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Sample records for cyp86a33-targeted gene silencing

  1. Chromatin, gene silencing and HIV latency

    PubMed Central

    Mok, Hoi-Ping; Lever, Andrew ML

    2007-01-01

    One of the cellular defenses against virus infection is the silencing of viral gene expression. There is evidence that at least two gene-silencing mechanisms are used against the human immuno-deficiency virus (HIV). Paradoxically, this cellular defense mechanism contributes to viral latency and persistence, and we review here the relationship of viral latency to gene-silencing mechanisms. PMID:18036274

  2. Nickel and epigenetic gene silencing.

    PubMed

    Sun, Hong; Shamy, Magdy; Costa, Max

    2013-01-01

    Insoluble nickel compounds are well-established human carcinogens. Occupational exposure to these compounds leads to increased incidence of lung and nasal cancer in nickel refinery workers. Apart from its weak mutagenic activity and hypoxia mimicking effect there is mounting experimental evidence indicating that epigenetic alteration plays an important role in nickel-induced carcinogenesis. Multiple epigenetic mechanisms have been identified to mediate nickel-induced gene silencing. Nickel ion is able to induce heterochromatinization by binding to DNA-histone complexes and initiating chromatin condensation. The enzymes required for establishing or removing epigenetic marks can be targeted by nickel, leading to altered DNA methylation and histone modification landscapes. The current review will focus on the epigenetic changes that contribute to nickel-induced gene silencing. PMID:24705264

  3. Advances in plant gene silencing methods.

    PubMed

    Pandey, Prachi; Senthil-Kumar, Muthappa; Mysore, Kirankumar S

    2015-01-01

    Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.

  4. Personalized gene silencing therapeutics for Huntington disease.

    PubMed

    Kay, C; Skotte, N H; Southwell, A L; Hayden, M R

    2014-07-01

    Gene silencing offers a novel therapeutic strategy for dominant genetic disorders. In specific diseases, selective silencing of only one copy of a gene may be advantageous over non-selective silencing of both copies. Huntington disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin gene (HTT). Silencing both expanded and normal copies of HTT may be therapeutically beneficial, but preservation of normal HTT expression is preferred. Allele-specific methods can selectively silence the mutant HTT transcript by targeting either the expanded CAG repeat or single nucleotide polymorphisms (SNPs) in linkage disequilibrium with the expansion. Both approaches require personalized treatment strategies based on patient genotypes. We compare the prospect of safe treatment of HD by CAG- and SNP-specific silencing approaches and review HD population genetics used to guide target identification in the patient population. Clinical implementation of allele-specific HTT silencing faces challenges common to personalized genetic medicine, requiring novel solutions from clinical scientists and regulatory authorities.

  5. Homology-dependent Gene Silencing in Paramecium

    PubMed Central

    Ruiz, Françoise; Vayssié, Laurence; Klotz, Catherine; Sperling, Linda; Madeddu, Luisa

    1998-01-01

    Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant. PMID:9529389

  6. MIGS: miRNA-induced gene silencing.

    PubMed

    Felippes, Felipe Fenselau de; Wang, Jia-wei; Weigel, Detlef

    2012-05-01

    Gene silencing is an important tool in the study of gene function. Virus-induced gene silencing (VIGS) and hairpin RNA interference (hpRNAi), both of which rely on small interfering RNAs, together with artificial microRNAs (amiRNA), are amongst the most popular methods for reduction of gene activity in plants. However, all three approaches have limitations. Here, we introduce miRNA-induced gene silencing (MIGS). This method exploits a special 22-nucleotide miRNA of Arabidopsis thaliana, miR173, which can trigger production of another class of small RNAs called trans-acting small interfering RNAs (tasiRNAs). We show that fusion of gene fragments to an upstream miR173 target site is sufficient for effective silencing of the corresponding endogenous gene. MIGS can be reliably used for the knockdown of a single gene or of multiple unrelated genes. In addition, we show that MIGS can be applied to other species by co-expression of miR173.

  7. Virus-induced gene silencing using begomovirus satellite molecules.

    PubMed

    Zhou, Xueping; Huang, Changjun

    2012-01-01

    Virus-induced gene silencing (VIGS) has emerged as a powerful method for studying gene function. VIGS is induced by infecting a plant with a plant virus that has had its genome modified to include a sequence from the host gene to be silenced. DNAβ and DNA1 are satellite and single-stranded DNA molecules associated with begomoviruses (family Geminiviridae). We converted DNAβ and DNA1 into gene-silencing vectors. The VIGS vectors can induce silencing efficiently in many solanaceous plants. Here, we describe procedures for the use of these two gene-silencing vectors for VIGS in different hosts. PMID:22678572

  8. Antisense Gene Silencing: Therapy for Neurodegenerative Disorders?

    PubMed Central

    Nielsen, Troels T.; Nielsen, Jørgen E.

    2013-01-01

    Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied to mammalian cells, the technology of RNAi expanded from being a valuable experimental tool to being an applicable method for gene-specific therapeutic regulation, and much effort has been put into further refinement of the technique. This review will focus on how RNAi has developed over the years and how the technique is exploited in a pre-clinical and clinical perspective in relation to neurodegenerative disorders. PMID:24705213

  9. Gene silencing by nuclear orphan receptors.

    PubMed

    Zhang, Ying; Dufau, Maria L

    2004-01-01

    Nuclear orphan receptors represent a large and diverse subgroup in the nuclear receptor superfamily. Although putative ligands for these orphan members remain to be identified, some of these receptors possess intrinsic activating, inhibitory, or dual regulatory functions in development, differentiation, homeostasis, and reproduction. In particular, gene-silencing events elicited by chicken ovalbumin upstream promoter-transcription factors (COUP-TFs); dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1); germ cell nuclear factor (GCNF); short heterodimer partner (SHP); and testicular receptors 2 and 4 (TR2 and TR4) are among the best characterized. These orphan receptors are critical in controlling basal activities or hormonal responsiveness of numerous target genes. They employ multiple and distinct mechanisms to mediate target gene repression. Complex cross-talk exists between these orphan receptors at their cognate DNA binding elements and an array of steroid?nonsteroid hormone receptors, other transcriptional activators, coactivators and corepressors, histone modification enzyme complexes, and components of basal transcriptional components. Therefore, perturbation induced by these orphan receptors at multiple levels, including DNA binding activities, receptor homo- or heterodimerization, recruitment of cofactor proteins, communication with general transcriptional machinery, and changes at histone acetylation status and chromatin structures, may contribute to silencing of target gene expression in a specific promoter or cell-type context. Moreover, the findings derived from gene-targeting studies have demonstrated the significance of these orphan receptors' function in physiologic settings. Thus, COUP-TFs, DAX-1, GCNF, SHP, and TR2 and 4 are known to be required for multiple physiologic and biologic functions, including neurogenesis and development of the heart and vascular system steroidogenesis and sex

  10. Gene silencing by DNA interference in fern gametophytes.

    PubMed

    Wada, Masamitsu; Tsuboi, Hidenori

    2015-01-01

    RNA interference is commonly used for posttranscriptional silencing of target gene transcripts. In fern gametophytes, however, sequence-specific gene silencing is possible by introducing double-stranded DNA fragments into gametophyte cells by particle bombardment. Silencing could be transmitted all over the gametophyte through live cells. Further, inheritance of the gene silencing to the progeny is depending on the gene used. Here we describe how to introduce the DNA fragments into the gametophyte cells and how to screen the DNA-transferred cells.

  11. Gene silencing below the immune radar.

    PubMed

    Hartmann, Gunther

    2009-03-01

    In vertebrates, the detection of viral nucleic acids is the first step toward innate and subsequent adaptive antiviral immune responses. A sophisticated,protein receptor-based sensor system has evolved to recognize viral nucleic acids and to trigger a variety of antiviral defense mechanisms. The more we learn about this elaborate sensor system, the more it becomes evident how difficult it is to introduce exogenous nucleic acids such as siRNA into cells without triggering antiviral immunoreceptors. In this issue of the JCI, Judge and colleagues provide evidence that siRNA can be designed and delivered in a way that allows specific and successful silencing of target genes in tumor cells in vivo, leading to tumor cell death and prolonged survival of tumor-bearing mice in the absence of immune activation. This study represents a major technological advance, setting new standards for well-controlled siRNA applications in vivo, and has the potential to guide clinical development toward siRNA therapeutics with well-defined and selective gene-silencing activities.

  12. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae.

    PubMed

    Steakley, David Lee; Rine, Jasper

    2015-06-16

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein-based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing.

  13. Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

    PubMed Central

    Méndez, Catalina; Ahlenstiel, Chantelle L; Kelleher, Anthony D

    2015-01-01

    While human immunodeficiency virus 1 (HIV-1) infection is controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference (RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by non-coding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing (TGS), as well as fine-tuning their expression through post-transcriptional gene silencing (PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells. PMID:26279984

  14. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  15. Rotavirus gene silencing by small interfering RNAs

    PubMed Central

    Déctor, Miguel Angel; Romero, Pedro; López, Susana; Arias, Carlos F.

