in the pituitary that contained GH producing adenoma. In addition, although the adenomas lacked the expression of Prop1, the non-neoplastic pituitary cells showed expression of Prop1. The Prop1 Tg mice also showed vacuolated cells with eccentric nuclei, which are characteristic of “signet-ring hypertrophic cells”. Using immunohistochemistry, these signet ring hypertrophic cells were found to be positive for gonadotropin. Taken together, our results suggest a (1) tumorigenic effect of Prop1 in the pituitaries, and (2) causative effects of signet ring-type gonadotropes. PMID:18636109

  3. Transgenic Fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish into which foreign DNA is artificially introduced and integrated into their genome are called transgenic fish. Since the development of the first transgenic fish in 1985, techniques to produce transgenic fish have improved tremendously, resulting in the production of genetically modified (GM) ...

  4. Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

    PubMed

    Luchetti, C G; Bevacqua, R J; Lorenzo, M S; Tello, M F; Willis, M; Buemo, C P; Lombardo, D M; Salamone, D F

    2016-08-01

    The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids. PMID:27260090

  5. Immunogenicity of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants.

    PubMed

    Wang, Lina; Webster, Diane E; Campbell, Alison E; Dry, Ian B; Wesselingh, Steve L; Coppel, Ross L

    2008-01-01

    Malaria is a major global health problem for which effective control measures are urgently needed. Considerable effort has been focused on the development of effective vaccines against the causative parasite and protective vaccine trials are now being reported. Due to the relative poverty and lack of infrastructure in malaria-endemic areas, a successful immunisation strategy will depend critically on cheap and scaleable methods of vaccine production, distribution and delivery. One promising technology is transgenic plants, both as a bioreactor for the vaccine-manufacturing process as well as a matrix for oral immunisation. In this study, we investigated the feasibility of using transgenic plants to induce protective immunity against malaria infection using Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) in a mouse model of malaria infection. Our data show that the PyMSP4/5 protein can be produced in plants in a configuration that reacts with protective antibodies. Optimisation of codon usage for the PyMSP4/5 gene resulted in significantly increased antigen expression in plants. PyMSP4/5 protein from the codon-optimised construct accumulated to 0.25% of total soluble protein, a sixfold increase over the native gene sequence. Tobacco-made PyMSP4/5 was able to induce antigen-specific antibodies in mice following parenteral delivery, as well as boost the antibody responses induced by DNA vaccination when delivered parenterally or orally. We believe this is the first report to show that plant-made malaria antigens are immunogenic. However, the antibody levels were not high enough to protect the immunised mice against a lethal challenge with P. yoelii. Further strategies are needed to achieve a protective dose, including improvements to antigen expression levels in plants and strategies to enhance the immunogenicity of the expressed antigen. PMID:17681344

  6. Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

    PubMed

    Lopez, M J; Upchurch, B H; Rindi, G; Leiter, A B

    1995-01-13

    We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells. PMID:7822327

  7. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

    PubMed

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-01-01

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894

  8. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

    PubMed Central

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-01-01

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894

  9. Targeted expression of SV40 T antigen in the hair follicle of transgenic mice produces an aberrant hair phenotype.

    PubMed

    Keough, R; Powell, B; Rogers, G

    1995-03-01

    Directed expression of SV40 large T antigen (TAg) in transgenic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultrastructure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormalities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy. PMID:7542671

  10. Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

    PubMed Central

    Kittur, Farooqahmed S.; Bah, Mamudou; Archer-Hartmann, Stephanie; Hung, Chiu-Yueh; Azadi, Parastoo; Ishihara, Mayumi; Sane, David C.; Xie, Jiahua

    2013-01-01

    Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production. PMID:24124563

  11. Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

    PubMed Central

    Tabashnik, Bruce E.; Dennehy, Timothy J.; Sims, Maria A.; Larkin, Karen; Head, Graham P.; Moar, William J.; Carrière, Yves

    2002-01-01

    Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended. PMID:12147473

  12. N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits

    PubMed Central

    Chevreux, Guillaume; Faid, Valegh; Scohyers, Jean-Marc; Bihoreau, Nicolas

    2013-01-01

    Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A2G2S1. Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)0, 1, 2 motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern. PMID:24092837

  13. Mechanical properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic silkworms.

    PubMed

    Zhu, Zhenghua; Kikuchi, Yuka; Kojima, Katsura; Tamura, Toshiki; Kuwabara, Nobuo; Nakamura, Takashi; Asakura, Tetsuo

    2010-01-01

    Regenerated silk fibroin fibers from the cocoons of silkworm, Bombyx mori, were prepared with hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or hexafluoroacetone-trihydrate (HFA), as dope solvents and methanol as coagulation solvent. The regenerated fiber prepared from the HFIP solution showed slightly larger tensile strength when the draw ratio is 1:3 than that of native silk fiber, but the strength of the regenerated fiber with draw ratio 1:3 from the HFA solution is much lower than that of native silk fiber. This difference in the tensile strength of the regenerated silk fibers between two dope solvents comes from the difference in the long-range orientation of the crystalline region rather than that of short-range structural environment such as the fraction of beta-sheet structure. The increase in the biodegradation was observed for the regenerated silk fiber compared with native silk fiber. Preparations of regenerated silk fibroin fibers containing spider silk sequences were obtained by mixing silk fibroins and silk-like proteins with characteristic sequences from a spider, Naphila clavipes, to produce drag-line silk in E. coli in the fluoro solvents. A small increase in the tensile strength was obtained by adding 5% (w/w) of the silk-like protein to the silk fibroin. The production of silk fibroin fibers with these spider silk sequences was also performed with transgenic silkworms. Small increase in the tensile strength of the fibers was obtained without significant change in the elongation-at-break. PMID:20178693

  14. A Cyclosporine-Sensitive Psoriasis-Like Disease Produced in Tie2 Transgenic Mice

    PubMed Central

    Voskas, Daniel; Jones, Nina; Van Slyke, Paul; Sturk, Celina; Chang, Wing; Haninec, Alex; Babichev, Yael Olya; Tran, Jennifer; Master, Zubin; Chen, Stephen; Ward, Nicole; Cruz, Maribelle; Jones, Jamie; Kerbel, Robert S.; Jothy, Serge; Dagnino, Lina; Arbiser, Jack; Klement, Giannoula; Dumont, Daniel J.

    2005-01-01

    Psoriasis is a common, persistent skin disorder characterized by recurrent erythematous lesions thought to arise as a result of inflammatory cell infiltration and activation of keratinocyte proliferation. Unscheduled angiogenic growth has also been proposed to mediate the pathogenesis of psoriasis although the cellular and molecular basis for this response remains unclear. Recently, a role for the angiopoietin signaling system in psoriasis has been suggested by studies that demonstrate an up-regulation of the tyrosine kinase receptor Tie2 (also known as Tek) as well as angiopoietin-1 and angiopoietin-2 in human psoriatic lesions. To examine temporal expression of Tie2, we have developed a binary transgenic approach whereby expression of Tie2 can be conditionally regulated by the presence of tetracycline analogs in double-transgenic mice. A psoriasis-like phenotype developed in double-transgenic animals within 5 days of birth and persisted throughout adulthood. The skin of affected mice exhibited many cardinal features of human psoriasis including epidermal hyperplasia, inflammatory cell accumulation, and altered dermal angiogenesis. These skin abnormalities resolved completely with tetracycline-mediated suppression of transgene expression, thereby illustrating a complete dependence on Tie2 signaling for disease maintenance and progression. Furthermore, the skin lesions in double-transgenic mice markedly improved after administration of the immunosuppressive anti-psoriatic agent cyclosporine, thus demonstrating the clinical significance of this new model. PMID:15743796

  15. Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

    PubMed Central

    Garrels, Wiebke; Holler, Stephanie; Cleve, Nicole; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A.

    2012-01-01

    Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome. PMID:24705079

  16. A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop.

    PubMed

    de Vetten, Nick; Wolters, Anne-Marie; Raemakers, Krit; van der Meer, Ingrid; ter Stege, Renaldo; Heeres, Els; Heeres, Paul; Visser, Richard

    2003-04-01

    It is generally thought that transformation of plant cells using Agrobacterium tumefaciens occurs at a very low frequency. Therefore, selection marker genes are used to identify the rare plants that have taken up foreign DNA. Genes encoding antibiotic and herbicide resistance are widely used for this purpose in plant transformation. Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic- and herbicide-resistance genes from an ecological and food safety perspective. Although no scientific basis has been determined for these concerns, generating marker-free plants would certainly contribute to the public acceptance of transgenic crops. Several methods have been reported to create marker gene-free transformed plants, for example co-transformation, transposable elements, site-specific recombination, or intrachromosomal recombination. Not only are most of these systems time-consuming and inefficient, but they are also employed on the assumption that isolation of transformants without a selective marker gene is not feasible. Here we present a method that permits the identification of transgenic plants without the use of selectable markers. This strategy relies on the transformation of tissue explants or cells with a virulent A. tumefaciens strain and selection of transformed cells or shoots after PCR analysis. Incubation of potato explants with A. tumefaciens strain AGL0 resulted in transformed shoots at an efficiency of 1-5% of the harvested shoots, depending on the potato genotype used. Because this system does not require genetic segregation or site-specific DNA-deletion systems to remove marker genes, it may provide a reliable and efficient tool for generating transgenic plants for commercial use, especially in vegetatively propagated species like potato and cassava. PMID:12627169

  17. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

    PubMed Central

    Chan, Anthony W. S.; Homan, E. Jane; Ballou, Linda U.; Burns, Jane C.; Bremel, Robert D.

    1998-01-01

    A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis. PMID:9826647

  18. Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-α or erythropoietin for a bovine mammary bioreactor.

    PubMed

    Kang, Hee Young; Hong, Eui-Ju; Hwang, Kyu-Chan; Kim, Nam-Hyung; Hwang, Woo-Suk; Jeung, Eui-Bae

    2015-07-01

    Interferon α (IFN-α) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia. In the production of recombinant proteins, a transgenic expression system in bovine species is an efficient strategy for pharmaceutical production. In the present study, recombinant constructs capable of producing recombinant human IFN-α and EPO proteins were established and were generated containing the mammary gland-specific αS1-casein promoter region (between -175 and + 796 nt), as this promoter was revealed to have the highest level of activity in a previous promoter study. In order to minimize developmental toxicity by constitutive exogenous expression, a doxycycline (dox)-inducible system was introduced to the IFN-α/EPO-expressing constructs. Therefore, a unitary tetracycline (tet)-on the IFN-α/EPO vector was established, which combined a tet-on activator cassette controlled by the αS1-casein promoter, with a responder cassette encoding the IFN-α/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet-controlled transactivator is affected by mammary gland-specific αS1-casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN-α/EPO genes in the presence of dox. To assess this, the unitary tet-on IFN-α/EPO vector was introduced into a bovine mammary gland cell line (MAC-T), and the cells were then treated with 0.1-1 µg/ml dox. A marked increase was observed in the expression levels of IFN-α/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland-specific and dox-inducible IFN

  19. Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

    PubMed

    Raybould, Alan; Kilby, Peter; Graser, Gerson

    2013-04-01

    Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence. PMID:23065372

  20. Efficient engineering of marker-free synthetic allotetraploids of Saccharomyces.

    PubMed

    Alexander, William G; Peris, David; Pfannenstiel, Brandon T; Opulente, Dana A; Kuang, Meihua; Hittinger, Chris Todd

    2016-04-01

    Saccharomyces interspecies hybrids are critical biocatalysts in the fermented beverage industry, including in the production of lager beers, Belgian ales, ciders, and cold-fermented wines. Current methods for making synthetic interspecies hybrids are cumbersome and/or require genome modifications. We have developed a simple, robust, and efficient method for generating allotetraploid strains of prototrophic Saccharomyces without sporulation or nuclear genome manipulation. S. cerevisiae×S. eubayanus, S. cerevisiae×S. kudriavzevii, and S. cerevisiae×S. uvarum designer hybrid strains were created as synthetic lager, Belgian, and cider strains, respectively. The ploidy and hybrid nature of the strains were confirmed using flow cytometry and PCR-RFLP analysis, respectively. This method provides an efficient means for producing novel synthetic hybrids for beverage and biofuel production, as well as for constructing tetraploids to be used for basic research in evolutionary genetics and genome stability. PMID:26555931

  1. Relevance of traditional integrated pest management (IPM) strategies for commercial corn producers in a transgenic agroecosystem: a bygone era?

    PubMed

    Gray, Michael E

    2011-06-01

    The use of transgenic Bt maize hybrids continues to increase significantly across the Corn Belt of the United States. In 2009, 59% of all maize planted in Illinois was characterized as a "stacked" gene variety. This is a 40% increase since 2006. Stacked hybrids typically express one Cry protein for corn rootworm control and one Cry protein for control of several lepidopteran pests; they also feature herbicide tolerance (to either glyphosate or glufosinate). Slightly more than 50 years has passed since Vernon Stern and his University of California entomology colleagues published (1959) their seminal paper on the integrated control concept, laying the foundation for modern pest management (IPM) programs. To assess the relevance of traditional IPM concepts within a transgenic agroecosystem, commercial maize producers were surveyed at a series of meetings in 2009 and 2010 regarding their perceptions on their use of Bt hybrids and resistance management. Special attention was devoted to two insect pests of corn, the European corn borer and the western corn rootworm. A high percentage of producers who participated in these meetings planted Bt hybrids in 2008 and 2009, 97 and 96.7%, respectively. Refuge compliance in 2008 and 2009, as mandated by the U.S. Environmental Protection Agency (EPA), was 82 and 75.7%, respectively, for those producers surveyed. A large majority of producers (79 and 73.3% in 2009 and 2010, respectively) revealed that they would, or had, used a Bt hybrid for corn rootworm (Diabrotica virgifera virgifera LeConte) or European corn borer (Ostrinia nubilalis Hübner) control even when anticipated densities were low. Currently, the EPA is evaluating the long-term use of seed blends (Bt and non-Bt) as a resistance management strategy. In 2010, a large percentage of producers, 80.4%, indicated they would be willing to use this approach. The current lack of integration of management tactics for insect pests of maize in the U.S. Corn Belt, due primarily to

  2. Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

    PubMed

    Amiri Yekta, Amir; Dalman, Azam; Eftekhari-Yazdi, Poopak; Sanati, Mohammad Hossein; Shahverdi, Abdol Hossein; Fakheri, Rahman; Vazirinasab, Hamed; Daneshzadeh, Mohammad Taghi; Vojgani, Mahdi; Zomorodipour, Alireza; Fatemi, Nayeralsadat; Vahabi, Zeinab; Mirshahvaladi, Shahab; Ataei, Fariba; Bahraminejad, Elmira; Masoudi, Najmehsadat; Rezazadeh Valojerdi, Mojtaba; Gourabi, Hamid

    2013-02-01

    There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to β-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats. PMID:22869287

  3. Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance.

    PubMed

    Dufourmantel, Nathalie; Dubald, Manuel; Matringe, Michel; Canard, Hélène; Garcon, Frédéric; Job, Claudette; Kay, Elisabeth; Wisniewski, Jean-Pierre; Ferullo, Jean-Marc; Pelissier, Bernard; Sailland, Alain; Tissot, Ghislaine

    2007-01-01

    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait. PMID:17207262

  4. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    PubMed

    Yamaguchi, Shigeyuki; Kazuki, Yasuhiro; Nakayama, Yuji; Nanba, Eiji; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2011-01-01

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression. PMID:21390305

  5. Continuous production of flexible fibers from transgenically produced honeybee silk proteins.

    PubMed

    Poole, Jacinta; Church, Jeffrey S; Woodhead, Andrea L; Huson, Mickey G; Sriskantha, Alagacone; Kyratzis, Ilias L; Sutherland, Tara D

    2013-10-01

    Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting. PMID:23881528

  6. Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.