    2002-01-01

    RNA interference is an evolutionarily conserved double-stranded RNA-triggered mechanism for suppressing gene expression. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, from which the spike protein VP4 projects. Here, we show that a small interfering RNA corresponding to the VP4 gene efficiently inhibits the synthesis of this protein in virus-infected cells. A large proportion of infected cells had no detectable VP4 and the yield of viral progeny was reduced. Most of the virus particles purified from these cells were triple-layered, but lacked VP4, and were poorly infectious. We also show that VP4 might not be required for the last step of virus morphogenesis. The VP4 gene silencing was specific, since the synthesis of VP4 from rotavirus strains that differ in the target sequence was not affected. These findings offer the possibility of carrying out reverse genetics in rotaviruses. PMID:12446562

  16. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes.

    PubMed

    Lunyak, Victoria V; Burgess, Robert; Prefontaine, Gratien G; Nelson, Charles; Sze, Sing-Hoi; Chenoweth, Josh; Schwartz, Phillip; Pevzner, Pavel A; Glass, Christopher; Mandel, Gail; Rosenfeld, Michael G

    2002-11-29

    The molecular mechanisms by which central nervous system-specific genes are expressed only in the nervous system and repressed in other tissues remain a central issue in developmental and regulatory biology. Here, we report that the zinc-finger gene-specific repressor element RE-1 silencing transcription factor/neuronal restricted silencing factor (REST/NRSF) can mediate extraneuronal restriction by imposing either active repression via histone deacetylase recruitment or long-term gene silencing using a distinct functional complex. Silencing of neuronal-specific genes requires the recruitment of an associated corepressor, CoREST, that serves as a functional molecular beacon for the recruitment of molecular machinery that imposes silencing across a chromosomal interval, including transcriptional units that do not themselves contain REST/NRSF response elements.

  17. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants.

    PubMed

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo; Liu, Yule

    2016-07-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  18. Virus-induced gene silencing in diverse maize lines using the Brome Mosaic virus-based silencing vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in monocots has been limited. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase gene, a genetically diverse set of maize inbred lines was ...

  19. PIAS1 Regulates Breast Tumorigenesis through Selective Epigenetic Gene Silencing

    PubMed Central

    Liu, Bin; Tahk, Samuel; Yee, Kathleen M.; Yang, Randy; Yang, Yonghui; Mackie, Ryan; Hsu, Cary; Chernishof, Vasili; O'Brien, Neil; Jin, Yusheng; Fan, Guoping; Lane, Timothy F.; Rao, Jianyu; Slamon, Dennis; Shuai, Ke

    2014-01-01

    Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing. PMID:24586797

  20. MORC Family ATPases Required for Heterochromatin Condensation and Gene Silencing#

    PubMed Central

    Moissiard, Guillaume; Cokus, Shawn J.; Cary, Joshua; Feng, Suhua; Billi, Allison C.; Stroud, Hume; Husmann, Dylan; Zhan, Ye; Lajoie, Bryan R.; McCord, Rachel Patton; Hale, Christopher J.; Feng, Wei; Michaels, Scott D.; Frand, Alison R.; Pellegrini, Matteo; Dekker, Job; Kim, John K.; Jacobsen, Steve

    2012-01-01

    Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause de-repression of DNA-methylated genes and TEs, but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes. PMID:22555433

  1. Structural diversity repertoire of gene silencing small interfering RNAs.

    PubMed

    Chang, Chan Il; Kim, Helena Andrade; Dua, Pooja; Kim, Soyoun; Li, Chiang J; Lee, Dong-ki

    2011-06-01

    Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells. PMID:21749289

  2. Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

    PubMed Central

    Sagi, Amir; Manor, Rivka; Ventura, Tomer

    2013-01-01

    Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice. PMID:24705266

  3. Phenotypic diversification by gene silencing in Phytophthora plant pathogens

    PubMed Central

    Vetukuri, Ramesh R; Åsman, Anna KM; Jahan, Sultana N; Avrova, Anna O; Whisson, Stephen C; Dixelius, Christina

    2013-01-01

    Advances in genome sequencing technologies have enabled generation of unprecedented information on genome content and organization. Eukaryote genomes in particular may contain large populations of transposable elements (TEs) and other repeated sequences. Active TEs can result in insertional mutations, altered transcription levels and ectopic recombination of DNA. The genome of the oomycete plant pathogen, Phytophthora infestans, contains vast numbers of TE sequences. There are also hundreds of predicted disease-promoting effector proteins, predominantly located in TE-rich genomic regions. Expansion of effector gene families is also a genomic signature of related oomycetes such as P. sojae. Deep sequencing of small RNAs (sRNAs) from P. infestans has identified sRNAs derived from all families of transposons, highlighting the importance of RNA silencing for maintaining these genomic invaders in an inactive form. Small RNAs were also identified from specific effector encoding genes, possibly leading to RNA silencing of these genes and variation in pathogenicity and virulence toward plant resistance genes. Similar findings have also recently been made for the distantly related species, P. sojae. Small RNA “hotspots” originating from arrays of amplified gene sequences, or from genes displaying overlapping antisense transcription, were also identified in P. infestans. These findings suggest a major role for RNA silencing processes in the adaptability and diversification of these economically important plant pathogens. Here we review the latest progress and understanding of gene silencing in oomycetes with emphasis on transposable elements and sRNA-associated events. PMID:24563702

  4. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    PubMed

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains.

  5. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    PubMed

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains. PMID:26930612

  6. Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway.

    PubMed

    Llave, C; Kasschau, K D; Carrington, J C

    2000-11-21

    Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA. PMID:11078509

  7. Different patterns of gene silencing in position-effect variegation.

    PubMed

    Lloyd, Vett K; Dyment, David; Sinclair, Donald A R; Grigliatti, Thomas A

    2003-12-01

    Position-effect variegation (PEV) results when a fully functional gene is moved from its normal position to a position near to a broken heterochromatic-euchromatic boundary. In this new position, the gene, while remaining unaltered at the DNA level, is transcriptionally silenced in some cells but active in others, producing a diagnostic mosaic phenotype. Many variegating stocks show phenotypic instability, in that the level of variegation is dramatically different in different isolates or when out crossed. To test if this phenotypic instability was due to segregation of spontaneously accumulated mutations that suppress variegation, four different and well-characterized strains showing PEV for the white+ gene (wm4, wmMc, wm51b, and wmJ) and representing both large and small spot variegators were repeatedly out crossed to a strain free of modifiers, and the phenotypes of these variegators were monitored for 30 generations. Once free of modifiers, these variegating strains were then allowed to reaccumulate modifiers. The spontaneous suppressors of variegation were found to include both dominant and recessive, autosomal and X-linked alleles selected to reduce the detrimental effects of silencing white+ and adjacent genes. The time of peak sensitivity to temperature during development was also determined for these four variegators. Although large and small spot variegators have previously been attributed to early and late silencing events, respectively, the variegators we examined all shared a common early period of peak sensitivity to temperature. Once free of their variegation suppressors, the different variegating strains showed considerable differences in the frequency of inactivation at a cellular level (the number of cells showing silencing of a given gene) and the extent of variegation within the cell (the number of silenced genes). These results suggest that large and small spot variegation may be a superficial consequence of spontaneous variegation suppressors

  8. Trans-specific gene silencing between host and parasitic plants.

    PubMed

    Tomilov, Alexey A; Tomilova, Natalia B; Wroblewski, Tadeusz; Michelmore, Richard; Yoder, John I

    2008-11-01

    Species of Orobanchaceae parasitize the roots of nearby host plants to rob them of water and other nutrients. Parasitism can be debilitating to the host plant, and some of the world's most pernicious agricultural pests are parasitic weeds. We demonstrate here that interfering hairpin constructs transformed into host plants can silence expression of the targeted genes in the parasite. Transgenic roots of the hemi-parasitic plant Triphysaria versicolor expressing the GUS reporter gene were allowed to parasitize transgenic lettuce roots expressing a hairpin RNA containing a fragment of the GUS gene (hpGUS). When stained for GUS activity, Triphysaria roots attached to non-transgenic lettuce showed full GUS activity, but those parasitizing transgenic hpGUS lettuce lacked activity in root tissues distal to the haustorium. Transcript quantification indicated a reduction in the steady-state level of GUS mRNA in Triphysaria when they were attached to hpGUS lettuce. These results demonstrate that the GUS silencing signal generated by the host roots was translocated across the haustorium interface and was functional in the parasite. Movement across the haustorium was bi-directional, as demonstrated in double-junction experiments in which non-transgenic Triphysaria concomitantly parasitized two hosts, one transgenic for hpGUS and the other transgenic for a functional GUS gene. Observation of GUS silencing in the second host demonstrated that the silencing trigger could be moved from one host to another using the parasite as a physiological bridge. Silencing of parasite genes by generating siRNAs in the host provides a novel strategy for controlling parasitic weeds. PMID:18643992

  9. RNA-Mediated Gene Silencing in Hematopoietic Cells

    PubMed Central

    Venturini, Letizia; Eder, Matthias; Scherr, Michaela

    2006-01-01

    In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis. PMID:17057372

  10. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    PubMed

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

  11. Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

    PubMed Central

    Gerke, Jennifer; Bayram, Özgür; Feussner, Kirstin; Landesfeind, Manuel; Shelest, Ekaterina; Feussner, Ivo

    2012-01-01

    The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs. PMID:23001671

  12. Virus-induced gene silencing in eggplant (Solanum melongena).

    PubMed

    Liu, Haiping; Fu, Daqi; Zhu, Benzhong; Yan, Huaxue; Shen, Xiaoying; Zuo, Jinhua; Zhu, Yi; Luo, Yunbo

    2012-06-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

  13. Virus-induced gene silencing in eggplant (Solanum melongena).

    PubMed

    Liu, Haiping; Fu, Daqi; Zhu, Benzhong; Yan, Huaxue; Shen, Xiaoying; Zuo, Jinhua; Zhu, Yi; Luo, Yunbo

    2012-06-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function. PMID:22268843

  14. Tissue homogeneity requires inhibition of unequal gene silencing during development.

    PubMed

    Le, Hai H; Looney, Monika; Strauss, Benjamin; Bloodgood, Michael; Jose, Antony M

    2016-08-01

    Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity. PMID:27458132

  15. Construction of mismatched inverted repeat (IR) silencing vectors for maximizing IR stability and effective gene silencing in plants.

    PubMed

    Rey, M E Chrissie; Harmse, Johan; Taylor, Sarah H; Arbuthnot, Patrick; Weinberg, Marc S

    2015-01-01

    Inverted repeat (IR) RNA silencing vectors containing homologous fragments of target endogenous plant genes, or pathogen genes, are the most widely used vectors to either study the function of genes involved in biotic stress or silence pathogens to induce plant resistance, respectively. RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of IR constructs during cloning and plant transformation. We have therefore developed a robust method for the production of long IR vector constructs by introducing base pair mismatches in the form of cytosine to thymine mutations on the sense arm by exposure to sodium bisulfite prior to assembly of the IR. PMID:25740374

  16. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    PubMed Central

    Nakashima, Nobutaka; Miyazaki, Kentaro

    2014-01-01

    Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering. PMID:24552876

  17. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications. PMID:27521696

  18. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications.

  19. Silencing of genes and alleles by RNAi in Anopheles gambiae.

    PubMed

    Lamacchia, Marina; Clayton, John R; Wang-Sattler, Rui; Steinmetz, Lars M; Levashina, Elena A; Blandin, Stéphanie A

    2013-01-01

    Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background. PMID:22990777

  20. Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

    PubMed

    Kazanets, Anna; Shorstova, Tatiana; Hilmi, Khalid; Marques, Maud; Witcher, Michael

    2016-04-01

    Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes.

  1. Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

    PubMed

    Kazanets, Anna; Shorstova, Tatiana; Hilmi, Khalid; Marques, Maud; Witcher, Michael

    2016-04-01

    Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes. PMID:27085853

  2. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  3. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  4. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  5. Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report

    SciTech Connect

    Grant, S. R.

    1999-02-05

    Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

  6. Virus-Induced Silencing of a Plant Cellulose Synthase Gene

    PubMed Central

    Burton, Rachel A.; Gibeaut, David M.; Bacic, Antony; Findlay, Kim; Roberts, Keith; Hamilton, Andrew; Baulcombe, David C.; Fincher, Geoffrey B.

    2000-01-01

    Specific cDNA fragments corresponding to putative cellulose synthase genes (CesA) were inserted into potato virus X vectors for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing. Plants infected with one group of cDNAs had much shorter internode lengths, small leaves, and a “dwarf” phenotype. Consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacent to vascular tissues. Linkage analyses of wall polysaccharides prepared from infected leaves revealed a 25% decrease in cellulose content. Transcript levels for at least one member of the CesA cellulose synthase gene family were lower in infected plants. The decrease in cellulose content in cell walls was offset by an increase in homogalacturonan, in which the degree of esterification of carboxyl groups decreased from ∼50 to ∼33%. The results suggest that feedback loops interconnect the cellular machinery controlling cellulose and pectin biosynthesis. On the basis of the phenotypic features of the infected plants, changes in wall composition, and the reduced abundance of CesA mRNA, we concluded that the cDNA fragments silenced one or more cellulose synthase genes. PMID:10810144

  7. Applications and advantages of virus-induced gene silencing for gene function studies in plants.

    PubMed

    Burch-Smith, Tessa M; Anderson, Jeffrey C; Martin, Gregory B; Dinesh-Kumar, S P

    2004-09-01

    Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes. PMID:15315635

  8. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  9. Downregulation of plant genes with miRNA-induced gene silencing.

    PubMed

    de Felippes, Felipe Fenselau

    2013-01-01

    In plants, some microRNAs (miRNAs) can trigger the production of secondary small interfering RNAs (siRNAs) from their targets. miRNA-induced gene silencing (MIGS) exploits this unique feature to efficiently downregulate gene expression. The simple flanking of a sequence of interest with the target site for the miR173 (an miRNA able to trigger transitivity) is sufficient to start the production of secondary siRNAs and, consequently, silencing of the target gene. This technique can be easily adapted to promote gene silencing of more than one gene, even with those that share no sequence similarities. This chapter describes the necessary steps for designing and implementing the use of MIGS in plants.

  10. Gene silencing in Caenorhabditis elegans by transitive RNA interference

    PubMed Central

    ALDER, MATTHEW N.; DAMES, SHALE; GAUDET, JEFFREY; MANGO, SUSAN E.

    2003-01-01

    When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5′ to 3′ synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi. PMID:12554873

  11. Systematic identification of cis-silenced genes by trans complementation

    PubMed Central

    Lee, Jae Hyun; Bugarija, Branimir; Millan, Enrique J.; Walton, Noah M.; Gaetz, Jedidiah; Fernandes, Croydon J.; Yu, Wei-Hua; Mekel-Bobrov, Nitzan; Vallender, Tammy W.; Snyder, Gregory E.; Xiang, Andy Peng; Lahn, Bruce T.

    2009-01-01

    A gene’s transcriptional output is the combined product of two inputs: diffusible factors in the cellular milieu acting in trans, and chromatin state acting in cis. Here, we describe a strategy for dissecting the relative contribution of cis versus trans mechanisms to gene regulation. Referred to as trans complementation, it entails fusing two disparate cell types and searching for genes differentially expressed between the two genomes of fused cells. Any differential expression can be causally attributed to cis mechanisms because the two genomes of fused cells share a single homogenized milieu in trans. This assay uncovered a state of transcriptional competency that we termed ‘occluded’ whereby affected genes are silenced by cis-acting mechanisms in a manner that blocks them from responding to the trans-acting milieu of the cell. Importantly, occluded genes in a given cell type tend to include master triggers of alternative cell fates. Furthermore, the occluded state is maintained during cell division and is extraordinarily stable under a wide range of physiological conditions. These results support the model that the occlusion of lineage-inappropriate genes is a key mechanism of cell fate restriction. The identification of occluded genes by our assay provides a hitherto unavailable functional readout of chromatin state that is distinct from and complementary to gene expression status. PMID:19050040

  12. A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim.

    PubMed

    Villanueva-Alonzo, Hernan J; Us-Camas, Rosa Y; López-Ochoa, Luisa A; Robertson, Dominique; Guerra-Peraza, Orlene; Minero-García, Yereni; Moreno-Valenzuela, Oscar A

    2013-05-01

    Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

  13. RNA silencing as a tool to uncover gene function and engineer novel traits in soybean

    PubMed Central

    Kasai, Megumi; Kanazawa, Akira

    2012-01-01

    RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed. PMID:23136487

  14. Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica

    PubMed Central

    Morf, Laura; Pearson, Richard J.; Wang, Angelia S.; Singh, Upinder

    2013-01-01

    RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects. PMID:23935116

  15. Molecular mechanisms of RNA-triggered gene silencing machineries.

    PubMed

    Li, Zhonghan; Rana, Tariq M

    2012-07-17

    Gene silencing by RNA triggers is an ancient, evolutionarily conserved, and widespread phenomenon. This process, known as RNA interference (RNAi), occurs when double-stranded RNA helices induce cleavage of their complementary mRNAs. Because these RNA molecules can be introduced exogenously as small interfering RNAs (siRNAs), RNAi has become an everyday experimental tool in laboratory research. In addition, the number of RNA-based therapeutics that are currently in clinical trials for a variety of human diseases demonstrate the therapeutic potential of RNAi. In this Account, we focus on our current understanding of the structure and function of various classes of RNAi triggers and how this knowledge has contributed to our understanding of the biogenesis and catalytic functions of siRNA and microRNA in mammalian cells. Mechanistic studies to understand the structure and function of small RNAs that induce RNAi have illuminated broad functions of the ancient RNAi machinery in animals and plants. In addition, such studies have provided insight to identify endogenous physiological gene silencing RNA triggers that engage functional machineries similar to siRNAs. Several endogenous small RNA species have been identified: small noncoding RNAs (microRNAs), piwi-interacting RNAs (piRNAs), and endogenous siRNAs (endo-siRNAs). microRNAs are the most widespread class of small RNAs in mammalian cells. Despite their importance in biology and medicine, the molecular and cellular mechanisms of microRNA biogenesis and function are not fully understood. We provide an overview of the current understanding of how these molecules are synthesized within cells and how they act on gene targets. Interesting questions remain both for understanding the effects of modifications and editing on microRNAs and the interactions between microRNAs and other cellular RNAs such as long noncoding RNAs. PMID:22304792

  16. RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Pompey, Justine M.; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a “trigger” to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing. PMID:25198343