    PubMed

    Dutt, Manjul; Li, Zhijian T; Dhekney, Sadanand A; Gray, Dennis J

    2012-01-01

    A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine. PMID:22351010

  7. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    PubMed

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein. PMID:25115849

  8. Marker-free registration of forest terrestrial laser scanner data pairs with embedded confidence metrics

    DOE PAGESBeta

    Van Aardt, Jan; Romanczyk, Paul; van Leeuwen, Martin; Kelbe, David; Cawse-Nicholson, Kerry

    2016-04-04

    Terrestrial laser scanning (TLS) has emerged as an effective tool for rapid comprehensive measurement of object structure. Registration of TLS data is an important prerequisite to overcome the limitations of occlusion. However, due to the high dissimilarity of point cloud data collected from disparate viewpoints in the forest environment, adequate marker-free registration approaches have not been developed. The majority of studies instead rely on the utilization of artificial tie points (e.g., reflective tooling balls) placed within a scene to aid in coordinate transformation. We present a technique for generating view-invariant feature descriptors that are intrinsic to the point cloud datamore » and, thus, enable blind marker-free registration in forest environments. To overcome the limitation of initial pose estimation, we employ a voting method to blindly determine the optimal pairwise transformation parameters, without an a priori estimate of the initial sensor pose. To provide embedded error metrics, we developed a set theory framework in which a circular transformation is traversed between disjoint tie point subsets. This provides an upper estimate of the Root Mean Square Error (RMSE) confidence associated with each pairwise transformation. Output RMSE errors are commensurate with the RMSE of input tie points locations. Thus, while the mean output RMSE=16.3cm, improved results could be achieved with a more precise laser scanning system. This study 1) quantifies the RMSE of the proposed marker-free registration approach, 2) assesses the validity of embedded confidence metrics using receiver operator characteristic (ROC) curves, and 3) informs optimal sample spacing considerations for TLS data collection in New England forests. Furthermore, while the implications for rapid, accurate, and precise forest inventory are obvious, the conceptual framework outlined here could potentially be extended to built environments.« less

  9. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    PubMed

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. PMID:26147010

  10. A marker-free system for highly efficient construction of vaccinia virus vectors using CRISPR Cas9

    PubMed Central

    Yuan, Ming; Gao, Xuefei; Chard, Louisa S; Ali, Zarah; Ahmed, Jahangir; Li, Yunqing; Liu, Pentao; Lemoine, Nick R; Wang, Yaohe

    2015-01-01

    The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (~90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application. PMID:26417609

  11. 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.; Rowe, D. W. (Principal Investigator)

    1995-01-01

    Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

  12. Pathological hallmarks of amyotrophic lateral sclerosis/frontotemporal lobar degeneration in transgenic mice produced with TDP-43 genomic fragments.

    PubMed

    Swarup, Vivek; Phaneuf, Daniel; Bareil, Christine; Robertson, Janice; Rouleau, Guy A; Kriz, Jasna; Julien, Jean-Pierre

    2011-09-01

    Transactive response DNA-binding protein 43 ubiquitinated inclusions are a hallmark of amyotrophic lateral sclerosis and of frontotemporal lobar degeneration with ubiquitin-positive inclusions. Yet, mutations in TARDBP, the gene encoding these inclusions are associated with only 3% of sporadic and familial amyotrophic lateral sclerosis. Recent transgenic mouse studies have revealed a high degree of toxicity due to transactive response DNA-binding protein 43 proteins when overexpressed under the control of strong neuronal gene promoters, resulting in early paralysis and death, but without the presence of amyotrophic lateral sclerosis-like ubiquitinated transactive response DNA-binding protein 43-positive inclusions. To better mimic human amyotrophic lateral sclerosis, we generated transgenic mice that exhibit moderate and ubiquitous expression of transactive response DNA-binding protein 43 species using genomic fragments that encode wild-type human transactive response DNA-binding protein 43 or familial amyotrophic lateral sclerosis-linked mutant transactive response DNA-binding protein 43 (G348C) and (A315T). These novel transgenic mice develop many age-related pathological and biochemical changes reminiscent of human amyotrophic lateral sclerosis including ubiquitinated transactive response DNA-binding protein 43-positive inclusions, transactive response DNA-binding protein 43 cleavage fragments, intermediate filament abnormalities, axonopathy and neuroinflammation. All three transgenic mouse models (wild-type, G348C and A315T) exhibited impaired learning and memory capabilities during ageing, as well as motor dysfunction. Real-time imaging with the use of biophotonic transactive response DNA-binding protein 43 transgenic mice carrying a glial fibrillary acidic protein-luciferase reporter revealed that the behavioural defects were preceded by induction of astrogliosis, a finding consistent with a role for reactive astrocytes in amyotrophic lateral sclerosis

  13. Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.

    PubMed

    Bukovinszki, Agnes; Divéki, Zoltán; Csányi, Márta; Palkovics, László; Balázs, Ervin

    2007-04-01

    In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species. PMID:17103215

  14. Marker-free plasmids for gene therapeutic applications--lack of antibiotic resistance gene substantially improves the manufacturing process.

    PubMed

    Mairhofer, Jürgen; Cserjan-Puschmann, Monika; Striedner, Gerald; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Grabherr, Reingard

    2010-04-01

    Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial

  15. Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

    NASA Astrophysics Data System (ADS)

    Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

    2014-02-01

    PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

  16. Contribution to a marker-free system for human motion analysis

    NASA Astrophysics Data System (ADS)

    Calais, Elodie F.; Legrand, Louis; Voisin, Yvon; Diou, Alan

    2002-07-01

    This paper presents a novel approach to human gait analysis using a marker-free system. The devised acquisition system is composed of three synchronized and calibrated charge coupled device cameras. The aim of this work is to recognize in gray level image sequences the leg of a walking human and to reconstruct it in the three-dimensional space. An articulated three- dimensional (3D) model of the human body, based on the use of tapered superquadric curves, is first introduced. A motion-based segmentation, using morphological operators, is then applied to the image sequences in order to extract the boundaries of the leg in motion. A reconstruction process, based on the use of a least median of squares regression is next performed, to determine the location of the human body in the 3D space. Finally, a spatial coherence is imposed on the reconstructed curves in order to better fit the anatomy of the leg and to take into account the articulated model. Each stage of the proposed methodology has been tested both on synthetic images and on real world images of walking humans. The obtained results suggest that this approach is quite promising and should be useful in the study of the gait.

  17. Analysis and tracking of human gait via a marker-free system

    NASA Astrophysics Data System (ADS)

    Calais, Elodie F.; Legrand, Louis

    2000-05-01

    This paper presents a marker-free methodology for facilitating the analysis and tracking of human motion during gait. It consists of recognition and reconstruction of the legs of a walking human. In order to study the gait, a video system composed of three synchronized CCD cameras has been devised. It provides three different grey level image sequences of the same scene. First of all, a model of the human, based on tapered superquadric curves, has been defined; the leg can be divided into three parts: the thigh, the calf, and the foot. The whole methodology can be split following this scheme: determination of the boundaries of the human in motion by using successively an optical flow process and a crest lines extraction algorithm, prediction of the location of the human body in the 3D space, direct reconstruction of each part of the leg with a Least Median of Squares regression, and finally application of a spatial coherence process. The method described has been tested on synthetic images (mean error of about 1.2 mm and a maximal error of about 5 mm along the coordinate axes) and on image sequences of a walking human.

  18. One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

    PubMed Central

    Matsuoka, Hiroyuki; Sano, Gen-ichiro; Hattori, Ryuta; Tomita, Hiroyuki; Yamamoto, Daisuke S.; Hirai, Makoto

    2012-01-01

    It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect. PMID:23097619

  19. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

    PubMed Central

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    Background 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). Results With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Conclusion Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. PMID:26469402

  20. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    PubMed

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. PMID:25554634

  1. Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels.

    PubMed

    Willis, Jonathan D; Mazarei, Mitra; Stewart, C Neal

    2016-01-01

    Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review. PMID:27303411

  2. Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels

    PubMed Central

    Willis, Jonathan D.; Mazarei, Mitra; Stewart, C. Neal

    2016-01-01

    Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review. PMID:27303411

  3. Removing the mustard oil bomb from seeds: transgenic ablation of myrosin cells in oilseed rape (Brassica napus) produces MINELESS seeds

    PubMed Central

    Borgen, Birgit Hafeld; Thangstad, Ole Petter; Ahuja, Ishita; Rossiter, John Trevor; Bones, Atle Magnar

    2010-01-01

    Many plant phytochemicals constitute binary enzyme–glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ and characterized as a ‘toxic mine’ in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named ‘MINELESS plants’. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants. PMID:20219777

  4. Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

    PubMed Central

    Floss, Doreen Manuela; Mockey, Michael; Zanello, Galliano; Brosson, Damien; Diogon, Marie; Frutos, Roger; Bruel, Timothée; Rodrigues, Valérie; Garzon, Edwin; Chevaleyre, Claire; Berri, Mustapha; Salmon, Henri; Conrad, Udo; Dedieu, Laurence

    2010-01-01

    This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis. PMID:20414351

  5. TRANSGENE INTEGRATION INTO THE SAME CHROMOSOME LOCATION CAN PRODUCE ALLELES THAT EXPRESS AT A PREDICTABLE LEVEL, OR ALLELES THAT ARE DIFFERENTIALLY SILENCED

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single-copy of the transgene at the designated targ...

  6. Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

    PubMed

    Rindi, G; Ratineau, C; Ronco, A; Candusso, M E; Tsai, M; Leiter, A B

    1999-09-01

    The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells. PMID:10457023

  7. Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines.

    PubMed

    De Marchis, Francesca; Balducci, Chiara; Pompa, Andrea; Riise Stensland, Hilde M F; Guaragno, Marco; Pagiotti, Rita; Menghini, Anna R; Persichetti, Emanuele; Beccari, Tommaso; Bellucci, Michele

    2011-12-01

    Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy. PMID:21645202

  8. Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

    PubMed

    Park, Sung-Chul; Kim, Yun-Hee; Kim, Sun Ha; Jeong, Yu Jeong; Kim, Cha Young; Lee, Joon Seol; Bae, Ji-Yeong; Ahn, Mi-Jeong; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2015-04-01

    The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity. PMID:25220246

  9. Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

    PubMed Central

    Merlo, A O; Cowen, N; Delate, T; Edington, B; Folkerts, O; Hopkins, N; Lemeiux, C; Skokut, T; Smith, K; Woosley, A; Yang, Y; Young, S; Zwick, M

    1998-01-01

    Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize. PMID:9761789

  10. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse

    PubMed Central

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 107 TCID50/mL 10 days after infection when using an MOI of 10−4. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system. PMID:26287531

  11. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

    PubMed

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system. PMID:26287531

  12. Glucagon gene 5'-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice.

    PubMed

    Lee, Y C; Asa, S L; Drucker, D J

    1992-05-25

    Glucagon and the glucagon-like peptides play important roles in the regulation of glucose homeostasis. Previous studies have demonstrated that approximately 1300 base pairs of rat glucagon gene 5'-flanking sequences direct transgene expression to the pancreas and brain, but not to the intestine, of transgenic mice. These observations suggested that different tissue-specific enhancer elements mediate activation of glucagon gene transcription in the pancreas and intestine. We have now generated mice that express SV40 large T antigen under the control of approximately 2000 base pairs of glucagon gene 5'-flanking sequences. Transgene expression was observed in the brain and pancreas in association with the development of pancreatic endocrine tumors. In contrast to the mice described previously, we also detected transgene expression throughout the gastrointestinal tract in endocrine cells of the stomach and small and large intestine. Focal areas of enteroendocrine cell hyperplasia in the large bowel invariably progressed to invasive and metastasizing plurihormonal endocrine carcinoma, which was clinically and pathologically evident by 4 weeks of age. In contrast, transgene expression in the small bowel and stomach was not associated with progression to either hyperplasia or carcinoma. The results of these studies provide functional evidence for the existence of an upstream cis-acting regulatory domain that directs glucagon gene transcription to the endocrine cells of the intestine in transgenic mice. PMID:1587847

  13. TRANSGENIC FISH: In Genomics and Genetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish into which foreign DNA is artificially introduced and integrated into their genome are called transgenic fish. Since the development of the first transgenic fish in 1985, techniques to produce transgenic fish have improved tremendously, resulting in the production of genetically modified (GM)...

  14. [Current status and industrialization of transgenic tomatoes].

    PubMed

    Wang, Ao-Xue; Chen, Xiu-Ling

    2011-09-01

    In this review, the progress in transgenic tomato research, including disease and insect resistance, herbicide resistance, stress tolerance, long-term storage, quality improvement, and male sterility, were described. The recent researches on producing heterologous proteins using transgenic tomatoes were also reviewed. Furthermore, the industrialization status and problems of transgenic tomatoes were analyzed and the prospects of both research and industrialization in transgenic tomatoes were discussed. PMID:21951797

  15. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites

    PubMed Central

    Manzoni, Giulia; Briquet, Sylvie; Risco-Castillo, Veronica; Gaultier, Charlotte; Topçu, Selma; Ivănescu, Maria Larisa; Franetich, Jean-François; Hoareau-Coudert, Bénédicte; Mazier, Dominique; Silvie, Olivier

    2014-01-01

    Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development. PMID:24755823

  16. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

    PubMed

    Manzoni, Giulia; Briquet, Sylvie; Risco-Castillo, Veronica; Gaultier, Charlotte; Topçu, Selma; Ivănescu, Maria Larisa; Franetich, Jean-François; Hoareau-Coudert, Bénédicte; Mazier, Dominique; Silvie, Olivier

    2014-01-01

    Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development. PMID:24755823

  17. A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis.

    PubMed

    Lin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongyou; Li, Weifen

    2013-11-01

    There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes. PMID:23911571

  18. Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block

    PubMed Central

    Hein, Lutz; Stevens, Mary E.; Barsh, Gregory S.; Pratt, Richard E.; Kobilka, Brian K.; Dzau, Victor J.

    1997-01-01

    Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the α-myosin heavy chain (αMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription–PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction. PMID:9177228

  19. Healthy Ready-to-Eat Expanded Snack with High Nutritional and Antioxidant Value Produced from Whole Amarantin Transgenic Maize and Black Common Bean.

    PubMed

    Espinoza-Moreno, Ramona J; Reyes-Moreno, Cuauhtémoc; Milán-Carrillo, Jorge; López-Valenzuela, José A; Paredes-López, Octavio; Gutiérrez-Dorado, Roberto

    2016-06-01

    The snack foods market is currently demanding healthier products. A ready-to-eat expanded snack with high nutritional and antioxidant value was developed from a mixture (70:30) of whole amarantin transgenic maize (Zea mays L.) and black common bean (Phaseolus vulgaris L.) by optimizing the extrusion process. Extruder operation conditions were: feed moisture content (FMC, 15-25 %, wet basis), barrel temperature (BT, 120-170 °C), and screw speed (SS, 50-240). The desirability numeric method of the response surface methodology (RSM) was applied as the optimization technique over four response variables [expansion ratio (ER), bulk density (BD), hardness (H), antioxidant activity (AoxA)] to obtain maximum ER and AoxA, and minimum BD, and H values. The best combination of extrusion process variables for producing an optimized expanded snack (OES, healthy snack) were: FMC = 15 %/BT = 157 °C/SS = 238 rpm. The OES had ER = 2.86, BD = 0.119 g/cm (3) , H = 1.818 N, and AoxA = 13,681 μmol Trolox equivalent (TE)/100 g, dry weight. The extrusion conditions used to produce the OES increased the AoxA (ORAC: +18 %, ABTS:+20 %) respect to the unprocessed whole grains mixture. A 50 g portion of OES had higher protein content (7.23 vs 2.32 g), total dietary fiber (7.50 vs 1.97 g), total phenolic content (122 vs 47 mg GAE), and AoxA (6626 vs 763 μmol TE), and lower energy (169 vs 264 kcal) than an expanded commercial snack (ECS = Cheetos™). Because of its high content of quality protein, dietary fiber and phenolics, as well as high AoxA and low energy density, the OES could be used for health promotion and chronic disease prevention and as an alternative to the widely available commercial snacks with high caloric content and low nutritional/nutraceutical value. PMID:27170034

  20. Field plot assessments demonstrate that transgenic plums expressing Plum pox virus (PPV) coat protein gene do not affect the PPV strain composition or produce PPV recombinants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serological and molecular variability of Plum pox virus (PPV) detected in transgenic plum trees harboring PPV capsid gene versus those found in conventional plums were analyzed. Strain characterization was serologically determined by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodie...

  1. Marker-free transgenic (MFT) near-isogenic introgression lines (NIILs) of 'golden' indica rice (cv. IR64) with accumulation of provitamin A in the endosperm tissue.

    PubMed

    Baisakh, Niranjan; Rehana, Sayda; Rai, Mayank; Oliva, Norman; Tan, Jing; Mackill, David J; Khush, Gurdev S; Datta, Karabi; Datta, Swapan K

    2006-07-01

    We have developed near-isogenic introgression lines (NIILs) of an elite indica rice cultivar (IR64) with the genes for beta-carotene biosynthesis from dihaploid (DH) derivatives of golden japonica rice (cv. T309). A careful analysis of the DH lines indicated the integration of the genes of interest [phytoene synthase (psy) and phytoene desaturase (crtI)] and the selectable marker gene (hygromycin phosphotransferase, hph) in two unlinked loci. During subsequent crossing, progenies could be obtained carrying only the locus with psy and crtI, which was segregated independently from the locus containing the hph gene during meiotic segregation. The NIILs (BC(2)F(2)) showed maximum similarity with the recurrent parent cultivar IR64. Further, progenies of two NIILs were devoid of any fragments beyond the left or right border, including the chloramphenicol acetyltransferase (cat) antibiotic resistance gene of the transformation vector. Spectrophotometric readings showed the accumulation of up to 1.06 microg total carotenoids, including beta-carotene, in 1 g of the endosperm. The accumulation of beta-carotene was also evident from the clearly visible yellow colour of the polished seeds. PMID:17177811

  2. Selectable marker genes in transgenic plants: applications, alternatives and biosafety.

    PubMed

    Miki, Brian; McHugh, Sylvia

    2004-02-01

    used. Many of the genes have specific limitations or have not been sufficiently tested to merit their widespread use. For research, a variety of selection systems are essential as no single selectable marker gene was found to be sufficient for all circumstances. Although, no adverse biosafety effects have been reported for the marker genes that have been adopted for widespread use, biosafety concerns should help direct which markers will be chosen for future crop development. Common sense dictates that marker genes conferring resistance to significant therapeutic antibiotics should not be used. An area of research that is growing rapidly but is still in its infancy is the development of strategies for eliminating selectable marker genes to generate marker-free plants. Among the several technologies described, two have emerged with significant potential. The simplest is the co-transformation of genes of interest with selectable marker genes followed by the segregation of the separate genes through conventional genetics. The more complicated strategy is the use of site-specific recombinases, under the control of inducible promoters, to excise the marker genes and excision machinery from the transgenic plant after selection has been achieved. In this review each of the genes and processes will be examined to assess the alternatives that exist for producing transgenic plants. PMID:14736458

  3. Transgenic Animals.

    ERIC Educational Resources Information Center

    Jaenisch, Rudolf

    1988-01-01

    Describes three methods and their advantages and disadvantages for introducing genes into animals. Discusses the predictability and tissue-specificity of the injected genes. Outlines the applications of transgenic technology for studying gene expression, the early stages of mammalian development, mutations, and the molecular nature of chromosomes.…

  4. A high throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S), that contain selective marke...

  5. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

    PubMed

    Panahi, Mitra; Alli, Zaman; Cheng, Xiongying; Belbaraka, Loubaba; Belgoudi, Jaafar; Sardana, Ravinder; Phipps, Jenny; Altosaar, Illimar

    2004-06-01

    Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been

  6. N-ACYL HOMOSERINE LACTONe LACTONASE, AiiA, INACTIVATION OF QUORUM-SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE(1).

    PubMed

    Rajamani, Sathish; Teplitski, Max; Kumar, Anil; Krediet, Cory J; Sayre, Richard T; Bauer, Wolfgang D

    2011-10-01

    Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N-acyl homoserine lactone (AHL) bacterial quorum-sensing (QS) signals and alter QS-regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal-mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR-stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal-mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL-producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS. PMID:27020200

  7. Repetitive, marker-free, site-specific integration as a novel tool for multiple chromosomal integration of DNA.

    PubMed

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

    2013-06-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB(min)), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB(min) site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps. PMID:23542630

  8. 2D/4D marker-free tumor tracking using 4D CBCT as the reference image

    PubMed Central

    Wang, Mengjiao; Rit, Simon; Delmon, Vivien; Wang, Guangzhi

    2014-01-01

    Tumor motion caused by respiration is an important issue in image guided radiotherapy. A 2D/4D matching method between 4D volumes derived from cone beam computed tomography (CBCT) and 2D fluoroscopic images was implemented to track the tumor motion without the use of implanted markers. In this method, firstly, 3DCBCT and phase-rebinned 4DCBCT are reconstructed from cone beam acquisition. Secondly, 4DCBCT volumes and streak free 3DCBCT volume are combined to improve the image quality of the DRRs. Finally, the 2D/4D matching problem is converted into a 2D/2D matching between incoming projections and DRR images from each phase of the 4DCBCT. The diaphragm is used as a target surrogate for matching instead of using the tumor position directly. This relies on the assumption that if a patient has the same breathing phase and diaphragm position as the reference 4DCBCT, then the tumor position is the same. From the matching results, the phase information, diaphragm position and tumor position at the time of each incoming projection acquisition can be derived. The accuracy of this method was verified using 16 candidate datasets, representing lung and liver applications and 1-minute and 2-minute acquisitions. The criteria for the eligibility of datasets were described: 11 eligible datasets were selected to verify the accuracy of diaphragm tracking, and one eligible dataset was chosen to verify the accuracy of tumor tracking. Diaphragm matching accuracy was 1.88±1.35mm in the isocenter plane, the 2D tumor tracking accuracy was 2.13±1.26mm in the isocenter plane. These features make this method feasible for real-time marker-free tumor motion tracking purpose. PMID:24710793

  9. 2D/4D marker-free tumor tracking using 4D CBCT as the reference image

    NASA Astrophysics Data System (ADS)

    Wang, Mengjiao; Sharp, Gregory C.; Rit, Simon; Delmon, Vivien; Wang, Guangzhi

    2014-05-01

    Tumor motion caused by respiration is an important issue in image-guided radiotherapy. A 2D/4D matching method between 4D volumes derived from cone beam computed tomography (CBCT) and 2D fluoroscopic images was implemented to track the tumor motion without the use of implanted markers. In this method, firstly, 3DCBCT and phase-rebinned 4DCBCT are reconstructed from cone beam acquisition. Secondly, 4DCBCT volumes and a streak-free 3DCBCT volume are combined to improve the image quality of the digitally reconstructed radiographs (DRRs). Finally, the 2D/4D matching problem is converted into a 2D/2D matching between incoming projections and DRR images from each phase of the 4DCBCT. The diaphragm is used as a target surrogate for matching instead of using the tumor position directly. This relies on the assumption that if a patient has the same breathing phase and diaphragm position as the reference 4DCBCT, then the tumor position is the same. From the matching results, the phase information, diaphragm position and tumor position at the time of each incoming projection acquisition can be derived. The accuracy of this method was verified using 16 candidate datasets, representing lung and liver applications and one-minute and two-minute acquisitions. The criteria for the eligibility of datasets were described: 11 eligible datasets were selected to verify the accuracy of diaphragm tracking, and one eligible dataset was chosen to verify the accuracy of tumor tracking. The diaphragm matching accuracy was 1.88 ± 1.35 mm in the isocenter plane and the 2D tumor tracking accuracy was 2.13 ± 1.26 mm in the isocenter plane. These features make this method feasible for real-time marker-free tumor motion tracking purposes.

  10. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    SciTech Connect

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. )

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  11. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

    PubMed

    Jessop-Fabre, Mathew M; Jakočiūnas, Tadas; Stovicek, Vratislav; Dai, Zongjie; Jensen, Michael K; Keasling, Jay D; Borodina, Irina

    2016-08-01

    Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available. PMID:27166612

  12. Transgenic mouse offspring generated by ROSI.

    PubMed

    Moreira, Pedro; Pérez-Cerezales, Serafín; Laguna, Ricardo; Fernández-Gonzalez, Raúl; Sanjuanbenito, Belén Pintado; Gutiérrez-Adán, Alfonso

    2016-01-01

    The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes. PMID:26498042

  13. Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

    PubMed

    Leibman, Diana; Prakash, Shanmugam; Wolf, Dalia; Zelcer, Aaron; Anfoka, Ghandi; Haviv, Sabrina; Brumin, Marina; Gaba, Victor; Arazi, Tzahi; Lapidot, Moshe; Gal-On, Amit

    2015-11-01

    Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved. PMID:26255053

  14. Accuracy assessment of a marker-free method for registration of CT and stereo images applied in image-guided implantology: a phantom study.

    PubMed

    Mohagheghi, Saeed; Ahmadian, Alireza; Yaghoobee, Siamak

    2014-12-01

    To assess the accuracy of a proposed marker-free registration method as opposed to the conventional marker-based method using an image-guided dental system, and investigating the best configurations of anatomical landmarks for various surgical fields in a phantom study, a CT-compatible dental phantom consisting of implanted targets was used. Two marker-free registration methods were evaluated, first using dental anatomical landmarks and second, using a reference marker tool. Six implanted markers, distributed in the inner space of the phantom were used as the targets; the values of target registration error (TRE) for each target were measured and compared with the marker-based method. Then, the effects of different landmark configurations on TRE values, measured using the Parsiss IV Guided Navigation system (Parsiss, Tehran, Iran), were investigated to find the best landmark arrangement for reaching the minimum registration error in each target region. It was proved that marker-free registration can be as precise as the marker-based method. This has a great impact on image-guided implantology systems whereby the drawbacks of fiducial markers for patient and surgeon are removed. It was also shown that smaller values of TRE could be achieved by using appropriate landmark configurations and moving the center of the landmark set closer to the surgery target. Other common factors would not necessarily decrease the TRE value so the conventional rules accepted in the clinical community about the ways to reduce TRE should be adapted to the selected field of dental surgery. PMID:25441868

  15. Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato.

    PubMed

    Cuellar, Wilmer; Gaudin, Amélie; Solórzano, Dennis; Casas, Armando; Nopo, Luis; Chudalayandi, Prakash; Medrano, Giuliana; Kreuze, Jan; Ghislain, Marc

    2006-09-01

    Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant. PMID:16912912

  16. Ambient insect pressure and recipient genotypes determine fecundity of transgenic crop-weed rice hybrid progeny: Implications for environmental biosafety assessment.

    PubMed

    Xia, Hui; Zhang, Hongbin; Wang, Wei; Yang, Xiao; Wang, Feng; Su, Jun; Xia, Hanbing; Xu, Kai; Cai, Xingxing; Lu, Bao-Rong

    2016-08-01

    Transgene introgression into crop weedy/wild relatives can provide natural selective advantages, probably causing undesirable environmental impact. The advantages are likely associated with factors such as transgenes, selective pressure, and genetic background of transgene recipients. To explore the role of the environment and background of transgene recipients in affecting the advantages, we estimated the fitness of crop-weed hybrid lineages derived from crosses between marker-free insect-resistant transgenic (Bt/CpTI) rice with five weedy rice populations under varied insect pressure. Multiway anova indicated the significant effect of both transgenes and weedy rice genotypes on the performance of crop-weed hybrid lineages in the high-insect environment. Increased fecundity was detected in most transgene-present F1 and F2 hybrid lineages under high-insect pressure, but varied among crop-weed hybrid lineages with different weedy rice parents. Increased fecundity of transgenic crop-weed hybrid lineages was associated with the environmental insect pressure and genotypes of their weedy rice parents. The findings suggest that the fitness effects of an insect-resistant transgene introgressed into weedy populations are not uniform across different environments and genotypes of the recipient plants that have acquired the transgene. Therefore, these factors should be considered when assessing the environmental impact of transgene flow to weedy or wild rice relatives. PMID:27468303

  17. Cryopreservation of transgenic mouse lines.

    PubMed

    Pomeroy, K O

    1993-01-01

    A transgenic animal represents an enormous investment in time and money. Animals can be destroyed through disease, fire, malfuncnons in the control of the environment, negligence, sabotage, or accidental disposal. Researchers can protect valuable transgenic lines from accrdental destruction by "banking" them in liquid nitrogen. Cryopreservation can also reduce animal costs by decreasing the number of live animals investigators must maintain. Often, when one is trying to produce a transgenic animal, some lines will be derived that may not initially appear interesting. These animals can be stored in liquid nitrogen for future recovery and study. The maintenance of just one line of mice, say 25 mice at 15 cents/d, can cost over $1000 (US) in a single year. PMID:21390665

  18. Production of homozygous transgenic rainbow trout with enhanced disease resistance.

    PubMed

    Chiou, Pinwen Peter; Chen, Maria J; Lin, Chun-Mean; Khoo, Jenny; Larson, Jon; Holt, Rich; Leong, Jo-Ann; Thorgarrd, Gary; Chen, Thomas T

    2014-06-01

    Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P₁ transgenic fish were crossed to non-transgenic fish to establish F₁ transgenic founder families, and subsequently generating F₂, and F₃ progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F₁ transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F₂ and F₃ transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F₃ heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis. PMID:24085608

  19. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  20. Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Földes-Papp, Zeno; Kostner, Gerhard M.; König, Karsten

    2010-02-01

    The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12 femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy. Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for optical manipulation.

  1. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

    PubMed

    Qi, Weicong; Tinnenbroek-Capel, Iris E M; Salentijn, Elma M J; Schaart, Jan G; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G F; Huang, Bangquan; Van Loo, Eibertus N; Krens, Frans A

    2015-01-01

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera. PMID:26358007

  2. Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

    PubMed Central

    Qi, Weicong; Tinnenbroek-Capel, Iris E. M.; Salentijn, Elma M. J.; Schaart, Jan G.; Cheng, Jihua; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, Richard G. F.; Huang, Bangquan; Van Loo, Eibertus N.; Krens, Frans A.

    2015-01-01

    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera. PMID:26358007

  3. Neuroanatomy and transgenic technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This is a short review that introduces recent advances of neuroanatomy and transgenic technologies. The anatomical complexity of the nervous system remains a subject of tremendous fascination among neuroscientists. In order to tackle this extraordinary complexity, powerful transgenic technologies a...

  4. Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

    PubMed Central

    2012-01-01

    Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants. PMID:23006412

  5. Efficient Generation of Mice with Consistent Transgene Expression by FEEST.

    PubMed

    Gao, Lei; Jiang, Yonghua; Mu, Libing; Liu, Yanbin; Wang, Fengchao; Wang, Peng; Zhang, Aiqun; Tang, Nan; Chen, Ting; Luo, Minmin; Yu, Lei; Gao, Shaorong; Chen, Liang

    2015-01-01

    Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale. PMID:26573149

  6. Molecular Analyses of Transgenic Plants.

    PubMed

    Trijatmiko, Kurniawan Rudi; Arines, Felichi Mae; Oliva, Norman; Slamet-Loedin, Inez Hortense; Kohli, Ajay

    2016-01-01

    One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue. PMID:26614292

  7. The effect of Bt-transgene introgression on plant growth and reproduction in wild Brassica juncea.

    PubMed

    Liu, Yong-Bo; Darmency, Henry; Stewart, C Neal; Wei, Wei; Tang, Zhi-Xi; Ma, Ke-Ping

    2015-06-01

    This study aims to investigate the relative plant growth and reproduction of insect-resistant and susceptible plants following the introgression of an insect-resistance Bt-transgene from Brassica napus, oilseed rape, to wild Brassica juncea. The second backcrossed generation (BC2) from a single backcross family was grown in pure and mixed stands of Bt-transgenic and non-transgenic siblings under two insect treatments. Various proportions of Bt-transgenic plants were employed in mixed stands to study the interaction between resistant and susceptible plants. In the pure stands, Bt-transgenic BC2 plants performed better than non-transgenic plants with or without insect treatments. In mixed stands, Bt-transgenic BC2 plants produced fewer seeds than their non-Bt counterparts at low proportions of Bt-transgenic BC2 plants in the absence of insects. Reproductive allocation of non-transgenic plants marginally increased with increasing proportions of Bt-transgenic plants under herbivore pressure, which resulted in increased total biomass and seed production per stand. The results showed that the growth of non-transgenic plants was protected by Bt-transgenic plants under herbivore pressure. The Bt-transgene might not be advantageous in mixed stands of backcrossed hybrids; thus transgene introgression would not be facilitated when herbivorous insects are not present. However, a relatively large initial population of Bt-transgenic plants might result in transgene persistence when target herbivores are present. PMID:25487040

  8. An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

    PubMed

    Zhang, Bo; Zhang, Lin; Dai, Ruixue; Yu, Meiying; Zhao, Guoping; Ding, Xiaoming

    2013-01-01

    Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes. PMID:23409083

  9. Generation of Transgenic Mice

    PubMed Central

    Cho, Andrew; Haruyama, Naoto; Kulkarni, Ashok B.