  17. RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

    PubMed

    Pompey, Justine M; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  18. Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

    PubMed

    Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

    2016-09-22

    Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. PMID:27662090

  19. Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

    PubMed Central

    2013-01-01

    Background Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Results Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. Conclusions These results indicate that F. oxysporum possesses

  20. RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

    PubMed Central

    2005-01-01

    The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos. PMID:15951844

  1. Simple gene silencing using the trans-acting siRNA pathway.

    PubMed

    Jacobs, Thomas B; Lawler, Noah J; LaFayette, Peter R; Vodkin, Lila O; Parrott, Wayne A

    2016-01-01

    In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis. Several ta-siRNA loci have been identified in soybean, but, prior to this work, few of the inducing miRNAs have been experimentally validated, much less used to silence genes. Nine ta-siRNA loci and their respective miRNA targets were identified, and the abundance of the inducing miRNAs varies dramatically in different tissues. The miRNA targets were experimentally verified by silencing a transgenic GFP gene and two endogenous genes in hairy roots and transgenic plants. Small RNAs were produced in patterns consistent with the utilization of the ta-siRNA pathway. A side-by-side experiment demonstrated that MIGS is as effective at inducing gene silencing as traditional hairpin vectors in soybean hairy roots. Soybean plants transformed with MIGS vectors produced siRNAs and silencing was observed in the T1 generation. These results complement previous reports in Arabidopsis by demonstrating that MIGS is an efficient way to produce siRNAs and induce gene silencing in other species, as shown with soybean. The miRNA targets identified here are simple to incorporate into silencing vectors and offer an effective and efficient alternative to other gene silencing strategies.

  2. Trans-Reactivation: A New Epigenetic Phenomenon Underlying Transcriptional Reactivation of Silenced Genes.

    PubMed

    Onorati, Maria Cristina; Arancio, Walter; Cavalieri, Vincenzo; Ingrassia, Antonia M R; Pavesi, Giulio; Corona, Davide F V

    2015-08-01

    In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA's and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes. PMID:26292210

  3. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    PubMed

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  4. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  5. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    PubMed

    Nakashima, Nobutaka; Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

    2014-01-01

    We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

  6. Heterologous virus-induced gene silencing as a promising approach in plant functional genomics.

    PubMed

    Hosseini Tafreshi, Seied Ali; Shariati, Mansour; Mofid, Mohammad Reza; Khayam Nekui, Mojtaba; Esmaeili, Abolghasem

    2012-03-01

    VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death. PMID:21655951

  7. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  8. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    PubMed

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

  9. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    PubMed

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  10. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

    PubMed Central

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang

    2016-01-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  11. Gene silencing triggered by non-LTR retrotransposons in the female germline of Drosophila melanogaster.

    PubMed Central

    Robin, Stéphanie; Chambeyron, Séverine; Bucheton, Alain; Busseau, Isabelle

    2003-01-01

    Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing. PMID:12807773

  12. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  13. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

    PubMed Central

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-01-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi. PMID:24594602

  14. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.

    PubMed

    Chantreau, Maxime; Chabbert, Brigitte; Billiard, Sylvain; Hawkins, Simon; Neutelings, Godfrey

    2015-12-01

    Flax (Linum usitatissimum) bast fibres are located in the stem cortex where they play an important role in mechanical support. They contain high amounts of cellulose and so are used for linen textiles and in the composite industry. In this study, we screened the annotated flax genome and identified 14 distinct cellulose synthase (CESA) genes using orthologous sequences previously identified. Transcriptomics of 'primary cell wall' and 'secondary cell wall' flax CESA genes showed that some were preferentially expressed in different organs and stem tissues providing clues as to their biological role(s) in planta. The development for the first time in flax of a virus-induced gene silencing (VIGS) approach was used to functionally evaluate the biological role of different CESA genes in stem tissues. Quantification of transcript accumulation showed that in many cases, silencing not only affected targeted CESA clades, but also had an impact on other CESA genes. Whatever the targeted clade, inactivation by VIGS affected plant growth. In contrast, only clade 1- and clade 6-targeted plants showed modifications in outer-stem tissue organization and secondary cell wall formation. In these plants, bast fibre number and structure were severely impacted, suggesting that the targeted genes may play an important role in the establishment of the fibre cell wall. Our results provide new fundamental information about cellulose biosynthesis in flax that should facilitate future plant improvement/engineering.

  15. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

    PubMed Central

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S.

    2016-01-01

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. PMID:26744421

  16. TRV Based Virus Induced Gene Silencing in Gladiolus (Gladiolus grandiflorus L.), A Monocotyledonous Ornamental Plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) has not yet successfully been used as a tool for gene functional analysis in non-grass monocotyledonous geophytes. We therefore tested VIGS in gladiolus (Gladiolus grandiflora L) using a Tobacco Rattle Virus (TRV) vector containing a fragment of the gladiolus gene...

  17. Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...

  18. Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

    PubMed

    Wang, J-E; Li, D-W; Gong, Z-H; Zhang, Y-L

    2013-07-24

    Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

  19. Virus-Induced Gene Silencing in Cultivated Cotton (Gossypium spp.) Using Tobacco Rattle Virus.

    PubMed

    Mustafa, Roma; Shafiq, Muhammad; Mansoor, Shahid; Briddon, Rob W; Scheffler, Brian E; Scheffler, Jodi; Amin, Imran

    2016-01-01

    The study described here has optimized the conditions for virus-induced gene silencing (VIGS) in three cultivated cotton species (Gossypium hirsutum, G. arboreum, and G. herbaceum) using a Tobacco rattle virus (TRV) vector. The system was used to silence the homolog of the Arabidopsis thaliana chloroplastos alterados 1 (AtCLA1) gene, involved in chloroplast development, in G. herbaceum, G. arboreum, and six commercial G. hirsutum cultivars. All plants inoculated with the TRV vector to silence CLA1 developed a typical albino phenotype indicative of silencing this gene. Although silencing in G. herbaceum and G. arboreum was complete, silencing efficiency differed for each G. hirsutum cultivar. Reverse transcriptase polymerase chain reaction (PCR) and real-time quantitative PCR showed a reduction in mRNA levels of the CLA1 homolog in all three species, with the highest efficiency (lowest CLA1 mRNA levels) in G. arboreum followed by G. herbaceum and G. hirsutum. The results indicate that TRV is a useful vector for VIGS in Gossypium species. However, selection of host cultivar is important. With the genome sequences of several cotton species recently becoming publicly available, this system has the potential to provide a very powerful tool for the rapid, large-scale reverse-genetic analysis of genes in Gossypium spp.

  20. Multiple silencer elements are involved in regulating the chicken vimentin gene.

    PubMed Central

    Garzon, R J; Zehner, Z E

    1994-01-01

    Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well. Images PMID:8289833

  1. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants1[OPEN

    PubMed Central

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo

    2016-01-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  2. RNAi mediated gene silencing against betasatellite associated with Croton yellow vein mosaic begomovirus.

    PubMed

    Sahu, Anurag Kumar; Marwal, Avinash; Nehra, Chitra; Choudhary, Devendra Kumar; Sharma, Pradeep; Gaur, Rajarshi Kumar

    2014-11-01

    Plant viruses encode suppressors of posttranscriptional gene silencing, an adaptive antiviral defense responses that confines virus infection. Previously, we identified single-stranded DNA satellite (also known as DNA-β) of ~1,350 nucleotides in length associated with Croton yellow vein mosaic begomovirus (CYVMV) in croton plants. The expression of genes from DNA-β requires the begomovirus for packaged, replication, insect transmission and movement in plants. The present study demonstrates the effect of the βC1 gene on the silencing pathway as analysed by using both transgenic systems and transient Agrobacterium tumefaciens based delivery. Plants that carry an intron-hairpin construct covering the βC1 gene accumulated cognate small-interfering RNAs and remained symptom-free after exposure to CYVMV and its satellite. These results suggest that βC1 interferes with silencing mechanism. PMID:25086625

  3. Gene transfer engineering for astrocyte-specific silencing in the CNS.

    PubMed

    Merienne, N; Delzor, A; Viret, A; Dufour, N; Rey, M; Hantraye, P; Déglon, N

    2015-10-01

    Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications. PMID:26109254

  4. Gene transfer engineering for astrocyte-specific silencing in the CNS.

    PubMed

    Merienne, N; Delzor, A; Viret, A; Dufour, N; Rey, M; Hantraye, P; Déglon, N

    2015-10-01

    Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.

  5. RNAi mediated gene silencing against betasatellite associated with Croton yellow vein mosaic begomovirus.

    PubMed

    Sahu, Anurag Kumar; Marwal, Avinash; Nehra, Chitra; Choudhary, Devendra Kumar; Sharma, Pradeep; Gaur, Rajarshi Kumar

    2014-11-01

    Plant viruses encode suppressors of posttranscriptional gene silencing, an adaptive antiviral defense responses that confines virus infection. Previously, we identified single-stranded DNA satellite (also known as DNA-β) of ~1,350 nucleotides in length associated with Croton yellow vein mosaic begomovirus (CYVMV) in croton plants. The expression of genes from DNA-β requires the begomovirus for packaged, replication, insect transmission and movement in plants. The present study demonstrates the effect of the βC1 gene on the silencing pathway as analysed by using both transgenic systems and transient Agrobacterium tumefaciens based delivery. Plants that carry an intron-hairpin construct covering the βC1 gene accumulated cognate small-interfering RNAs and remained symptom-free after exposure to CYVMV and its satellite. These results suggest that βC1 interferes with silencing mechanism.