    2009-01-01

    This unit describes detailed step-by-step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. PMID:19283729

  10. WEEDING IN TRANSGENES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenes promise to reduce insecticide and fungicide use, but relatively little has been done to significantly reduce herbicide use through genetic engineering. Three strategies for transgene utilization are discussed which have the potential to change this: 1) improvement of weed-specific biocon...

  11. Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple.

    PubMed

    Smolka, Anders; Li, Xue-Yuan; Heikelt, Catrin; Welander, Margareta; Zhu, Li-Hua

    2010-12-01

    Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants. PMID:20135223

  12. Transgenic phenolic production in corn silks moderately enhances insect resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some phenolic compounds produced in corn silks, such as maysin, can promote resistance to caterpillar pests. We evaluated transgenic maize engineered to express a maize cDNA controlled by a putative silk specific promoter for secondary metabolite production and corn earworm resistance. Transgene e...

  13. Principles and application of transgenic technology in marine organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marine organisms into which a foreign gene or noncoding DNA fragment is artificially introduced and stably integrated in their genomes are termed transgenic marine organisms. Since the first report in 1985, a wide range of transgenic fish and marine bivalve mollusks have been produced by microinjec...

  14. Marker-free phase nanoscopy

    NASA Astrophysics Data System (ADS)

    Cotte, Yann; Toy, Fatih; Jourdain, Pascal; Pavillon, Nicolas; Boss, Daniel; Magistretti, Pierre; Marquet, Pierre; Depeursinge, Christian

    2013-02-01

    We introduce a microscopic method that determines quantitative optical properties beyond the optical diffraction limit and allows direct imaging of unstained living biological specimens. In established holographic microscopy, complex fields are measured using interferometric detection, allowing diffraction-limited phase measurements. Here, we show that non-invasive optical nanoscopy can achieve a lateral resolution of 90 nm by using a quasi-2π-holographic detection scheme and complex deconvolution. We record holograms from different illumination directions on the sample plane and observe subwavelength tomographic variations of the specimen. Nanoscale apertures serve to calibrate the tomographic reconstruction and to characterize the imaging system by means of the coherent transfer function. This gives rise to realistic inverse filtering and guarantees true complex field reconstruction. The observations are shown for nanoscopic porous cell frustule (diatoms), for the direct study of bacteria (Escherichia coli), and for a time-lapse approach to explore the dynamics of living dendritic spines (neurones).

  15. Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

    SciTech Connect

    Chakravarthy, Balu; Gaudet, Chantal; Menard, Michel; Brown, Leslie; Atkinson, Trevor; LaFerla, Frank M.; Ito, Shingo; Armato, Ubaldo; Dal Pra, Ilaria; Whitfield, James

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.

  16. Transgenic plants protected from insect attack

    NASA Astrophysics Data System (ADS)

    Vaeck, Mark; Reynaerts, Arlette; Höfte, Herman; Jansens, Stefan; de Beuckeleer, Marc; Dean, Caroline; Zabeau, Marc; Montagu, Marc Van; Leemans, Jan

    1987-07-01

    The Gram-positive bacterium Bacillus thuringiensis produces proteins which are specifically toxic to a variety of insect species. Modified genes have been derived from bt2, a toxin gene cloned from one Bacillus strain. Transgenic tobacco plants expressing these genes synthesize insecticidal proteins which protect them from feeding damage by larvae of the tobacco hornworm.

  17. A transgenic perspective on plant functional genomics.

    PubMed

    Pereira, A

    2000-01-01

    Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops. PMID:11131004

  18. A transgenic, visual screenable marker for soybean seeds.

    PubMed

    Kanizay, Lisa; Jacobs, Thomas; Hancock, C Nathan

    2016-04-01

    Most soybean cultivars produce buff colored seeds due to a seed coat specific siRNA mechanism. This phenomenon is specifically limited to the seed coat and produces a strong visual effect, thus, a strategy to evade the silencing was used to produce a maternal transgenic marker for soybeans. Expression of a rice chalcone synthase transgene with little DNA sequence homology to the soybean siRNAs resulted in dark colored seed coats. This phenotype is the result of anthocyanin pigment production and does not appear to affect other tissues. This novel approach for producing an easily scored transgenic marker for soybean will facilitate high-throughput screening and analysis of transgenic soybean. PMID:26660729

  19. Screening for transgenic Japanese quail offspring.

    PubMed

    Poynter, Greg; Huss, David; Lansford, Rusty

    2009-01-01

    After mosaic founder breeding pairs of Japanese quail start to produce fertile eggs, the hatchlings must be screened for germ-line transmission to the subsequent G1 generation. This article describes how to isolate hatchling genomic DNA from the chorioallantoic membrane (CAM), which remains inside the egg after hatching. Collecting genomic DNA from the CAM decreases the hatchling's stress during handling and eliminates the need for a blood draw. By following this protocol, the CAM of a single egg will provide 50 microg or more of high-quality genomic DNA. The article also describes how to screen the genomic DNA samples for the transgene by the polymerase chain reaction (PCR). PCR genotyping should be used for screening hatchlings with a nonfluorescent transgene or with a fluorescently labeled transgene that does not lend itself well to phenotypic screening. PMID:20147014

  20. Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

    PubMed Central

    Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

    2012-01-01

    The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

  1. BAC TransgeneOmics

    PubMed Central

    Poser, Ina; Sarov, Mihail; Hutchins, James R A; Hériché, Jean-Karim; Toyoda, Yusuke; Pozniakovsky, Andrei; Weigl, Daniela; Nitzsche, Anja; Hegemann, Björn; Bird, Alexander W; Pelletier, Laurence; Kittler, Ralf; Hua, Sujun; Naumann, Ronald; Augsburg, Martina; Sykora, Martina M; Hofemeister, Helmut; Zhang, Youming; Nasmyth, Kim; White, Kevin P; Dietzel, Steffen; Mechtler, Karl; Durbin, Richard; Stewart, A Francis; Peters, Jan-Michael; Buchholz, Frank; Hyman, Anthony A

    2009-01-01

    The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems. PMID:18391959

  2. Effect of transgene number of spontaneous and radiation-induced micronuclei in lacl transgenic mice

    SciTech Connect

    O`Loughlin, K.G.; Hamer, J.D.; Winegar, R.A.; Mirsalis, J.C.; Short, J.M.

    1994-12-31

    Lacl transgenic mice are widely used for the measurement of mutations in specific target issues. The lacl transgene is present in mice as 40 tandem repeats; this sequence is homozygous (contained in both copies of chromosome 5) in C57Bl/6 mice, and is hemizygous in B6C3F1 mice. Previous reports have indicated that tandem repeats can produce chromosome instability, fragile sites, and other effects. To determine whether the presence of the transgene effects micronucleus induction we compared the response of nontransgenic (NTR) to hemizygous (HEMI) transgenic B6C3F1 mice and to hemizygous and homozygous (HOMO) transgenic C57Bl/6 mice. Five mice/group were irradiated with 500 cGy from a {sup 137}Cs source. Bone marrow was harvested 24 hr after treatment and 2000 polychromatic erythrocytes (PCE) were analyzed per animal. The presence or absence of the lacl transgene had no effect in unirradiated mice on the percent of micronucleated PCE (MN) or on the ratio of PCE to total red blood cells for either strain: B6C3F1 mice had MN frequencies of 0.26% and 0.20% for NTR and HEMI mice, respectively; C57Bl/6 mice had MN frequencies of 0.34%, 0.32%, and 0.38% for NTR, HEMI, and HOMO mice, respectively. Radiation-induced micronucleus frequencies were significantly higher in HEMI lacl B6C3F1 mice (2.85%) than in NTR litter mates (1.59%); the converse was true in C57Bl/6 mice: NTR were 2.45%, HEMI were 1.25%, HOMO were 1.65%. These data suggest that the lacl transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

  3. Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them

    SciTech Connect

    Bohmert-Tatarev, Karen; McAvoy, Susan; Peoples, Oliver P.; Snell, Kristi D.

    2015-08-04

    Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.

  4. [Oncogenesis in transgenic mice].

    PubMed

    Shvemberger, I N; Ermilov, A N

    1994-01-01

    Oncogenesis in transgenic mice is at present a model, most adequately reflecting the natural conditions of tumor development. One of more important traits of this model is that it allows to study malignant growth simultaneously at all the structure-function levels in the context of the whole organism. This paper is a review of results of a series of experiments in which the localization of tumors was dependent or independent on the tissue specificity of a promoter, as well as development of multiple tumors with the use of viral regulatory sequences in genetic constructions. It has been shown that although a transgene is expressed in most of the tissues, tumors develop in some particular tissues only. These observations are interpreted by some authors in favour of the concept of multistep cancerogenesis. In this view, of primary importance are the results of studies on oncogenesis in transgenic mice, which contradict this concept and are regarded by their authors as an evidence of the possibility of a one-step transformation of normal cell into malignant one. The analysis of the obtained material enabled us to put forward an assumption that the key role in oncogenesis is played not only by certain genetic disturbances, but also by multi-level homeostatic mechanisms. Apparently, it is just the transgenic mice with cellular or viral oncogenes in their genome that represent a more adequate model for the detection of certain molecular-biological mechanisms underlying these disturbances. Also, of much importance is abundant material accumulated by now on oncogenesis of transgenic mice which shows a possibility of the effective use of various genetic constructions with prokaryotic and eukaryotic regulatory sequences, a possibility to induce not only tumors of some particular tissues, but also multiple hyperplastic and neoplastic changes in one and the same mouse. Development of tumors in such transgenic mice can be regarded as a model of different types of cancer disease

  5. Transgenic Crops for Herbicide Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their introduction in 1995, crops made resistant to the broad-spectrum herbicides glyphosate and glufosinate with transgenes are widely available and used in much of the world. As of 2008, over 80% of the transgenic crops grown world-wide have this transgenic trait. This technology has had m...

  6. Transgenic Farm Animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of recombinant DNA technology has enabled scientists to isolate single genes, analyze and modify their nucleotide structure(s), make copies of these isolated genes, and insert copies of these genes into the genome of plants and animals. The transgenic technology of adding genes to li...

  7. Illegal gene flow from transgenic creeping bentgrass: the saga continues.

    PubMed

    Snow, Allison A

    2012-10-01

    Ecologists have paid close attention to environmental effects that fitness-enhancing transgenes might have following crop-to-wild gene flow (e.g. Snow et al. 2003). For some crops, gene flow also can lead to legal problems,especially when government agencies have not approved transgenic events for unrestricted environmental release.Creeping bentgrass (Agrostis stolonifera), a common turf grass used in golf courses, is the focus of both areas of concern. In 2002, prior to expected deregulation (still pending), The Scotts Company planted creeping bentgrass with transgenic resistance to the herbicide glyphosate,also known as RoundUp, on 162 ha in a designated control area in central Oregon (Fig. 1).Despite efforts to restrict gene flow, wind-dispersed pollen carried transgenes to florets of local A. stolonifera and A. gigantea as far as 14 km away, and to sentinel plants placed as far as 21 km away (Watrud et al. 2004).Then, in August 2003, a strong wind event moved transgenic seeds from wind rows of cut bentgrass into nearby areas. The company’s efforts to kill all transgenic survivors in the area failed: feral glyphosate-resistant populations of A. stolonifera were found by Reichman et al.(2006), and 62% of 585 bentgrass plants had the telltale CP4 EPSPS transgene in 2006 (Zapiola et al. 2008; Fig. 2).Now, in this issue, the story gets even more interesting as Zapiola & Mallory-Smith (2012) describe a transgenic,intergeneric hybrid produced on a feral, transgenic creeping bentgrass plant that received pollen from Polypogon monspeliensis (rabbitfoot grass). Their finding raises a host of new questions about the prevalence and fitness of intergeneric hybrids, as well as how to evaluate the full extent of gene flow from transgenic crops. PMID:23009646

  8. Transgenic plants with increased calcium stores

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Robertson, Dominique (Inventor); Boss, Wendy (Inventor)

    2004-01-01

    The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

  9. Transgenic Mouse Model of Chronic Beryllium Disease

    SciTech Connect

    Gordon, Terry

    2009-05-26

    Animal models provide powerful tools for dissecting dose-response relationships and pathogenic mechanisms and for testing new treatment paradigms. Mechanistic research on beryllium exposure-disease relationships is severely limited by a general inability to develop a sufficient chronic beryllium disease animal model. Discovery of the Human Leukocyte Antigen (HLA) - DPB1Glu69 genetic susceptibility component of chronic beryllium disease permitted the addition of this human beryllium antigen presentation molecule to an animal genome which may permit development of a better animal model for chronic beryllium disease. Using FVB/N inbred mice, Drs. Rubin and Zhu, successfully produced three strains of HLA-DPB1 Glu 69 transgenic mice. Each mouse strain contains a haplotype of the HLA-DPB1 Glu 69 gene that confers a different magnitude of odds ratio (OR) of risk for chronic beryllium disease: HLA-DPB1*0401 (OR = 0.2), HLA-DPB1*0201 (OR = 15), HLA-DPB1*1701 (OR = 240). In addition, Drs. Rubin and Zhu developed transgenic mice with the human CD4 gene to permit better transmission of signals between T cells and antigen presenting cells. This project has maintained the colonies of these transgenic mice and tested the functionality of the human transgenes.

  10. A dominant-negative mutation within AtMYB90 blocks flower pigment production in transgenic tobacco.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During de novo shoot induction in cultured transgenic tobacco callus a spontaneous mutation within the coding region of a AtMYB90 transgene produced a plant line in which the original transgene-induced over-pigmented phenotype (dark red/purple from anthocyanin overproduction in most tissues) was los...

  11. Transgenics in crops

    NASA Technical Reports Server (NTRS)

    Li, Y.; Wu, Y. H.; McAvoy, R.; Duan, H.

    2001-01-01

    With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.

  12. Hybridization of downregulated-COMT transgenic switchgrass lines with field selected switchgrass for improved biomass traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. Downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In the present study we sought to further...

  13. Design and Management of Field Trials of Transgenic Cereals

    NASA Astrophysics Data System (ADS)

    Bedő, Zoltán; Rakszegi, Mariann; Láng, László

    The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment.

  14. Efficient production of transgenic cassava using negative and positive selection.

    PubMed

    Zhang, P; Potrykus, I; Puonti-Kaerlas, J

    2000-12-01

    In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system. PMID:11206969

  15. Transgenic algae engineered for higher performance

    DOEpatents

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  16. Cryopreservation of Xenopus transgenic lines.

    PubMed

    Buchholz, Daniel R; Fu, Liezhen; Shi, Yun-Bo

    2004-01-01

    Xenopus laevis has been widely used for molecular, cellular, and developmental studies. With the development of the sperm-mediated transgenic method, it is now possible to study gene function during vertebrate development by using this popular model. On the other hand, like other animal species, it is labor intensive, and the maintenance of transgenic lines is expensive. In this article, we investigated the possibility of using sperm-cryopreservation as a means to preserve transgenic frog lines. We demonstrated that cryopreserved sperms are viable but not fertile under our in vitro fertilization (IVF) conditions. However, by microinjecting cryopreserved sperm nuclei, we successfully regenerated a transgenic line carrying a double promoter transgene construct, where the marker gene encoding the green fluorescent protein (GFP) is driven by the gamma-crystallin gene promoter and a gene of interest, encoding a fusion protein of GFP with the matrix metalloproteinase stromelysin-3 (ST3-GFP), is driven by a heat shock-inducible promoter. We demonstrated the functional transmission of the ST3-GFP transgene by analyzing the phenotype of the F1 animals after heat-shock to induce its expression. Our method thus provides an inexpensive means to preserve transgenic frog lines and a convenient way for distribution of transgenic lines. Furthermore, the ease with which to microinject nuclei compared to the technically demanding transgenesis procedure with variable outcome should facilitate more laboratories to use transgenic Xenopus laevis for functional studies in vivo. Mol. Reprod. Dev. 67: 65-69, 2004. PMID:14648875

  17. Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

    PubMed

    Chong-Pérez, Borys; Kosky, Rafael G; Reyes, Maritza; Rojas, Luis; Ocaña, Bárbara; Tejeda, Marisol; Pérez, Blanca; Angenon, Geert

    2012-06-30

    Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species. PMID:21839123

  18. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    SciTech Connect

    Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  19. Transgenic fish systems and their application in ecotoxicology.

    PubMed

    Lee, Okhyun; Green, Jon M; Tyler, Charles R

    2015-02-01

    The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4-UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities. PMID:25394772

  20. Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

    DOEpatents

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

    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.

  1. Designer proton-channel transgenic algae for photobiological hydrogen production

    DOEpatents

    Lee, James Weifu

    2011-04-26

    A designer proton-channel transgenic alga for photobiological hydrogen production that is specifically designed for production of molecular hydrogen (H.sub.2) through photosynthetic water splitting. The designer transgenic alga includes proton-conductive channels that are expressed to produce such uncoupler proteins in an amount sufficient to increase the algal H.sub.2 productivity. In one embodiment the designer proton-channel transgene is a nucleic acid construct (300) including a PCR forward primer (302), an externally inducible promoter (304), a transit targeting sequence (306), a designer proton-channel encoding sequence (308), a transcription and translation terminator (310), and a PCR reverse primer (312). In various embodiments, the designer proton-channel transgenic algae are used with a gas-separation system (500) and a gas-products-separation and utilization system (600) for photobiological H.sub.2 production.

  2. Transgenic farm animals: present and future.

    PubMed

    Niemann, H; Kues, W; Carnwath, J W

    2005-04-01

    Until recently, pronuclear microinjection of deoxyribonucleic acid (DNA) was the standard method for producing transgenic animals. This technique is now being replaced by more efficient protocols based on somatic nucleartransferthat also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid technology are also becoming important tools for transgenesis. Transgenic farm animals are important in human medicine as sources of biologically active proteins, as donors in xenotransplantation, and for research in cell and gene therapy. Typical agricultural applications include improved carcass composition, lactational performance and wool production, as well as enhanced disease resistance and reduced environmental impact. Product safety can be ensured by standardisation of procedures and monitored by polymerase chain reaction and array technology. As sequence information and genomic maps of farm animals are refined, it becomes increasingly practical to remove or modify individual genes. This approach to animal breeding will be instrumental in meeting global challenges in agricultural production in the future. PMID:16110896

  3. Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-α(1-3) Galactosyl Epitope

    PubMed Central

    Magre, Saema; Takeuchi, Yasuhiro; Langford, Gillian; Richards, Andrew; Patience, Clive; Weiss, Robin

    2004-01-01

    Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-α(1-3) galactosyl sugar structure (α-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing α-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out α-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which α-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed α-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation. PMID:15140979

  4. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  5. Transgenic horticultural crops in Asia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modern biotechnology applications, including genetic engineering, are a powerful tool to complement the conventional methods of crop improvement. Asia currently has three countries cultivating biotech/transgenic crops – China, India, and the Philippines, but only China commercially grows a transgen...

  6. Hybridization and backcrossing between transgenic oilseed rape and two related weed species under field conditions.

    PubMed

    Halfhill, Matthew D; Zhu, Bin; Warwick, Suzanne I; Raymer, Paul L; Millwood, Reginald J; Weissinger, Arthur K; Stewart, C Neal

    2004-01-01

    Determining the frequency of crop-wild transgene flow under field conditions is a necessity for the development of regulatory strategies to manage transgenic hybrids. Gene flow of green fluorescent protein (GFP) and Bacillus thuringiensis (Bt) transgenes was quantified in three field experiments using eleven independent transformed Brassica napus L. lines and the wild relatives, B. rapa L. and Raphanus raphanistrum L. Under a high crop to wild relative ratio (600:1), hybridization frequency with B. rapa differed among the individual transformed B. napus lines (ranging from ca. 4% to 22%), however, this difference could be caused by the insertion events or other factors, e.g., differences in the hybridization frequencies among the B. rapa plants. The average hybridization frequency over all transformed lines was close to 10%. No hybridization with R. raphanistrum was detected. Under a lower crop to wild relative ratio (180:1), hybridization frequency with B. rapa was consistent among the transformed B. napus lines at ca. 2%. Interspecific hybridization was higher when B. rapa occurred within the B. napus plot (ca. 37.2%) compared with plot margins (ca. 5.2%). No significant differences were detected among marginal plants grown at 1, 2, and 3 m from the field plot. Transgene backcrossing frequency between B. rapa and transgenic hybrids was determined in two field experiments in which the wild relative to transgenic hybrid ratio was 5-15 plants of B. rapa to 1 transgenic hybrid. As expected, ca. 50% of the seeds produced were transgenic backcrosses when the transgenic hybrid plants served as the maternal parent. When B. rapa plants served as the maternal parent, transgene backcrossing frequencies were 0.088% and 0.060%. Results show that transgene flow from many independent transformed lines of B. napus to B. rapa can occur under a range of field conditions, and that transgenic hybrids have a high potential to produce transgenic seeds in backcrosses. PMID:15612504

  7. Tongue Epithelium Cells from shRNA Mediated Transgenic Goat Show High Resistance to Foot and Mouth Disease Virus

    PubMed Central

    Li, Wenting; Wang, Kejun; Kang, Shimeng; Deng, Shoulong; Han, Hongbing; Lian, Ling; Lian, Zhengxing

    2015-01-01

    Foot and mouth disease induced by foot and mouth disease virus (FMDV) is severe threat to cloven-hoofed domestic animals. The gene 3Dpol in FMDV genome encodes the viral RNA polymerase, a vital element for FMDV replication. In this study, a conserved 3D-7414shRNA targeting FMDV-3Dpol gene was designed and injected into pronuclear embryos to produce the transgenic goats. Sixty-one goats were produced, of which, seven goats positively integrated 3D-7414shRNA. Loss of function assay demonstrated that siRNA effectively knockdown 3Dpol gene in skin epithelium cells of transgenic goats. Subsequently, the tongue epithelium cells from transgenic and non-transgenic goats were infected with FMDV O/YS/CHA/05 strain. A significant decrease of virus titres and virus copy number was observed in cells of transgenic goats compared with that of non-transgenic goats, which indicated that 3D-7414siRNA inhibited FMDV replication by interfering FMDV-3Dpol gene. Furthermore, we found that expression of TLR7, RIG-I and TRAF6 was lower in FMDV infected cells from transgenic goats compared to that from non-transgenic goats, which might result from lower virus copy number in transgenic goats’ cells. In conclusion, we successfully produced transgenic goats highly expressing 3D-7414siRNA targeting 3Dpol gene, and the tongue epithelium cells from the transgenic goats showed effective resistance to FMDV. PMID:26671568

  8. [Transgenics without Manichaeism].

    PubMed

    Valle, S

    2000-01-01

    We live in an era characterized by the hegemony of science and technology, an era fraught with questions awaiting answers which would enable a safe and sustainable future for humankind. The development of agro-industrial processes - food products in particular - through recombinant DNA technology has enhanced the profit prospects of the few big biotechnology companies and of large-scale farmers who have access to the latest technological developments. We thus oppose a moratorium on recombinant DNA technology. Moreover, hasty statements about risk-free transgenics may be misleading in the absence of extensive safety tests. There is a pressing need for the establishment of biosafety policy in this country involving the organized civil society and every government agency responsible for monitoring such matters. There is also the need to put in place a bio-surveillance and a code of ethics regarding genetic manipulation. PMID:16680900

  9. Transgenic oil palm: production and projection.

    PubMed

    Parveez, G K; Masri, M M; Zainal, A; Majid, N A; Yunus, A M; Fadilah, H H; Rasid, O; Cheah, S C

    2000-12-01

    Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020. PMID:11171275

  10. Approaches for improving present laboratory and field methodology for evaluation efficacy of transgenic technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Assessing the efficacy of transgenic plants under new environmental and management regimes is of prime importance to the companies which produce new or improved existing transgenic products, breeders which create different varieties stacked with Bt endotoxins, and growers who use them for production...

  11. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine

    1997-01-01

    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  12. Quantitative Genetics of Transgenic Mice: Components of Phenotypic Variation in Body Weights and Weight Gains

    PubMed Central

    Clutter, A. C.; Pomp, D.; Murray, J. D.

    1996-01-01

    Transgenic mice possessing an ovine growth hormone gene were used to study the effects of elevated growth hormone on quantitative genetic variation. Males hemizygous for the transgene were mated to wild-type females to produce half- and full-sib families in which approximately half the progeny were transgenic and half were wild type. Analyses of body weights at 3-10 weeks, and weight gains from 3 to 6, and 6 to 10 weeks produced estimates of the proportion of total variance due to additive genetic effects (h(2)) and common litter effects (c(2)), and the genetic correlation between transgenic and wild-type expression of each trait. At 10 weeks, body weight of transgenics exceeded that of wild types by 26 and 49% in males and females, respectively. Estimated genetic variances in the transgenic group were significantly greater than zero for body weights at most ages and for both measurements of gain. Common litter effects accounted for a similar proportion of variation in the wild-type and transgenic groups. Additive genetic correlations between wild-type and transgenic expression of body weights tended to decline with age, indicating that a partially different array of genes may have begun to affect body weight in the transgenic group. PMID:8844161

  13. Production of pharmaceutical proteins by transgenic animals.

    PubMed

    Houdebine, Louis-Marie

    2009-03-01

    Proteins started being used as pharmaceuticals in the 1920s with insulin extracted from pig pancreas. In the early 1980s, human insulin was prepared in recombinant bacteria and it is now used by all patients suffering from diabetes. Several other proteins and particularly human growth hormone are also prepared from bacteria. This success was limited by the fact that bacteria cannot synthesize complex proteins such as monoclonal antibodies or coagulation blood factors which must be matured by post-translational modifications to be active or stable in vivo. These modifications include mainly folding, cleavage, subunit association, gamma-carboxylation and glycosylation. They can be fully achieved only in mammalian cells which can be cultured in fermentors at an industrial scale or used in living animals. Several transgenic animal species can produce recombinant proteins but presently two systems started being implemented. The first is milk from farm transgenic mammals which has been studied for 20 years and which allowed a protein, human antithrombin III, to receive the agreement from EMEA (European Agency for the Evaluation of Medicinal Products) to be put on the market in 2006. The second system is chicken egg white which recently became more attractive after essential improvement of the methods used to generate transgenic birds. Two monoclonal antibodies and human interferon-beta 1a could be recovered from chicken egg white. A broad variety of recombinant proteins were produced experimentally by these systems and a few others. This includes monoclonal antibodies, vaccines, blood factors, hormones, growth factors, cytokines, enzymes, milk proteins, collagen, fibrinogen and others. Although these tools have not yet been optimized and are still being improved, a new era in the production of recombinant pharmaceutical proteins was initiated in 1987 and became a reality in 2006. In the present review, the efficiency of the different animal systems to produce

  14. An efficient method for organic acetylation and use of DL-phosphinothricin as a negative selection agent in argE transgenic rice.

    PubMed

    Byeon, Ji-Hui; Cho, Joon-Hyeong

    2013-11-01

    We present an efficient method for the production of N-acetyl-L-phosphinothricin (N-AcPt) from commercial DL-phosphinothricin (DL-PPT) by organic acetylation for use as a negative selection agent (NSA) that induces cell death in argE transgenic rice. DL-PPT was efficiently converted into N-AcPt with tetrahydrofuran (THF) and acetic anhydride (Ac2O). Chemical changes were confirmed using NMR and ATR-FTIR analyses. DL-PPT was toxic but N-AcPt did not show cytotoxic effects on leaf discs or seed germination of wild-type rice. Conversely, in argE-hpt transgenic rice, non-toxic N-AcPt showed the negative selection (NS) effect by inducing cell destruction in leaf discs and restricting seed germination. For inducing NS, ≥0.1 mg ml(-1) and ≥0.5 mg ml(-1) of N-AcPt were effective in leaf and seed assays, respectively. Further, the NS effect occurred faster in the leaf assay compared with the seed germination assay, again indicating the leaf assay was a more sensitive indicator of N-AcPt as an NSA to argE transgenic rice than the seed germination assay. This negative selection approach could be useful for the development of selectable marker free transgenic plants in the economically important monocot species and its commercialization for multiple gene transformation. PMID:24141117

  15. Post-mortem re-cloning of a transgenic red fluorescent protein dog

    PubMed Central

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

    2011-01-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

  16. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    PubMed

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption. PMID:27411901

  17. Transgenic hepatocarcinogenesis in the rat.

    PubMed Central

    Hully, J. R.; Su, Y.; Lohse, J. K.; Griep, A. E.; Sattler, C. A.; Haas, M. J.; Dragan, Y.; Peterson, J.; Neveu, M.; Pitot, H. C.

    1994-01-01

    Although transgenic hepatocarcinogenesis has been accomplished in the mouse with a number of genetic constructs targeting the oncogene to expression primarily in the liver, no example of this process has yet been developed in the rat. Because our understanding of the multistage nature of hepatocarcinogenesis is most advanced in the rat, we have developed a strain of transgenic rats carrying the promoter-enhancer sequences of the mouse albumin gene linked 5' to the simian virus-40 T antigen gene. A line of transgenic rats bearing this transgene has been developed from a single founder female. Five to six copies of the transgene, possibly in tandem, occur within the genome of the transgenic animals, which are maintained by heterozygous matings. Livers of transgenic animals are histologically normal after weaning; at 2 months of age, small foci of vacuolated cells appear in this organ. By 4 months of age, all animals exhibit focal lesions and nodules consisting primarily of small basophilic cells, many of which exhibit considerable cytoplasmic vacuolization. Mating of animals each bearing the transgene results in rats with a demyelinating condition that develops acutely in pregnant females and more chronically in males. Ultrastructural studies of these cells indicate that the vacuoles contain substantial amounts of glycogen, with the cells resembling hepatoblasts. Malignant neoplasms with both a glandular and a hepatoblastoma/hepatocellular carcinoma pattern arise from the nodules. Enzyme and immunohistochemical studies of all lesions reveal many similarities in gene expression to comparable lesions in rats subjected to chemically induced hepatocarcinogenesis, with certain exceptions. The placental form of glutathione-S-transferase is absent from all lesions in the transgenic animal, as is the expression of connexin 32. A significant number of lesions express serum albumin, and many, but not all, exhibit the T antigen. Lesions expressing the T antigen also contain

  18. RESOLUTION OF COMPLEX INTEGRATION PATTERNS TO OBTAIN SINGLE COPY TRANSGENES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present invention provides methods for producing a transgenic cell having a stably integrated, single copy of an exogenous polynucleotide sequence. The method, which resolves repeated insertions of the introduced polynucleotide sequence into a single copy, involves introducing into a genetic loc...

  19. Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids

    PubMed Central

    Dou, Hongwei; Chen, Lei; Chen, Longxin; Lin, Lin; Tan, Pingping; Vajta, Gabor; Gao, Jianfeng; Du, Yutao; Ma, Runlin Z.

    2013-01-01

    Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n−3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n−6) into n−3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n  = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n−3 fatty acid desaturase, accompanied by more than 2-folds reduction of n−6/n−3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation. PMID:23437077

  20. Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

    PubMed

    Halfhill, M D; Millwood, R J; Weissinger, A K; Warwick, S I; Stewart, C N

    2003-11-01

    The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid

  1. [Biofuels, food security and transgenic crops].