  6. Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

    PubMed Central

    Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

    2013-01-01

    Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum. PMID:24023833

  7. Induction of stable epigenetic gene silencing in plants using a virus vector.

    PubMed

    Kanazawa, Akira; Kasai, Megumi

    2015-01-01

    Gene silencing through transcriptional repression can be induced by double-stranded RNA targeted to a gene promoter, a process known as RNA-mediated transcriptional gene silencing (TGS). This phenomenon is associated with epigenetic changes involving cytosine methylation of the promoter. Plant virus vectors have been used to induce RNA-mediated TGS. Here, we describe methods relevant to the induction of epigenetic changes and RNA-mediated TGS in plants using a virus vector, which include inoculation of recombinant virus, detection of short interfering RNAs, bisulfite sequencing analysis, and nuclear run-on transcription assay. PMID:25740361

  8. Silencing of six hydrophobins in Cladosporium fulvum: complexities of simultaneously targeting multiple genes.

    PubMed

    Lacroix, Hélène; Spanu, Pietro D

    2009-01-01

    In this study, we have constructed and expressed inverted repeat chimeras from the first exons of the six known hydrophobins of the fungus Cladosporium fulvum, the causal agent of tomato leaf mold. We used quantitative PCR to measure specifically the expression levels of the hydrophobins. The targeted genes are silenced to different degrees, but we also detected clear changes in the expression levels of nontargeted genes. This work highlights the difficulties that are likely to be encountered when attempting to silence more than one gene in a multigene family.

  9. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  10. Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing.

    PubMed

    Yamamoto-Hino, Miki; Yoshida, Hideki; Ichimiya, Tomomi; Sakamura, Sho; Maeda, Megumi; Kimura, Yoshinobu; Sasaki, Norihiko; Aoki-Kinoshita, Kiyoko F; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Ueda, Ryu; Nishihara, Shoko; Goto, Satoshi

    2015-06-01

    Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes. PMID:25940448

  11. Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing

    PubMed Central

    Yamamoto-Hino, Miki; Yoshida, Hideki; Ichimiya, Tomomi; Sakamura, Sho; Maeda, Megumi; Kimura, Yoshinobu; Sasaki, Norihiko; Aoki-Kinoshita, Kiyoko F; Kinoshita-Toyoda, Akiko; Toyoda, Hidenao; Ueda, Ryu; Nishihara, Shoko; Goto, Satoshi

    2015-01-01

    Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes. PMID:25940448

  12. Cationic liposome-nucleic acid nanoparticle assemblies with applications in gene delivery and gene silencing.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-07-28

    Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'. PMID:27298431

  13. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    PubMed

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  14. Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions.

    PubMed

    Bucher, Etienne; Sijen, Titia; De Haan, Peter; Goldbach, Rob; Prins, Marcel

    2003-01-01

    Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the BUNYAVIRIDAE: Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS(S) gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS(S)-expressing leaf sectors suggests that the tospoviral NS(S) protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing. PMID:12502849

  15. RNA-mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus.

    PubMed

    Leng, Yueqiang; Wu, Chengxiang; Liu, Zhaohui; Friesen, Timothy L; Rasmussen, Jack B; Zhong, Shaobin

    2011-04-01

    A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.

  16. Dissection of Tomato Lycopene Biosynthesis through Virus-Induced Gene Silencing1[C][W][OPEN

    PubMed Central

    Fantini, Elio; Falcone, Giulia; Frusciante, Sarah; Giliberto, Leonardo; Giuliano, Giovanni

    2013-01-01

    Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and ζ-carotene desaturase [ZDS]), and two cis-trans isomerases (ζ-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9’-di-cis-ζ-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-ζ-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes. PMID:24014574

  17. RAP1 Is Essential for Silencing Telomeric Variant Surface Glycoprotein Genes in Trypanosoma brucei

    PubMed Central

    Yang, Xiaofeng; Figueiredo, Luisa M.; Espinal, Amin; Okubo, Eiji; Li, Bibo

    2009-01-01

    SUMMARY Trypanosoma brucei expresses Variant Surface Glycoprotein (VSG) genes in a strictly monoallelic fashion in its mammalian hosts, but it is unclear how this important virulence mechanism is enforced. Telomere position effect (TPE), an epigenetic phenomenon, has been proposed to play a critical role in VSG regulation, yet no telomeric protein has been identified whose disruption led to VSG derepression. We now identify tbRAP1 as an intrinsic component of the T. brucei telomere complex and a major regulator for silencing VSG expression sites (ESs). Knockdown of tbRAP1 led to derepression of all VSGs in silent ESs, but not VSGs located elsewhere, and resulted in stronger derepression of genes located within 10 kb from telomeres than genes located further upstream. This graduated silencing pattern suggests that telomere integrity plays a key role in tbRAP1-dependent silencing and VSG regulation. PMID:19345190

  18. Autonomous silencing of the imprinted Cdkn1c gene in stem cells

    PubMed Central

    Wood, Michelle D.; Hiura, Hitoshi; Tunster, Simon; Arima, Takahiro; Shin, Jong-Yeon; Higgins, Michael; John, Rosalind M.

    2010-01-01

    Parent-of-origin specific expression of imprinted genes relies on the differential DNA methylation of specific genomic regions. Differentially methylated regions (DMRs) acquire DNA methylation either during gametogenesis (primary DMR) or after fertilization when allele-specific expression is established (secondary DMR). Little is known about the function of these secondary DMRs. We investigated the DMR spanning Cdkn1c in mouse embryonic stem cells, androgenetic stem cells and embryonic germ stem cells. In all cases, expression of Cdkn1c was appropriately repressed in in vitro differentiated cells. However, stem cells failed to de novo methylate the silenced gene even after sustained differentiation. In the absence of maintained DNA methylation (Dnmt1−/−), Cdkn1c escapes silencing demonstrating the requirement for DNA methylation in long term silencing in vivo. We propose that post-fertilization differential methylation reflects the importance of retaining single gene dosage of a subset of imprinted loci in the adult. PMID:20372090

  19. Persistent virus-induced gene silencing in asymptomatic accessions of Arabidopsis.

    PubMed

    Flores, Miguel A; Reyes, Maria I; Robertson, Dominique Niki; Kjemtrup, Susanne

    2015-01-01

    Coupled with the advantages afforded by the model plant Arabidopsis, virus-induced gene silencing (VIGS) offers a rapid means to assess gene function. The geminivirus vector based on Cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. Here, we show that genetic variation in the vast collection of Arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the VIGS procedure. The plasticity of phenotypes under different day lengths or temperature conditions can be exploited to achieve maximum silencing efficacy in either vegetative or inflorescence tissue, according to the question being asked. Protocols and vectors for Agro-infiltration of primary leaves, subapical pricking in older plants, and microprojectile bombardment are described. PMID:25757779

  20. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

  1. Efficient gene silencing in mesenchymal stem cells by substrate-mediated RNA interference.

    PubMed

    Hsu, Shan-Hui; Huang, Guo-Shiang; Ho, Tung-Tso; Feng, Fuh

    2014-11-01

    We described a novel substrate-mediated RNA interference (RNAi) technology to investigate the effect of neural crest marker expression on the multipotency of human gingival fibroblasts (HGFs). HGFs showed significantly higher neural and chondrogenic differentiation potentials compared with adult bone-marrow-derived mesenchymal stem cells and stem cells from human exfoliated deciduous teeth. By sending target-specific RNAi agents with the conventional vehicle (PolyFect), we observed that the multipotency of HGFs was closely associated with the expression of neural crest marker gene Forkhead box D3 (FoxD3). Using the novel chitosan substrate-mediated method, we successfully delivered short-hairpin RNA constructs to HGFs grown on chitosan without the use of conventional vehicles. The delivery efficiency measured by flow cytometry showed a 10-fold increase for HGFs on chitosan versus those on culture dish, and the cell viability was >95%. Moreover, HGFs with FoxD3 gene knockdown did not form spheroids on chitosan. Based on this working principle, we further selected the gene-silenced population from HGFs. The nonsilenced HGFs showed much higher neural differentiation ability with the nestin expression 40-fold greater than FoxD3-silenced population after induction, suggesting the feasibility of the method to silence genes. The new substrate-mediated gene silencing platform that combines the use of substrate and RNAi can be used to clarify the functions of important genes without suffering the toxicity. PMID:24624901

  2. Silencing of host genes directed by virus-derived short interfering RNAs in Caenorhabditis elegans.

    PubMed

    Guo, Xunyang; Li, Wan-Xiang; Lu, Rui

    2012-11-01

    Small interfering RNAs (siRNAs) processed from viral replication intermediates by RNase III-like enzyme Dicer guide sequence-specific antiviral silencing in fungi, plants, and invertebrates. In plants, virus-derived siRNAs (viRNAs) can target and silence cellular transcripts and, in some cases, are responsible for the induction of plant diseases. Currently it remains unclear whether viRNAs are also capable of modulating the expression of cellular genes in the animal kingdom, although animal virus-encoded microRNAs (miRNAs) are known to guide efficient silencing of host genes, thereby facilitating virus replication. In this report, we showed that viRNAs derived from a modified nodavirus triggered potent silencing of homologous cellular transcripts produced by the endogenous gene or transgene in the nematode worm Caenorhabditis elegans. Like that found in plants, virus-induced gene silencing (VIGS) in C. elegans also involves RRF-1, a worm RNA-dependent RNA polymerase (RdRP) that is known to produce single-stranded secondary siRNAs in a Dicer-independent manner. We further demonstrated that VIGS in C. elegans is inheritable, suggesting that VIGS has the potential to generate profound epigenetic consequences in future generations. Altogether, these findings, for the first time, confirmed that viRNAs have the potential to modulate host gene expression in the animal kingdom. Most importantly, the success in uncoupling the trigger and the target of the antiviral silencing would allow for the exploration of novel features of virus-host interactions mediated by viRNAs in the animal kingdom. PMID:22896621

  3. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    PubMed Central

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  4. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    PubMed

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  5. Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to P. pachyrhizi, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression i...