    PubMed

    Acosta, Orlando; Chaparro-Giraldo, Alejandro

    2009-01-01

    Soaring global food prices are threatening to push more poor people back below the poverty line; this will probably become aggravated by the serious challenge that increasing population and climate changes are posing for food security. There is growing evidence that human activities involving fossil fuel consumption and land use are contributing to greenhouse gas emissions and consequently changing the climate worldwide. The finite nature of fossil fuel reserves is causing concern about energy security and there is a growing interest in the use of renewable energy sources such as biofuels. There is growing concern regarding the fact that biofuels are currently produced from food crops, thereby leading to an undesirable competition for their use as food and feed. Nevertheless, biofuels can be produced from other feedstocks such as lingo-cellulose from perennial grasses, forestry and vegetable waste. Biofuel energy content should not be exceeded by that of the fossil fuel invested in its production to ensure that it is energetically sustainable; however, biofuels must also be economically competitive and environmentally acceptable. Climate change and biofuels are challenging FAO efforts aimed at eradicating hunger worldwide by the next decade. Given that current crops used in biofuel production have not been domesticated for this purpose, transgenic technology can offer an enormous contribution towards improving biofuel crops' environmental and economic performance. The present paper critically presents some relevant relationships between biofuels, food security and transgenic plant technology. PMID:19722000

  2. THE POTENTIAL ROLE OF REMOTE SENSING IN TRANSGENIC CROP MONITORING PROGRAMS

    EPA Science Inventory

    Sustainable agriculture combines efficient production with wise stewardship of the earth's resources. Development of environmentally benign production techniques is one focus of sustainable agriculture. The new transgenic crops producing toxic proteins that target specific crop p...

  3. Development and application of transgenic technologies in cassava.

    PubMed

    Taylor, Nigel; Chavarriaga, Paul; Raemakers, Krit; Siritunga, Dimuth; Zhang, Peng

    2004-11-01

    The capacity to integrate transgenes into the tropical root crop cassava (Manihot esculenta Crantz) is now established and being utilized to generate plants expressing traits of agronomic interest. The tissue culture and gene transfer systems currently employed to produce these transgenic cassava have improved significantly over the past 5 years and are assessed and compared in this review. Programs are underway to develop cassava with enhanced resistance to viral diseases and insects pests, improved nutritional content, modified and increased starch metabolism and reduced cyanogenic content of processed roots. Each of these is described individually for the underlying biology the molecular strategies being employed and progress achieved towards the desired product. Important advances have occurred, with transgenic plants from several laboratories being prepared for field trails. PMID:15630627

  4. Identification and quantification of anthocyanins in transgenic purple tomato.

    PubMed

    Su, Xiaoyu; Xu, Jianteng; Rhodes, Davina; Shen, Yanting; Song, Weixing; Katz, Benjamin; Tomich, John; Wang, Weiqun

    2016-07-01

    Anthocyanins are natural pigments derived from the phenylpropanoid pathway. Most tomatoes produce little anthocyanins, but the transgenic purple tomato biosynthesizes a high level of anthocyanins due to expression of two transcription factors (Del and Ros1). This study was to identify and quantify anthocyanins in this transgenic tomato line. Seven anthocyanins, including two new anthocyanins [malvidin-3-(p-coumaroyl)-rutinoside-5-glucoside and malvidin-3-(feruloyl)-rutinoside-5-glucoside], were identified by LC-MS/MS. Petunidin-3-(trans-coumaroyl)-rutinoside-5-glucoside and delphinidin-3-(trans-coumaroyl)-rutinoside-5-glucoside were the most abundant anthocyanins, making up 86% of the total anthocyanins. Compared to undetectable anthocyanins in the wild type, the contents of anthocyanins in the whole fruit, peel, and flesh of the Del/Ros1-transgenic tomato were 5.2±0.5, 5.1±0.5, and 5.8±0.3g/kg dry matter, respectively. Anthocyanins were undetectable in the seeds of both wide-type and transgenic tomato lines. Such novel and high levels of anthocyanins obtained in this transgenic tomato may provide unique functional products with potential health benefits. PMID:26920283

  5. Overexpression of mouse follistatin causes reproductive defects in transgenic mice.

    PubMed

    Guo, Q; Kumar, T R; Woodruff, T; Hadsell, L A; DeMayo, F J; Matzuk, M M

    1998-01-01

    Follistatin is an activin-binding protein that can act as an activin antagonist in vitro. Follistatin also binds heparin sulfate proteoglycans and may function as a reservoir for activins in vivo. In the mouse, follistatin mRNA is first detected in the deciduum on embryonic day 5.5 and later in the developing hindbrain, somites, vibrissae, teeth, epidermis, and muscle. We have previously shown that follistatin-deficient mice have numerous embryonic defects including shiny, taut skin, growth retardation, and cleft palate leading to death within hours of birth. To further define the roles of follistatin during mammalian reproduction and development, we created gain-of-function mutant mice in which mouse follistatin is overexpressed. The mouse metallothionein (MT)-I promoter was placed upstream of the six-exon mouse follistatin (FS) gene. To distinguish wild-type and transgenic follistatin mRNA, the 3'-untranslated region of the mouse follistatin gene was replaced with the SV40 untranslated and polyA sequences. Three male and two female founder transgenic mice were produced, were fertile, and transmitted the transgene to offspring. Northern blot analysis demonstrated that the transgene mRNA was expressed at varying levels in the livers of offspring from four of five of the transgenic lines and was expressed in the testes in all five lines. In MT-FS line 4, which had the highest expression of the transgene mRNA in the liver, the transgene transcripts were also present in multiple other tissues. Phenotypically, the MT-FS transgenic lines had defects in the testis, ovary, and hair. Mice from MT-FS lines 7 and 10 had slightly decreased testis size, whereas mice from lines 4, 5, and 9 had much smaller testes and shiny, somewhat irregular, fur. Histological analysis of the adult testes from line 5 and 9 males showed variable degrees of Leydig cell hyperplasia, an arrest of spermatogenesis, and seminiferous tubular degeneration leading to infertility. Female transgenic mice

  6. Establishment of a novel, eco-friendly transgenic pig model using porcine pancreatic amylase promoter-driven fungal cellulase transgenes.

    PubMed

    Lin, Y S; Yang, C C; Hsu, C C; Hsu, J T; Wu, S C; Lin, C J; Cheng, W T K

    2015-02-01

    Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of byproducts and alternative ingredients in pig diets has the potential to reduce feed costs. Moreover, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down nonstarch polysaccharides into simple sugars. Here, we investigated the feasibility of a transgenic strategy in which expression of the fungal cellulase transgene was driven by the porcine pancreatic amylase promoter in pigs. A 2,488 bp 5'-flanking region of the porcine pancreatic amylase gene was cloned by the genomic walking technique, and its structural features were characterized. Using GFP as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Transgenic pigs were generated by pronuclear microinjection. Founders and offspring were identified by PCR and Southern blot analyses. Cellulase mRNA and protein showed tissue-specific expression in the pancreas of F1 generation pigs. Cellulolytic enzyme activity was also identified in the pancreas of transgenic pigs. These results demonstrated the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene. Transgenic pigs expressing exogenous cellulase may represent a way to increase the intake of low-cost, fiber-rich feeds. PMID:25063310

  7. [Transgenic animals and animal welfare

    PubMed

    Reinhardt, Christoph

    1998-01-01

    Under the pressure of a public vote in Switzerland (7 June 1998) on an initiative to ban the production, use and patenting of transgenic animals, their value for biomedical research and development is intensely debated. In addition, the Swiss legislation has adopted (1992) a constitutional obligation to "take into account the dignity of creatures". The term "dignity of creatures", however, can be interpreted in anthropocentric or biocentric ways. The government has now formulated the legal implications of this term for transgenic animals and plants in various laws including the animal and environmental protection laws. This paper gives arguments for a fair evaluation of trangenic animals from an animal welfare point of view where not only the costs of animal suffering must be considered but also the probability of potential benefit for man. A self-confident research community should allow such an evaluation procedure even in view of an outcome which could ban many uses of transgenic animals PMID:11208266

  8. Osteogenic capacity of transgenic flax scaffolds.

    PubMed

    Gredes, Tomasz; Wróbel-Kwiatkowska, Magdalena; Dominiak, Marzena; Gedrange, Tomasz; Kunert-Keil, Christiane

    2012-02-01

    The modification of flax fibers to create biologically active dressings is of undoubted scientific and practical interest. Flax fibers, derived from transgenic flax expressing three bacterial genes for the synthesis of poly-3-hydroxybutyric acid (PHB), have better mechanical properties than unmodified flax fibers; do not show any inflammation response after subcutaneous insertion; and have a good in vitro and in vivo biocompatibility. The aim of this study was to examine the applicability of composites containing flax fibers of genetically modified (M50) or non-modified (wt-Nike) flax within a polylactide (PLA) matrix for bone regeneration. For this, the mRNA expression of genes coding for growth factors (insulin-like growth factor IGF1, IGF2, vascular endothelial growth factor), for osteogenic differentiation (alkaline phosphatase, osteocalcin, Runx2, Phex, type 1 and type 2 collagen), and for bone resorption markers [matrix metalloproteinase 8 (MMP8), acid phosphatase type 5] were analyzed using quantitative real-time polymerase chain reaction. We found a significant elevated mRNA expression of IGF1 with PLA and PLA-wt-Nike composites. The mRNA amount of MMP8 and osteocalcin was significantly decreased in all biocomposite-treated cranial tissue samples compared to controls, whereas the expression of all other tested transcripts did not show any differences. It is assumed that both flax composites are able to stimulate bone regeneration, but composites from transgenic flax plants producing PHB showed faster bone regeneration than composites of non-transgenic flax plants. The application of these linen membranes for bone tissue engineering should be proved in further studies. PMID:22718592

  9. Consistent transcriptional silencing of 35S-driven transgenes in gentian.

    PubMed

    Mishiba, Kei-ichiro; Nishihara, Masahiro; Nakatsuka, Takashi; Abe, Yoshiko; Hirano, Hiroshi; Yokoi, Takahide; Kikuchi, Akiko; Yamamura, Saburo

    2005-11-01

    In this study, no transgenic gentian (Gentiana triflora x Gentiana scabra) plants produced via Agrobacterium-mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5' coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing. PMID:16262705

  10. Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety.

    PubMed

    Rukavtsova, Elena B; Rudenko, Natalya V; Puchko, Elena N; Zakharchenko, Natalya S; Buryanov, Yaroslav I

    2015-06-10

    Oral immunogenicity of the hepatitis B surface antigen (HBsAg) synthesized in the tubers of marker-free potato plants has been demonstrated. Experiments were performed in the two groups of outbred NMRI mice. At the beginning of investigations, the mice of experimental group were fed the tubers of transgenic potato synthesizing the HBsAg three times. The mice of control group were fed nontransgenic potato. Intraperitoneal injection of the commercial vaccine against hepatitis B (0.5μg/mouse) was made on day 71 of the experiment. Enzyme-linked immunoassay (ELISA) of the serum of immunized animals showed an increase in the level of HBsAg antibodies significantly above the protective value, which was maintained for 1 year after the immunization. In 1 year, the experimental group of mice underwent additional oral immunization with HBsAg-containing potato tubers. As a result, the level of antibodies against the HBsAg increased and remained at a high protective level for several months. The findings show the possibility of using transgenic plants as a substance for obtaining a safe edible vaccine against hepatitis B. PMID:25840367

  11. TRANSGENIC PLANT CONTAINMENT

    EPA Science Inventory

    The new technology using plant genetics to produce chemicals, pharmaceuticals, and therapeuitics in a wide array of new plant forms requires sufficient testing to ensure that these new plant introductions are benign in the environment. A recent effort to provide necessary guidan...

  12. Transgenic plants as factories for biopharmaceuticals.

    PubMed

    Giddings, G; Allison, G; Brooks, D; Carter, A

    2000-11-01

    Plants have considerable potential for the production of biopharmaceutical proteins and peptides because they are easily transformed and provide a cheap source of protein. Several biotechnology companies are now actively developing, field testing, and patenting plant expression systems, while clinical trials are proceeding on the first biopharmaceuticals derived from them. One transgenic plant-derived biopharmaceutical, hirudin, is now being commercially produced in Canada for the first time. Product purification is potentially an expensive process, and various methods are currently being developed to overcome this problem, including oleosin-fusion technology, which allows extraction with oil bodies. In some cases, delivery of a biopharmaceutical product by direct ingestion of the modified plant potentially removes the need for purification. Such biopharmaceuticals and edible vaccines can be stored and distributed as seeds, tubers, or fruits, making immunization programs in developing countries cheaper and potentially easier to administer. Some of the most expensive biopharmaceuticals of restricted availability, such as glucocerebrosidase, could become much cheaper and more plentiful through production in transgenic plants. PMID:11062432

  13. Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa.

    PubMed

    Chandrashekran, Anil; Sarkar, Rupa; Thrasher, Adrian; Fraser, Scott E; Dibb, Nicholas; Casimir, Colin; Winston, Robert; Readhead, Carol

    2014-02-01

    Transgenic technologies conventionally rely on the oocyte as a substrate for genetic modification. Owing to their accessibility, however, male germ cells, including mature sperm, have material advantages for use in transgenesis. Here we have exploited lentiviruses to generate transgenic animals via the male germline. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally transduced mouse spermatozoa were used in in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥ 42% of founders were found to be transgenic for GFP. Inverse PCR strategy for integration site analysis demonstrated integration of at least 1 or 2 copies of GFP in the transgenics, mapping to different chromosomes. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals. This relatively simple, yet highly efficient, technique for generating transgenic animals by transducing spermatozoa with lentiviral vectors in vitro is a powerful tool for the study of fertilization/preimplantation development, vertical viral gene transmission, gene function and regulation, and epigenetic inheritance. PMID:24297703

  14. Human health and transgenic crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Under the joint auspices of the Agrochemical and the Agricultural and Food Chemistry Divisions of the American Chemical Society, we organized a short symposium on “Human Health and Transgenic Crops” at the 244th ACS national meeting, held August 19-23, 2012 in Philadelphia, PA, to examine an array o...

  15. Improved antioxidant activity in transgenic Perilla frutescens plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene.

    PubMed

    Ghimire, Bimal Kumar; Seong, Eun Soo; Lee, Chan Ok; Lee, Jae Geun; Yu, Chang Yeon; Kim, Seung Hyun; Chung, Ill Min

    2015-09-01

    The main goal of this study was to generate transgenic Perilla frutescens with enhanced antioxidant properties by overexpressing the γ-tocopherol methyltransferase (γ-tmt) gene. In this study, the antioxidant activity of methanolic crude extracts of transgenic and non-transgenic control plants was investigated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. Free radical scavenging activity was evaluated using α-tocopherol and butylated hydroxyl toluene as standard antioxidants. In general, the ethyl acetate fraction of transgenic P. frutescens showed stronger DPPH radical scavenging activity than the ethyl acetate fraction from non-transgenic control plants (IC50 2.00 ± 0.10 and 5.53 ± 0.40 μg ∙ ml(-1), respectively). High-performance liquid chromatography analysis of phenolic acids in leaf extracts confirmed increased levels of 16 individual phenolic compounds in two transgenic lines (pf47-5 and pf47-8) compared with control plants. Changes in the phenolic compound profile and α-tocopherol content were correlated with the antioxidant properties of transgenic plants, indicating that the introduction of transgene γ-tmt influenced the metabolism of phenolic compounds and subsequently produced biochemical changes in the transformants. There were no significant differences in photosynthetic rate in the transgenic plants as compared to the non-transgenic control plants, suggesting that the alteration of phenolic compounds and tocopherol composition had little impact on photosynthesis. PMID:25604637

  16. Heat-inducible Cre-lox system for marker excision in transgenic rice.

    PubMed

    Khattri, Abhilasha; Nandy, Soumen; Srivastava, Vibha

    2011-03-01

    The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination 'footprint'. The presence of the 'footprint' was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice. PMID:21451246

  17. Development and validation of an automated and marker-free CT-based spatial analysis method (CTSA) for assessment of femoral hip implant migration In vitro accuracy and precision comparable to that of radiostereometric analysis (RSA)

    PubMed Central

    Scheerlinck, Thierry; Polfliet, Mathias; Deklerck, Rudi; Van Gompel, Gert; Buls, Nico; Vandemeulebroucke, Jef

    2016-01-01

    Background and purpose — We developed a marker-free automated CT-based spatial analysis (CTSA) method to detect stem-bone migration in consecutive CT datasets and assessed the accuracy and precision in vitro. Our aim was to demonstrate that in vitro accuracy and precision of CTSA is comparable to that of radiostereometric analysis (RSA). Material and methods — Stem and bone were segmented in 2 CT datasets and both were registered pairwise. The resulting rigid transformations were compared and transferred to an anatomically sound coordinate system, taking the stem as reference. This resulted in 3 translation parameters and 3 rotation parameters describing the relative amount of stem-bone displacement, and it allowed calculation of the point of maximal stem migration. Accuracy was evaluated in 39 comparisons by imposing known stem migration on a stem-bone model. Precision was estimated in 20 comparisons based on a zero-migration model, and in 5 patients without stem loosening. Results — Limits of the 95% tolerance intervals (TIs) for accuracy did not exceed 0.28 mm for translations and 0.20° for rotations (largest standard deviation of the signed error (SDSE): 0.081 mm and 0.057°). In vitro, limits of the 95% TI for precision in a clinically relevant setting (8 comparisons) were below 0.09 mm and 0.14° (largest SDSE: 0.012 mm and 0.020°). In patients, the precision was lower, but acceptable, and dependent on CT scan resolution. Interpretation — CTSA allows detection of stem-bone migration with an accuracy and precision comparable to that of RSA. It could be valuable for evaluation of subtle stem loosening in clinical practice. PMID:26634843

  18. Development and validation of an automated and marker-free CT-based spatial analysis method (CTSA) for assessment of femoral hip implant migration In vitro accuracy and precision comparable to that of radiostereometric analysis (RSA).