  6. Structure Tuning of Cationic Oligospermine-siRNA Conjugates for Carrier-Free Gene Silencing.

    PubMed

    Nothisen, Marc; Bagilet, Jérémy; Behr, Jean-Paul; Remy, Jean-Serge; Kotera, Mitsuharu

    2016-08-01

    Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand. PMID:27398779

  7. Modification of Seed Oil Composition in Arabidopsis by Artificial microRNA-Mediated Gene Silencing

    PubMed Central

    Belide, Srinivas; Petrie, James Robertson; Shrestha, Pushkar; Singh, Surinder Pal

    2012-01-01

    Various post transcriptional gene silencing strategies have been developed and exploited to study gene function or engineer disease resistance. The recently developed artificial microRNA strategy is an alternative method of effectively silencing target genes. The Δ12-desaturase (FAD2), Fatty acid elongase (FAE1), and Fatty acyl-ACP thioesterase B (FATB) were targeted with amiR159b-based constructs in Arabidopsis thaliana to evaluate changes in oil composition when expressed with the seed-specific Brassica napus truncated napin (FP1) promoter. Fatty acid profiles from transgenic homozygous seeds reveal that the targeted genes were silenced. The down-regulation of the AtFAD-2 gene substantially increased oleic acid from the normal levels of ∼15% to as high as 63.3 and reduced total PUFA content (18:2Δ9,12 + 18:3Δ9,12,15 + 20:2Δ11,14 + 20:3Δ11,14,17) from 46.8 to 4.8%. Δ12-desaturase activity was reduced to levels as low as those in the null fad-2-1 and fad-2-2 mutants. Silencing of the FAE1 gene resulted in the reduction of eicosenoic acid (20:1Δ11) to 1.9 from 15.4% and silencing of FATB resulted in the reduction of palmitic acid (16:0) to 4.4% from 8.0%. Reduction in FATB activity is comparable with a FATB knock-out mutant. These results demonstrate for the first time amiR159b constructs targeted against three endogenous seed-expressed genes are clearly able to down-regulate and generate genotypic changes that are inherited stably over three generations. PMID:22866055

  8. Gene duplication in tetraploid fish: model for gene silencing at unlinked duplicated loci.

    PubMed Central

    Bailey, G S; Poulter, R T; Stockwell, P A

    1978-01-01

    Several groups of fishes, including salmonids and catastomids, appear to have originated through genome duplication events. However, these two groups retain approximately 50% of the loci examined as functioning duplicates, despite the passage of 50 million years or more of mutation and selection. Although other effects are not excluded, this apparently slow rate of duplicate silencing can be explained in terms of the effects of selection against defective double homozygotes to unlinked duplicates. We have derived a computer simulation of genetic drift that affords direct evaluation of the effects of population size (N), mutation rate (micron), initial allele frequencies, back mutation, fitness, and time on the probability of fixation for null alleles at unlinked duplicate loci. The results show that this probability is approximately linearly related to population size for N greater than or equal to 10(3). Specifically, for naive populations, the time for 50% probability of gene silencing is approximately equal to 15N + micron-3/4 generations. The retention of 50% of the loci as functional duplicates may therefore result from the large effective size of salmonid and catastomid populations. The results also show that, under most conditions for populations of 2000--3000 or larger, unlinked duplicate loci will be sustained in the functional state longer than tandem (linked) duplicates and hence are available for evolution of new functions for a longer time. PMID:281706

  9. RNA silencing of genes involved in Alzheimer's disease enhances mitochondrial function and synaptic activity.

    PubMed

    Manczak, Maria; Reddy, P Hemachandra

    2013-12-01

    An age-dependent increase in mRNA levels of the amyloid precursor protein (APP), the microtubule-associated protein Tau, and voltage-dependent anion channel 1 (VDAC1) genes are reported to be toxic to neurons affected by Alzheimer's disease (AD). However, the underlying toxic nature of these genes is not completely understood. The purpose of our study was to determine the effects of RNA silencing of APP, Tau, and VDAC1 genes in AD pathogenesis. Using human neuroblastoma (SHSY5Y) cells, we first silenced RNA for APP, Tau, and VDAC1 genes, and then performed real-time RT-PCR analysis to measure mRNA levels of 34 genes that are involved in AD pathogenesis. Using biochemical assays, we also assessed mitochondrial function by measuring levels of H2O2 production, lipid peroxidation, cytochrome c oxidase activity, ATP production, and GTPase enzymatic activity. We found that increased mRNA expression of synaptic function and mitochondrial fission genes, and reduced levels of mitochondrial fusion genes in RNA silenced the SHSY5Y cells for APP, Tau and VDAC1 genes relative to the control SHSY5Y cells. In addition, RNA-silenced APP, Tau, and VDAC1 genes in SHSY5Y cells showed reduced levels of H2O2 production, lipid peroxidation, fission-linked GTPase activity, and increased cytochrome oxidase activity and ATP production. These findings suggest that a reduction of human APP, Tau, and VDAC1 may enhance synaptic activity, may improve mitochondrial maintenance and function, and may protect against toxicities of AD-related genes. Thus, these findings also suggest that the reduction of APP, Tau, and VDAC1 mRNA expressions may have therapeutic value for patients with AD.

  10. Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

    PubMed

    Barry, Gerald; Alberdi, Pilar; Schnettler, Esther; Weisheit, Sabine; Kohl, Alain; Fazakerley, John K; Bell-Sakyi, Lesley

    2013-03-01

    Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

  11. Systematic knockdown of morphine pathway enzymes in opium poppy using virus-induced gene silencing.

    PubMed

    Wijekoon, Champa P; Facchini, Peter J

    2012-03-01

    Opium poppy (Papaver somniferum) remains the sole commercial source for several pharmaceutical alkaloids including the narcotic analgesics codeine and morphine, and the semi-synthetic drugs oxycodone, buprenorphine and naltrexone. Although most of the biosynthetic genes have been identified, the post-transcriptional regulation of the morphinan alkaloid pathway has not been determined. We have used virus-induced gene silencing (VIGS) as a functional genomics tool to investigate the regulation of morphine biosynthesis via a systematic reduction in enzyme levels responsible for the final six steps in the pathway. Specific gene silencing was confirmed at the transcript level by real-time quantitative PCR (polymerase chain reaction), and at the protein level by immunoblot analysis using antibodies raised against salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridine 7-O-acetyltransferase (SalAT), thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), and codeine O-demethylase (CODM). In some cases, silencing a specific biosynthetic gene resulted in a predictable accumulation of the substrate for the corresponding enzyme. Reduced SalSyn, SalR, T6ODM and CODM protein levels correlated with lower morphine levels and a substantial increase in the accumulation of reticuline, salutaridine, thebaine and codeine, respectively. In contrast, the silencing of genes encoding SalAT and COR resulted in the accumulation of salutaridine and reticuline, respectively, which are not the corresponding enzymatic substrates. The silencing of alkaloid biosynthetic genes using VIGS confirms the physiological function of enzymes previously characterized in vitro, provides insight into the biochemical regulation of morphine biosynthesis, and demonstrates the immense potential for metabolic engineering in opium poppy.

  12. A viral satellite DNA vector-induced transcriptional gene silencing via DNA methylation of gene promoter in Nicotiana benthamiana.

    PubMed

    Ju, Zheng; Wang, Lei; Cao, Dongyan; Zuo, Jinhua; Zhu, Hongliang; Fu, Daqi; Luo, Yunbo; Zhu, Benzhong

    2016-09-01

    Virus-induced gene silencing (VIGS) has been widely used for plant functional genomics study at the post-transcriptional level using various DNA or RNA viral vectors. However, while virus-induced transcriptional gene silencing (VITGS) via DNA methylation of gene promoter was achieved using several plant RNA viral vectors, it has not yet been done using a satellite DNA viral vector. In this study, a viral satellite DNA associated with tomato yellow leaf curl China virus (TYLCCNV), which has been modified as a VIGS vector in previous research, was developed as a VITGS vector. Firstly, the viral satellite DNA VIGS vector was further optimized to a more convenient p1.7A+2mβ vector with high silencing efficiency of the phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. Secondly, the constructed VITGS vector (TYLCCNV:35S), which carried a portion of the cauliflower mosaic virus 35S promoter, could successfully induce heritable transcriptional gene silencing (TGS) of the green fluorescent protein (GFP) gene in the 35S-GFP transgenic N. benthamiana line 16c plants. Moreover, bisulfite sequencing results revealed higher methylated cytosine residues at CG, CHG and CHH sites of the 35S promoter sequence in TYLCCNV:35S-inoculated plants than in TYLCCNV-inoculated line 16c plants (control). Overall, these results demonstrated that the viral satellite DNA vector could be used as an effective VITGS vector to study DNA methylation in plant genomes. PMID:27422476

  13. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  14. Gene silencing of VP9 gene impairs WSSV infectivity on Macrobrachium rosenbergii.

    PubMed

    Alenton, Rod Russel R; Kondo, Hidehiro; Hirono, Ikuo; Maningas, Mary Beth B

    2016-03-01

    White Spot Syndrome Virus (WSSV) remains the most widespread and devastating infectious agent that hit the shrimp aquaculture industry worldwide. To date, there are no known effective strategies yet to combat WSSV infection. Hence, functional studies on genes critical for viral infection is essential in elucidating shrimp-virus interaction. Here we report the function of a gene from WSSV coding for a non-structural protein, VP9, utilizing RNA interference. Silencing of VP9 gene also effectively suppressed other gene region in the WSSV genome (wsv168 gene) as early as day 1 post infection (dpi). Three set-ups using Macrobrachium rosenbergii shrimp were prepared for treatment using VP9-dsRNA, GFP-dsRNA, and PBS. Each shrimp was challenge with WSSV, and survival rate was recorded. VP9- and GFP-dsRNA injected shrimps showed a significant survival rate of 80% and 70%, respectively, in contrast to 0% of the PBS injected shrimps at 25dpi. Re-infection of shrimp survivors using a higher viral titer concentration, concurrent with the infection of new shrimp samples for the PBS control group, resulted in a significant 67% survival rate for VP9-dsRNA compared to 0% with that of GFP-dsRNA and PBS group. Challenge test on two more species, Penaeus monodon and Marsupenaeus japonicus, also significantly increased survival after VP9-dsRNA treatment. Our results provided evidence that VP9 gene plays an essential role in WSSV replication and it can be a potent target gene in the development of RNAi therapeutics for shrimps.

  15. Biological and clinical significance of epigenetic silencing of MARVELD1 gene in lung cancer

    PubMed Central

    Shi, Ming; Wang, Shan; Yao, Yuanfei; Li, Yiqun; Zhang, Hao; Han, Fang; Nie, Huan; Su, Jie; Wang, Zeyu; Yue, Lei; Cao, Jingyan; Li, Yu

    2014-01-01

    Epigenetic silence in cancer frequently altered signal-transduction pathways during the early stages of tumor development. Recent progress in the field of cancer epigenetics has led to new opportunities for diagnosis and treatment of cancer. We previously demonstrated that novel identified nuclear factor MARVELD1 was widely expressed in human tissues, but down-regulated by promoter methylation in multiple cancers. This study was carried out to determine the biological and clinical significance of MARVELD1 gene silencing in lung cancer. Here, we found the reduced MARVELD1 expression significantly correlated with diagnostic histopathology and malignant degree of lung cancers. DNA hypermethylation and histone deacetylation synergistically inactivated MARVELD1 gene in lung cancer cells. Moreover, MARVELD1 modulated the efficiency of nonsense-mediated mRNA decay (NMD) through interaction with NMD core factor SMG1. The decreased MARVELD1 level in lung cancer reduces NMD efficiency through diminishing the association between NMD complex component UPF1/SMG1 and premature termination codons containing mRNA (PTC-mRNA). The results suggested that MARVELD1 silencing is an appealing diagnostic biomarker for lung cancer and epigenetic silencing of MARVELD1 gene links with the regulatory mechanism of NMD pathway in lung cancer, which may be required for tumorigenesis. PMID:25520033

  16. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

    PubMed Central

    2014-01-01

    Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred

  17. Host-induced silencing of Fusarium culmorum genes protects wheat from infection.

    PubMed

    Chen, Wanxin; Kastner, Christine; Nowara, Daniela; Oliveira-Garcia, Ely; Rutten, Twan; Zhao, Yusheng; Deising, Holger B; Kumlehn, Jochen; Schweizer, Patrick

    2016-09-01

    Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant-fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the β-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes. PMID:27540093

  18. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

    PubMed Central

    Mei, Yu; Kernodle, Bliss M.; Hill, John H.

    2016-01-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  19. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

    PubMed

    Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

    2016-06-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  20. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

    PubMed

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

  1. Host-induced silencing of Fusarium culmorum genes protects wheat from infection

    PubMed Central

    Chen, Wanxin; Kastner, Christine; Nowara, Daniela; Oliveira-Garcia, Ely; Rutten, Twan; Zhao, Yusheng; Deising, Holger B.; Kumlehn, Jochen; Schweizer, Patrick

    2016-01-01

    Plants producing antisense or double-stranded RNA molecules that target specific genes of eukaryotic pests or pathogens can become protected from their attack. This beneficial effect was also reported for plant–fungus interactions and is believed to reflect uptake of the RNAs by the fungus via an as yet unknown mechanism, followed by target gene silencing. Here we report that wheat plants pre-infected with Barley stripe mosaic virus (BSMV) strains containing antisense sequences against target genes of the Fusarium head blight (FHB) fungus F. culmorum caused a reduction of corresponding transcript levels in the pathogen and reduced disease symptoms. Stable transgenic wheat plants carrying an RNAi hairpin construct against the β-1, 3-glucan synthase gene FcGls1 of F. culmorum or a triple combination of FcGls1 with two additional, pre-tested target genes also showed enhanced FHB resistance in leaf and spike inoculation assays under greenhouse and near-field conditions, respectively. Microscopic evaluation of F. culmorum development in plants transiently or stably expressing FcGls1 silencing constructs revealed aberrant, swollen fungal hyphae, indicating severe hyphal cell wall defects. The results lead us to propose host-induced gene silencing (HIGS) as a plant protection approach that may also be applicable to highly FHB-susceptible wheat genotypes. PMID:27540093

  2. Transvection and silencing of the Scr homeotic gene of Drosophila melanogaster.

    PubMed

    Southworth, Jeffrey W; Kennison, James A

    2002-06-01

    The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.

  3. Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

    PubMed

    Hansen, Klavs R; Burns, Gavin; Mata, Juan; Volpe, Thomas A; Martienssen, Robert A; Bähler, Jürg; Thon, Geneviève

    2005-01-01

    Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing. PMID:15632061

  4. Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

    SciTech Connect

    Filichkin, Sergei A; DiFazio, Steven P; Brunner, Amy M; Davis, John M; Yang, Zamin Koo; Kalluri, Udaya C; Arias, Renee S; Etherington, Elizabeth; Tuskan, Gerald A; Strauss, S

    2007-01-01

    We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.

  5. Cytosine Methylation Associated with Repeat-Induced Point Mutation Causes Epigenetic Gene Silencing in Neurospora Crassa

    PubMed Central

    Irelan, J. T.; Selker, E. U.

    1997-01-01

    Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-1) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(-4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns. PMID:9178002

  6. Templated assembly of albumin-based nanoparticles for simultaneous gene silencing and magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Mertz, Damien; Affolter-Zbaraszczuk, Christine; Barthès, Julien; Cui, Jiwei; Caruso, Frank; Baumert, Thomas F.; Voegel, Jean-Claude; Ogier, Joelle; Meyer, Florent

    2014-09-01

    In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing. Electronic supplementary information (ESI) available: Experimental details and supporting Fig. S1-S4. See DOI: 10.1039/c4nr02623c

  7. Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion

    PubMed Central

    Chen, Haiying; Sun, Manni; Liu, Jing; Tong, Chunxiao; Meng, Tao

    2015-01-01

    Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1. PMID:26680220

  8. Role of Inner Nuclear Membrane Protein Complex Lem2-Nur1 in Heterochromatic Gene Silencing.

    PubMed

    Banday, Shahid; Farooq, Zeenat; Rashid, Romana; Abdullah, Ehsaan; Altaf, Mohammad

    2016-09-16

    Heterochromatin in the fission yeast Schizosaccharomyces pombe is clustered at the nuclear periphery and interacts with a number of nuclear membrane proteins. However, the significance and the factors that sequester heterochromatin at the nuclear periphery are not fully known. Here, we report that an inner nuclear membrane protein complex Lem2-Nur1 is essential for heterochromatin-mediated gene silencing. We found that Lem2 is physically associated with another inner nuclear membrane protein, Nur1, and deletion of either lem2 or nur1 causes silencing defect at centromeres, telomeres, and rDNA loci. We analyzed the genome-wide association of Lem2 using ChIP sequencing and we found that it binds to the central core region of centromeres, in striking contrast to Chp1, a component of pericentromeric heterochromatin, which binds H3K9me-rich chromatin in neighboring sequences. The recruitment of Lem2 and Nur1 to silent regions of the genome is dependent on H3K9 methyltransferase, Clr4. Finally, we show that the Lem2-Nur1 complex regulates the local balance between the underln]Snf2/HDAC-containing repressor complex (SHREC) histone deacetylase complex and the anti-silencing protein Epe1. These findings uncover a novel role for Lem2-Nur1 as a key functional link between localization at the nuclear periphery and heterochromatin-mediated gene silencing.