    PubMed

    Scheerlinck, Thierry; Polfliet, Mathias; Deklerck, Rudi; Van Gompel, Gert; Buls, Nico; Vandemeulebroucke, Jef

    2016-04-01

    Background and purpose - We developed a marker-free automated CT-based spatial analysis (CTSA) method to detect stem-bone migration in consecutive CT datasets and assessed the accuracy and precision in vitro. Our aim was to demonstrate that in vitro accuracy and precision of CTSA is comparable to that of radiostereometric analysis (RSA). Material and methods - Stem and bone were segmented in 2 CT datasets and both were registered pairwise. The resulting rigid transformations were compared and transferred to an anatomically sound coordinate system, taking the stem as reference. This resulted in 3 translation parameters and 3 rotation parameters describing the relative amount of stem-bone displacement, and it allowed calculation of the point of maximal stem migration. Accuracy was evaluated in 39 comparisons by imposing known stem migration on a stem-bone model. Precision was estimated in 20 comparisons based on a zero-migration model, and in 5 patients without stem loosening. Results - Limits of the 95% tolerance intervals (TIs) for accuracy did not exceed 0.28 mm for translations and 0.20° for rotations (largest standard deviation of the signed error (SDSE): 0.081 mm and 0.057°). In vitro, limits of the 95% TI for precision in a clinically relevant setting (8 comparisons) were below 0.09 mm and 0.14° (largest SDSE: 0.012 mm and 0.020°). In patients, the precision was lower, but acceptable, and dependent on CT scan resolution. Interpretation - CTSA allows detection of stem-bone migration with an accuracy and precision comparable to that of RSA. It could be valuable for evaluation of subtle stem loosening in clinical practice. PMID:26634843

  19. [Transgenics - Plant-Based Drugs (PBD)].

    PubMed

    Rocha, Daniele Rachidi da; Marin, Victor Augustus

    2011-07-01

    Plant-Based Drugs - PBD - represent the 4(th) generation of genetically-modified plants and in this case the technology is used to develop and produce pharmaceuticals vaccines and/or products from transgenic seeds. This technology, like all scientific innovations, has inherent risks. However, the current knowledge available about the use of this technology means that no firm conclusions can be drawn about the nature of the risks involved, as well as their significance and the likelihood of causing serious damage or not. Risk analysis should be the starting premise prior to any implementation of techno-scientific innovations. The parameters must be evaluated and precautions taken and research must be conducted in a detailed and broad-ranging manner with respect to the potential risks of any innovation. This article analyzed the applicability of this new technology, as well as risk management and containment in order to guarantee safe use, handling and consumption by human beings. PMID:21808921

  20. A Transgenic Mouse Model of Poliomyelitis.

    PubMed

    Koike, Satoshi; Nagata, Noriyo

    2016-01-01

    Transgenic mice (tg mice) that express the human poliovirus receptor (PVR), CD155, are susceptible to poliovirus and develop a neurological disease that resembles human poliomyelitis. Assessment of the neurovirulence levels of poliovirus strains, including mutant viruses produced by reverse genetics, circulating vaccine-derived poliovirus, and vaccine candidates, is useful for basic research of poliovirus pathogenicity, the surveillance of circulating polioviruses, and the quality control of oral live poliovirus vaccines, and does not require the use of monkeys. Furthermore, PVR-tg mice are useful for studying poliovirus tissue tropism and host immune responses. PVR-tg mice can be bred with mice deficient in the genes involved in viral pathogenicity. This report describes the methods used to analyze the pathogenicity and immune responses of poliovirus using the PVR-tg mouse model. PMID:26983733

  1. Germline transmission in transgenic Huntington’s disease monkeys

    PubMed Central

    Moran, Sean; Chi, Tim; Prucha, Melinda S.; Ahn, Kwang Sung; Connor-Stroud, Fawn; Jean, Sherrie; Gould, Kenneth; Chan, Anthony W. S.

    2015-01-01

    Transgenic nonhuman primate models are increasingly popular model for neurological and neurodegenerative disease because their brain functions and neural anatomies closely resemble those of humans [1–6]. Transgenic Huntington’s disease monkeys (HD monkeys) developed clinical features similar to those seen in HD patients, making the monkeys suitable for preclinical study of HD [6–12]. However, until HD monkey colonies can be readily expanded, their use in preclinical studies will be limited [1, 13, 14]. In the present study, we confirmed germline transmission of the mutant huntingtin (mHTT) transgene in both embryonic stem cells (ESCs) generated from three male HD monkey founders (F0), as well as in second-generation offspring (F1) produced via artificial insemination by using intrauterine insemination (IUI) technique. A total of five offspring were produced from fifteen females that were inseminated by IUI using semen collected from the three HD founders (5/15; 33%). Thus far, sperm collected from HD founder (rHD8) has led to two F1 transgenic HD moenkys with germline transmission rate at 100% (2/2). mHTT expression was confirmed by quantitative real-time PCR (qPCR) using skin fibroblasts from the F1 HD monkeys, as well as induced pluripotent stem cells (iPSCs) established from one of the F1 HD monkeys (rHD8-2). Here we report the stable germline transmission and expression of the mHTT transgene in HD monkeys, which suggest possible expansion of HD monkey colonies for preclinical and biomedical researches. PMID:25917881

  2. Measuring gene flow in the cultivation of transgenic cotton (Gossypium hirsutum L.).

    PubMed

    Zhang, Bao-Hong; Pan, Xiao-Ping; Guo, Teng-Long; Wang, Qing-Lian; Anderson, Todd A

    2005-09-01

    Transgenic Bt cotton NewCott 33B and transgenic tfd A cotton TFD were chosen to evaluate pollen dispersal frequency and distance of transgenic cotton (Gossypium hirsutum L.) in the Huanghe Valley Cotton-producing Zone, China. The objective was to evaluate the efficacy of biosafety procedures used to reduce pollen movement. A field test plot of transgenic cotton (6 x 6 m) was planted in the middle of a nontransgenic field measuring 210 x 210 m. The results indicated that the pollen of Bt cotton or tfd A cotton could be dispersed into the environment. Out-crossing was highest within the central test plot where progeny from nontransgenic plants, immediately adjacent to transgenic plants, had resistant plant progeny at frequencies up to 10.48%. Dispersal frequency decreased significantly and exponentially as dispersal distance increased. The flow frequency and distance of tfd A and Bt genes were similar, but the pollen-mediated gene flow of tfd A cotton was higher and further to the transgenic block than that of Bt cotton (chi2 = 11.712, 1 degree of freedom, p < 0.001). For the tfd A gene, out-crossing ranged from 10.13% at 1 m to 0.04% at 50 m from the transgenic plants. For the Bt gene, out-crossing ranged from 8.16% at 1 m to 0.08% at 20 m from the transgenic plants. These data were fit to a power curve model: y = 10.1321x-1.4133 with a correlation coefficient of 0.999, and y = 8.0031x-1.483 with a correlation coefficient of 0.998, respectively. In this experiment, the farthest distance of pollen dispersal from transgenic cotton was 50 m. These results indicate that a 60-m buffer zone would serve to limit dispersal of transgenic pollen from small-scale field tests. PMID:16118411

  3. Biolistic-mediated production of transgenic oil palm.

    PubMed

    Parveez, Ghulam Kadir Ahmad; Bahariah, Bohari

    2012-01-01

    The effectiveness of mannose (using phosphomannose isomerase [pmi] gene) as a positive selection agent to preferably allow the growth of transformed oil palm embryogenic calli was successfully evaluated. Using the above selection agent in combination with the previously optimized physical and biological parameters and the best constitutive promoter, oil palm embryogenic calli were transformed with pmi gene for producing transgenic plants. Bombarded embryogenic calli were exposed to embryogenic calli medium containing 30:0 g/L mannose to sucrose 3 weeks postbombardment. Selectively, proliferating embryogenic calli started to emerge around 6 months on the above selection medium. The proliferated embryogenic calli were individually isolated once they reached a specific size and regenerated to produce complete plantlets. The complete regenerated plantlets were evaluated for the presence of transgenes by PCR and Southern analyses. PMID:22351007

  4. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  5. Preventing Spontaneous Genetic Rearrangements in the Transgene Cassettes of Adenovirus Vectors

    PubMed Central

    Cottingham, Matthew G.; Carroll, Fionnadh; Morris, Susan J.; Turner, Alison V.; Vaughan, Aisling M.; Kapulu, Melissa C.; Colloca, Stefano; Siani, Loredana; Gilbert, Sarah C.; Hill, Adrian V.S.

    2016-01-01

    First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture. PMID:22252512

  6. Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1

    PubMed Central

    Wang, Meizhen; Liu, Chen; Li, Shixue; Zhu, Dengyun; Zhao, Qian; Yu, Jingjuan

    2013-01-01

    Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9–10 and 19–11, ZmMYC1 in line 19–11 and ZmSDR in line 19–11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt

  7. Transgenic rabbit that expresses a functional human lipoprotein (a)

    DOEpatents

    Rouy, Didier; Duverger, Nicolas; Emmanuel, Florence; Denefle, Patrice; Houdebine, Louis-Marie; Viglietta, Celine; Rubin, Edward M.; Hughes, Steven D.

    2003-01-01

    A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

  8. Expression of human protamine P1 in sperm of transgenic mice

    SciTech Connect

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X.; Anderson, G.

    1994-12-31

    Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.

  9. Transgenic plants and biosafety: science, misconceptions and public perceptions.

    PubMed

    Stewart, C N; Richards, H A; Halfhill, M D

    2000-10-01

    One usually thinks of plant biology as a non-controversial topic, but the concerns raised over the biosafety of genetically modified (GM) plants have reached disproportionate levels relative to the actual risks. While the technology of changing the genome of plants has been gradually refined and increasingly implemented, the commercialization of GM crops has exploded. Today's commercialized transgenic plants have been produced using Agrobacterium tumefaciens-mediated transformation or gene gun-mediated transformation. Recently, incremental improvements of biotechnologies, such as the use of green fluorescent protein (GFP) as a selectable marker, have been developed. Non-transformation genetic modification technologies such as chimeraplasty will be increasingly used to more precisely modify germplasm. In spite of the increasing knowledge about genetic modification of plants, concerns over ecological and food biosafety have escalated beyond scientific rationality. While several risks associated with GM crops and foods have been identified, the popular press, spurred by colorful protest groups, has left the general public with a sense of imminent danger. Reviewed here are the risks that are currently under research. Ecological biosafety research has identified potential risks associated with certain crop/transgene combinations, such as intra- and interspecific transgene flow, persistence and the consequences of transgenes in unintended hosts. Resistance management strategies for insect resistance transgenes and non-target effects of these genes have also been studied. Food biosafety research has focused on transgenic product toxicity and allergenicity. However, an estimated 3.5 x 10(12) transgenic plants have been grown in the U.S. in the past 12 years, with over two trillion being grown in 1999 and 2000 alone. These large numbers and the absence of any negative reports of compromised biosafety indicate that genetic modification by biotechnology poses no immediate or

  10. Postmortem findings in cloned and transgenic piglets dead before weaning.

    PubMed

    Schmidt, M; Winther, K D; Secher, J O; Callesen, H

    2015-10-01

    Important factors contributing to the well-known high mortality of piglets produced by SCNT are gross malformations of vital organs. The aim of the present retrospective study was to describe malformations found in cloned piglets, transgenic or not, dying or culled before weaning on Day 28. Large White (LW) embryos were transferred to 78 LW recipients, while 72 recipients received Göttingen embryos (67 transgenic and five not transgenic) and 56 received Yucatan embryos (43 transgenic and 13 not transgenic). Overall pregnancy rate was 76%, and there were more abortions in recipients with minipig embryos than in those with LW embryos (26% and 24% vs. 6%). Piglets (n = 815) were born from 128 sows with 6.5 ± 0.4 full-born piglets per litter. The overall rate of stillborn piglets was 21% of all born with the number of stillborn piglets ranging from one to nine in a litter. The mortality of the surviving piglets during the first month was 48%. Thus, altogether 58% of the full-born piglets died before weaning. In 87 of the 128 litters (68%), one to 12 of the piglets showed major or minor malformations. Malformations were found in 232 piglets (29.5% of all born). A single malformation was registered in 152 piglets, but several piglets showed two (n = 58) or more (n = 23) malformations (7.4% and 2.8% of all born, respectively). A significantly higher malformation rate was found in transgenic Göttingen and Yucatan piglets (32% and 46% of all born, respectively) than in nontransgenic LW (17%). There was a gender difference in the transgenic minipigs because male piglets had a higher rate of malformations (49.1%) than females (29.7%). The most common defects in the cloned piglets were in the digestive (12.2%), circulatory (9.4%), reproductive (11.3%), and musculoskeletal (9.1%) systems. Malformations of the musculoskeletal system were most frequent in Göttingen (16.3% vs. approximately 5.5% in the two other breeds), whereas abnormal cardiopulmonary systems were most

  11. A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice.

    PubMed

    Lin, Chaoyang; Fang, Jun; Xu, Xiaoli; Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

    2008-01-01

    Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

  12. A Built-In Strategy for Containment of Transgenic Plants: Creation of Selectively Terminable Transgenic Rice

    PubMed Central

    Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

    2008-01-01

    Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

  13. Production of Transgenic Calves Expressing an shRNA Targeting Myostatin

    PubMed Central

    Tessanne, K; Golding, MC; Long, CR; Peoples, MD; Hannon, G; Westhusin, ME

    2012-01-01

    Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing. PMID:22139943

  14. Transgenic mouse model for estrogen-regulated lipoprotein metabolism: studies on apoVLDL-II expression in transgenic mice.

    PubMed

    Zsigmond, E; Nakanishi, M K; Ghiselli, F E; Chan, L

    1995-07-01

    We have produced transgenic mice that express an estrogen-responsive avian apolipoprotein, apoVLDL-II. An apoVLDL-II natural gene construct containing 4.7 kb of 5' flanking and 19 bp of 3' flanking sequences together with the 4 exon/3 intron structural gene was expressed in a liver-specific manner in transgenic mice. A single injection of estrogen caused a 5.9- to 7.5-fold stimulation of apoVLDL-II mRNA in the liver. The transgene mRNA had the same initiation sites of transcription as the native mRNA isolated from laying hen liver, and the same sites were used before and after estrogen treatment. The number of hepatocytes that stain positive for immunoreactive apoVLDL-II increased from < 1% to 40-60% in 24 h after estrogen treatment. Thus, in trangenic mice as in the cockerel, hepatocytes are biochemically heterogeneous and induction of apoVLDL-II synthesis occurs by recruitment of hepatocytes. In the plamsa compartment, compared to controls, transgenic mice have a 3- to 5-fold higher basal total plasma triglyceride which was accounted for by a 5.4-fold high basal VLDL triglyceride. Estrogen treatment results in a approximately 2-fold increase in the VLDL triglycerides over basal levels and 8.5-fold increase over nontransgenic mice, which did not show any change in VLDL in response to estrogen. Transgenic mice with the integrated apoVLDL-II gene provide a useful model for the study of the regulation of lipoprotein metabolism by estrogen. PMID:7595069

  15. Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg

    PubMed Central

    Gao, Yi; Ma, Ying; Li, Mei; Cheng, Tong; Li, Shao-Wei; Zhang, Jun; Xia, Ning-Shao

    2003-01-01

    AIM: To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests. METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice’s serum were examined by ELISA every week. RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo, level of rHBsAg expressed in leaves was the highest with the yield up to 300 ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight. In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response. CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale. PMID:12717845

  16. Oviduct-specific expression of human neutrophil defensin 4 in lentivirally generated transgenic chickens.

    PubMed

    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

  17. Transgenic Models in Retinoblastoma Research.

    PubMed

    Nair, Rohini M; Vemuganti, Geeta K

    2015-04-01

    Understanding the mechanism of retinoblastoma (Rb) tumor initiation, development, progression and metastasis in vivo mandates the use of animal models that mimic this intraocular tumor in its genetic, anatomic, histologic and ultrastructural features. An early setback for developing mouse Rb models was that Rb mutations did not cause tumorigenesis in murine retinas. Subsequently, the discovery that the p107 protein takes over the role of pRb in mice led to the development of several animal models that phenotypically and histologically resemble the human form. This paper summarizes the transgenic models that have been developed over the last three decades. PMID:27171579

  18. Transgenic Models in Retinoblastoma Research

    PubMed Central

    Nair, Rohini M.; Vemuganti, Geeta K.

    2015-01-01

    Understanding the mechanism of retinoblastoma (Rb) tumor initiation, development, progression and metastasis in vivo mandates the use of animal models that mimic this intraocular tumor in its genetic, anatomic, histologic and ultrastructural features. An early setback for developing mouse Rb models was that Rb mutations did not cause tumorigenesis in murine retinas. Subsequently, the discovery that the p107 protein takes over the role of pRb in mice led to the development of several animal models that phenotypically and histologically resemble the human form. This paper summarizes the transgenic models that have been developed over the last three decades. PMID:27171579

  19. Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization.

    PubMed

    Newhouse, Andrew E; Schrodt, Franziska; Liang, Haiying; Maynard, Charles A; Powell, William A

    2007-07-01

    The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm. PMID:17310333

  20. Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity

    PubMed Central

    Wang, Shaohua; Zhang, Yuyu; Yang, Mingying; Ye, Lupeng; Gong, Lu; Qian, Qiujie; Shuai, Yajun; You, Zhengying; Chen, Yuyin; Zhong, Boxiong

    2016-01-01

    Silk fibers have many inherent properties that are suitable for their use in biomaterials. In this study, the silk fibroin was genetically modified by including a Ca-binding sequence, [(AGSGAG)6ASEYDYDDDSDDDDEWD]2 from shell nacreous matrix protein. It can be produced as fibers by transgenic silkworm. The Ca-binding activity and mineralization of the transgenic silk fibroin were examined in vitro. The results showed that this transgenic silk fibroin had relatively higher Ca-binding activity than unmodified silk fibroin. The increased Ca-binding activity could promote the usage of silk fibroin as a biomaterial in the pharmaceutical industry. This study shows the possibility of using silk fibroin as a mineralization accelerating medical material by generating genetically modified transgenic silkworm. PMID:27414647

  1. Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken

    PubMed Central

    Byun, Sung June; Ji, Mi-Ran; Jang, Ye-Jin; Hwang, A-In; Chung, Hee Kyoung; Kim, Jeom Sun; Kim, Kyung-Woon; Chung, Hak-Jae; Yang, Byoung-Chul; Jeon, Iksoo; Park, Jin-Ki; Yoo, Jae Gyu; Kim, Tae-Yoon

    2013-01-01

    Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409] PMID:23977988

  2. Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation.

    PubMed

    Lu, Chaofu; Kang, Jinling

    2008-02-01

    Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products. PMID:17899095

  3. Antifungal traits of a 14 kDa maize kernel trypsin inhibitor protein in transgenic cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic cotton plants expressing the maize kernel trypsin inhibitor (TI) protein were produced and evaluated for antifungal traits. This 14 kD trypsin inhibitor protein has been previously associated with resistance to aflatoxin-producing fungus Aspergillus flavus. Successful transformation of ...

  4. RHIZOSPHERE COLONIZATION OF TRANSGENIC AND WILD-TYPE PSEUDOMONAS FLUORESCENS IS MODULATED BY THE CROP SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas fluorescens strain Q8r1-96 (genotype D) produces 2,4-diacetylphloroglucinol (DAPG). Transgenic strain Z30-97, constructed by stably inserting the phenazine-1-carboxylic acid (PCA) biosynthetic locus from P. fluorescens 2-79 into Q8r1-96, produces both antibiotics in vitro and in situ. Th...

  5. Construction and long term preservation of clonal transgenic silkworms using a parthenogenetic strain.

    PubMed

    Zabelina, Valeriya; Uchino, Keiro; Mochida, Yuji; Yonemura, Naoyuki; Klymenko, Vyacheslav; Sezutsu, Hideki; Tamura, Toshiki; Sehnal, František

    2015-10-01

    For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace's medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene. PMID:26112978

  6. Transgenic plants in the biopharmaceutical market.

    PubMed

    Twyman, Richard M; Schillberg, Stefan; Fischer, Rainer

    2005-02-01

    Many of our 'small-molecule-drugs' are natural products from plants, or are synthetic compounds based on molecules found naturally in plants. However, the vast majority of the protein therapeutics (or biopharmaceuticals) we use are from animal or human sources, and are produced commercially in microbial or mammalian bioreactor systems. Over the last few years, it has become clear that plants have great potential for the production of human proteins and other protein-based therapeutic entities. Plants offer the prospect of inexpensive biopharmaceutical production without sacrificing product quality or safety, and following the success of several plant-derived technical proteins, the first therapeutic products are now approaching the market. In this review, the different plant-based production systems are discussed and the merits of transgenic plants are evaluated compared with other platforms. A detailed discussion is provided of the development issues that remain to be addressed before plants become an acceptable mainstream production technology. The many different proteins that have already been produced using plants are described, and a sketch of the current market and the activities of the key players is provided. Despite the currently unclear regulatory framework and general industry inertia, the benefits of plant-derived pharmaceuticals are now bringing the prospect of inexpensive veterinary and human medicines closer than ever before. PMID:15757412

  7. Transgenic Papaya: Development, Release, Impact, and Challenges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the technology for developing virus-resistant transgenic plants through the use of the coat protein of a virus was unveiled twenty years ago, it is surprising to note that only a three virus-resistant plants (squash, potato, and papaya) have been commercialized in the U.S. The transgenic p...

  8. Transgenic Biofuel Feedstocks and Strategies for Biocontainment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are several reasons to believe that transgenic plant feedstocks will be required to realize the full economic and environmental benefits of cellulosic and other biofuels. Much of the commercialization potential for the use of transgenic plant cellulosic feedstocks may be impacted by regulatio...

  9. 2008 FHB Analysis of Transgenic Barley Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic lines have been developed with the goal of reducing FHB and DON in barley. Replicated field trials for FHB reaction of 48 Conlon transgenic lines were conducted in 2008 in Langdon, ND and Rosemount, MN. The Langdon trials consisted of three replicates in hill plots in an inoculated misted...

  10. Accumulation of nickel in transgenic tobacco

    NASA Astrophysics Data System (ADS)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TF<1) at all levels of metal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  11. Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter

    PubMed Central

    Bodnar, Anastasia L.; Schroder, Megan N.; Scott, M. Paul

    2016-01-01

    High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected control populations. These higher levels of the 27kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression. PMID:26895451

  12. Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

    PubMed Central

    Kolb, Andreas F.; Pewe, Lecia; Webster, John; Perlman, Stanley; Whitelaw, C. Bruce A.; Siddell, Stuart G.

    2001-01-01

    Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis. PMID:11222704

  13. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    PubMed

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-07-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. PMID:26771455

  14. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

    PubMed Central

    Praitis, V; Casey, E; Collar, D; Austin, J

    2001-01-01

    In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing. PMID:11238406

  15. Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zi, Ha; Qiao, Jun; Wang, Pengyang; Sheng, Jinliang; Chen, Chuangfu

    2013-01-01

    Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production. PMID:23526994

  16. Disruption of neutrophil migration in a conditional transgenic model: evidence for CXCR2 desensitization in vivo.

    PubMed

    Wiekowski, M T; Chen, S C; Zalamea, P; Wilburn, B P; Kinsley, D J; Sharif, W W; Jensen, K K; Hedrick, J A; Manfra, D; Lira, S A

    2001-12-15

    We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the beta-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, beta-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine. PMID:11739532

  17. Using inositol as a biocompatible ligand for efficient transgene expression.

    PubMed

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

  18. Using inositol as a biocompatible ligand for efficient transgene expression

    PubMed Central

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

  19. Sustainability of insect resistance management strategies for transgenic Bt corn.

    PubMed

    Glaser, John A; Matten, Sharlene R

    2003-12-01

    Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies. An arena ripe for application of such analysis is the deployment of transgenic crops. The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content. However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance. The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act. Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B. thuringiensis (Bt) insecticidal proteins. PMID:14623043

  20. Transformation of pecan and regeneration of transgenic plants.

    PubMed

    McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E

    1993-09-01

    A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis. PMID:24201878

  1. Generation of transgenic Hydra by embryo microinjection.

    PubMed

    Juliano, Celina E; Lin, Haifan; Steele, Robert E

    2014-01-01

    As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost. PMID:25285460

  2. In vitro production of cloned and transgenically cloned embryos from Guangxi Huanjiang Xiang pig.

    PubMed

    Zhu, Xiangxing; Nie, Junyu; Quan, Shouneng; Xu, Huiyan; Yang, Xiaogan; Lu, Yangqing; Lu, Kehuan; Lu, Shengsheng

    2016-02-01

    Guangxi Huanjiang Xiang pig is a unique miniature pig strain that is originally from Huanjiang Maonan Autonomous County of Guangxi province, China, and shows great potential in agricultural and biomedical research. Although cloning and genetic modification of this pig would enhance its application value, cloning of this strain has not yet been reported. We sought to establish appropriate cloning procedures and produce transgenic embryos in Huanjiang Xiang pigs through the following methods. We isolated fibroblasts from tails of Huanjiang Xiang pig and genetically modified them using Xfect transfection. Fibroblasts, either in non-transgenic or transgenic forms, were used as donor cells for reconstructed embryos by somatic cell nuclear transfer (SCNT), and in vitro development was monitored after the reconstruction. We found no difference in blastocyst formation rate between non-transgenic and transgenic embryos (10.8% vs. 10.3%; P ≥ 0.05). In addition, we tested whether Scriptaid, a widely used histone deacetylase inhibitor, could enhance the in vitro development of Huanjiang Xiang pig cloned embryos. Treatment with 500 nM Scriptaid for 16 h post-activation significantly increased the blastocyst formation rate (26.1% vs. 10.8% for non-transgenic nuclear transfer groups with vs. without the Scriptaid treatment and 28.5% vs. 10.3% for transgenic nuclear transfer groups with vs. without the Scriptaid treatment; P < 0.05). This study provided a basis for further generation of cloned and transgenically cloned Huanjiang Xiang pigs used in agricultural and biomedical research. PMID:26559066

  3. Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm.

    PubMed

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Xu, Hanfu; Yuan, Lin; Ding, Huan; Ma, Sanyuan; Zhou, You; Zhao, Ping; Xia, Qingyou

    2015-06-01

    Deletion of transposable elements post-genomic integration holds great promise for stability of the transgene in the host genome and has an essential role for the practical application of transgenic animals. In this study, a modified piggyBac vector that mediated deletion of the transposon sequence post-integration for transgene stability in the economically important silkworm Bombyx mori was constructed. The piggyBac vector architecture contains inversed terminal repeat sequences L1, L2 and R1, which can form L1/R1 and L2/R1 types of transposition cassettes. hsp70-PIG as the piggyBac transposase expression cassette for initial transposition, further remobilization and transgene stabilization test was transiently expressed in a helper vector or integrated into the modified vector to produce a transgenic silkworm. Shortening L2 increased the transformation frequency of L1/R1 into the silkworm genome compared to L2/R1. After the integration of L1/R1 into the genome, the remobilization of L2/R1 impaired the transposon structure and the resulting transgene linked with an impaired transposon was stable in the genome even in the presence of exogenously introduced transposase, whereas those flanked by the intact transposon were highly mobile in the genome. Our results demonstrated the feasibility of post-integration deletion of transposable elements to guarantee true transgene stabilization in silkworm. We suggest that the modified vector will be a useful resource for studies of transgenic silkworms and other piggyBac-transformed organisms. PMID:25589404

  4. Glyphostate-drift but not herbivory alters the rate of transgene flow from single and stacked trait transgenic canola (Brassica napus L.) to non-transgenic B. napus and B. rapa

    EPA Science Inventory

    While transgenic plants can offer agricultural benefits, the escape of transgenes out of crop fields is a major environmental concern. Escape of transgenic herbicide resistance has occurred between transgenic Brassica napus (canola) and weedy species in numerous locations. In t...

  5. A transgenic embryonic sexing system for Anastrepha suspensa (Diptera: Tephritidae).

    PubMed

    Schetelig, Marc F; Handler, Alfred M

    2012-10-01

    The Sterile Insect Technique (SIT) is a highly successful biologically-based strategy to control pest insect populations that relies on the large-scale release of sterilized males to render females in the field non-reproductive. For medfly, a mutant-based sexing system is available as well as a transgenic system where a tetracycline-suppressible (Tet-off) toxic molecule is female-specifically produced. However, the former classical genetic system took many years to refine, and the latter system results in female death by a poorly understood mechanism, primarily in the pupal stage after rearing costs have been incurred. Here we describe a Tet-off transgenic embryonic sexing system (TESS) for Anastrepha suspensa that uses a driver construct having the promoter from the embryo-specific A. suspensa serendipity α gene, linked to the Tet-transactivator. This was used to drive the expression of a phospho-mutated variant of the pro-apoptotic cell death gene, Alhid, from Anastrepha ludens. The system uses a sex-specific intron splicing cassette linked to a cell death gene lethal effector. Progeny from TESS strains heterozygous for the transgene combination were 80-100% males, whereas four double homozygous TESS strains had 100% male-only progeny, with female death limited primarily to embryogenesis. In a large-scale test, more than 30,000 eggs from two strains resulted in 100% male-only progeny. The transgenic sexing approach described here is highly effective and cost-efficient by eliminating most, if not all, female insects early in embryogenesis using a well-characterized apoptotic mechanism. PMID:22858603

  6. Optical modulation of transgene expression in retinal pigment epithelium

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Lavinsky, D.; Chalberg, T.; Mandel, Y.; Huie, P.; Dalal, R.; Marmor, M.

    2013-03-01

    Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a "biofactory" that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.

  7. Transgenic fish: present status and future directions.

    PubMed

    Hew, C L

    1989-06-01

    Successful production of transgenic fish by gene transfer technology is a very important breakthrough in the techniques of genetic manipulation in animals. This will have an impact of an unprecedented scale in fish biology, aquaculture and mariculture. This is a summary of the workshop on the Transgenic Fish presented at this Symposium. The Workshop discussed the current knowledge, experimental difficulties and related topics of the transgenic fish. It recommended further research on better gene constructs, methods development, safety containment and the closer collaboration of researchers of different disciplines. PMID:24221801

  8. [Transgenic technology and soybean quality improvement].

    PubMed

    Cheng, Hao; Jin, Hang-Xia; Gai, Jun-Yi; Yu, De-Yue

    2011-05-01

    Soybean is an important source of edible oil, protein and protein diet. The breeding process of high quality soybean can be accelerated via employment of transgenic technology, by which the key genes for soybean quality traits could be directly manipulated. Thus, various soybean varieties could be bred to fulfill different needs for specific consumers. Here, we reviewed the contribution of transgenic technology to improvement of soybean qualities in recent years. We also introduce some newly developed safe transgenic technologies and hope this information could relieve some concerns on the GM food. PMID:21586389

  9. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    PubMed

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

  10. Expression of a bacterial alpha-amylase gene in transgenic rice seeds.

    PubMed

    Xu, Xiaoli; Fang, Jun; Wang, Wei; Guo, Jianli; Chen, Pinnan; Cheng, Jiaan; Shen, Zhicheng

    2008-08-01

    An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction. PMID:17926139