  9. An efficient virus-induced gene silencing vector for maize functional genomics research.

    PubMed

    Wang, Rong; Yang, Xinxin; Wang, Nian; Liu, Xuedong; Nelson, Richard S; Li, Weimin; Fan, Zaifeng; Zhou, Tao

    2016-04-01

    Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize. PMID:26921244

  10. Gene Silencing and Haploinsufficiency of Csk Increase Blood Pressure

    PubMed Central

    Kim, Sung-Moon; Ji, Su-Min; Park, So-Yon; Kim, Marina E.; Jigden, Baigalmaa; Lim, Ji Eun; Hwang, Sue-Yun; Lee, Young-Ho; Oh, Bermseok

    2016-01-01

    Objective Recent genome-wide association studies have identified 33 human genetic loci that influence blood pressure. The 15q24 locus is one such locus that has been confirmed in Asians and Europeans. There are 21 genes in the locus within a 1-Mb boundary, but a functional link of these genes to blood pressure has not been reported. We aimed to identify a causative gene for blood pressure change in the 15q24 locus. Methods and Results CSK and ULK3 were selected as candidate genes based on eQTL analysis studies that showed the association between gene transcript levels and the lead SNP (rs1378942). Injection of siRNAs for mouse homologs Csk, Ulk3, and Cyp1a2 (negative control) showed reduced target gene mRNA levels in vivo. However, Csk siRNA only increased blood pressure while Ulk3 and Cyp1a2 siRNA did not change it. Further, blood pressure in Csk+/- heterozygotes was higher than in wild-type, consistent with what we observed in Csk siRNA-injected mice. We confirmed that haploinsufficiency of Csk increased the active form of Src in Csk+/- mice aorta. We also showed that inhibition of Src by PP2, a Src inhibitor decreased high blood pressure in Csk+/- mice and the active Src in Csk+/- mice aorta and in Csk knock-down vascular smooth muscle cells, suggesting blood pressure regulation by Csk through Src. Conclusions Our study demonstrates that Csk is a causative gene in the 15q24 locus and regulates blood pressure through Src, and these findings provide a novel therapeutic target for the treatment of hypertension. PMID:26751575

  11. Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity

    PubMed Central

    Yamoutpour, Farnaz; Bodempudi, Vidya; Park, Shay E.; Pan, Weihong; Mauzy, Mary Jean; Kratzke, Robert A.; Dudek, Arkadiusz; Potter, David A.; Woo, Richard A.; O’Rourke, Donald M.; Tindall, Donald J.; Farassati, Faris

    2009-01-01

    Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87Δ) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87Δ cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signal-regulated kinase. A lentivirus capable of expressing anti-EGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87Δ cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Δ cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, β-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Δ cells with a decrease in G1 and increase in S and G2 fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Δ cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. PMID:19001441

  12. GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

    PubMed

    Matsumoto, Yukiko; Hattori, Makoto

    2016-03-01

    RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species. PMID:26728387

  13. GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

    PubMed

    Matsumoto, Yukiko; Hattori, Makoto

    2016-03-01

    RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species.

  14. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    PubMed

    Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

    2015-01-01

    In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  15. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    PubMed Central

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  16. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles.

    PubMed

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2008-02-18

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed that the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 microg siRNA/kg). This effective formulation also showed very little immunotoxicity.

  17. Efficient Gene Silencing in Metastatic Tumor by siRNA Formulated in Surface-modified Nanoparticles

    PubMed Central

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2009-01-01

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene, in a lung metastasis model. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 μg siRNA/kg). This effective formulation also showed very little immunotoxicity. PMID:18083264

  18. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    PubMed

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  19. Role of RUNX family members in transcriptional repression and gene silencing.

    PubMed

    Durst, Kristie L; Hiebert, Scott W

    2004-05-24

    RUNX family members are DNA-binding transcription factors that regulate the expression of genes involved in cellular differentiation and cell cycle progression. The RUNX family includes three mammalian RUNX proteins (RUNX1, -2, -3) and two homologues in Drosophila. Experiments in Drosophila and mouse indicate that the RUNX proteins are required for gene silencing of engrailed and CD4, respectively. RUNX-mediated repression involves recruitment of corepressors such as mSin3A and Groucho as well as histone deacetylases. Furthermore, RUNX1 and RUNX3 associate with SUV39H1, a histone methyltransferase involved in gene silencing. RUNX1 is frequently targeted in human leukemia by chromosomal translocations that fuse the DNA-binding domain of RUNX1 to other transcription factors and corepressor molecules. The resulting leukemogenic fusion proteins are transcriptional repressors that form stable complexes with corepressors, histone deacetylases and histone methyltransferases. Thus, transcriptional repression and gene silencing through RUNX1 contribute to the mechanisms of leukemogenesis of the fusion proteins. Therapies directed at the associated cofactors may be beneficial for treatment of these leukemias. PMID:15156176

  20. Gene silencing by artificial microRNAs in Chlamydomonas.

    PubMed

    Zhao, Tao; Wang, Wei; Bai, Xue; Qi, Yijun

    2009-04-01

    Chlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of the chloroplast, basal body and flagella. The completion of the Chlamydomonas genome sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down-regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA-based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant phenotypes. We further showed that the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome-wide approaches.

  1. The insulation of genes from external enhancers and silencing chromatin

    PubMed Central

    Burgess-Beusse, Bonnie; Farrell, Catherine; Gaszner, Miklos; Litt, Michael; Mutskov, Vesco; Recillas-Targa, Felix; Simpson, Melanie; West, Adam; Felsenfeld, Gary

    2002-01-01

    Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5′ end of the chicken β-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore. PMID:12154228

  2. MR VIGS: microRNA-based virus-induced gene silencing in plants.

    PubMed

    Chen, Weiwei; Zhang, Qi; Kong, Junhua; Hu, Feng; Li, Bin; Wu, Chaoqun; Qin, Cheng; Zhang, Pengcheng; Shi, Nongnong; Hong, Yiguo

    2015-01-01

    In plants, microRNA (miRNA)-based virus-induced gene silencing, dubbed MR VIGS, is a powerful technique to delineate the biological functions of genes. By targeting to a specific sequence, miRNAs can knock down expression of genes with fewer off-target effects. Here, using a modified Cabbage leaf curling virus (CaLCuV) and Tobacco rattle virus (TRV) as vectors, we describe two virus-based miRNA expression systems to perform MR VIGS for plant functional genomics assays. PMID:25740363

  3. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

    PubMed

    Garbian, Yael; Maori, Eyal; Kalev, Haim; Shafir, Sharoni; Sela, Ilan

    2012-12-01

    The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

  4. Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population

    PubMed Central

    Kalev, Haim; Shafir, Sharoni; Sela, Ilan

    2012-01-01

    The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control. PMID:23308063

  5. Silencing myostatin gene by RNAi in sheep embryos.

    PubMed

    Tang, Dayun; Zhu, Huabin; Wu, Jianmin; Chen, Hanzhong; Zhang, Yan; Zhao, Xueming; Chen, Xiaoliang; Du, Weihua; Wang, Dong; Lin, Xiukun

    2012-04-15

    Myostatin (MSTN) gene is described as a negative regulator of the skeletal muscle growth. Controlling MSTN gene expression by genetic manipulation could accelerate the muscle growth and meat production of livestock animals. In the present study, several siRNAs targeting sheep MSTN gene were designed and their interfering efficiency was evaluated in vitro. The present study showed that one of the siRNAs, PSL1, could down-regulate the expression of MSTN significantly. PSL1 was ligated into lentivirus vector, GP-Supersilencing, to construct a siRNA expression lentivirus vector. Fibroblast cells were infected by lentivirus particles and positive cells were isolated by flow cytometry. Nucleus of the positive cell was transferred into enucleated oocytes of sheep. The present study showed that 99.4% of the sorted cells displayed green fluorescence. After enucleation of oocytes with microinjection, about 20% of reconstructed embryos can be developed into morulas, and strong green fluorescence could be observed using a fluorescence microscope. This method can be available to produce transgenic cell line for somatic cell nucleus transfer for transgenic animals.

  6. Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans

    PubMed Central

    Jahan, Sultana N.; Åsman, Anna K. M.; Corcoran, Pádraic; Fogelqvist, Johan; Vetukuri, Ramesh R.; Dixelius, Christina

    2015-01-01

    Phytophthora infestans is an oomycete that causes severe damage to potato, and is well known for its ability to evolve rapidly in order to overcome resistant potato varieties. An RNA silencing strategy was evaluated here to clarify if small interfering RNA homologous to selected genes in P. infestans could be targeted from the plant host to reduce the magnitude of the infection. As a proof-of-concept, a hairpin RNA (hp-RNA) construct using the GFP marker gene was designed and introduced in potato. At 72 hpi, a 55-fold reduction of the signal intensity of a corresponding GFP expressing P. infestans strain on leaf samples of transgenic plants, compared with wild-type potato, was detected. This suggests that an RNA interference construct in the potato host could be processed and target a transcript of the pathogen. Three genes important in the infection process of P. infestans, PiGPB1, PiCESA2, and PiPEC, together with PiGAPDH taking part in basic cell maintenance were subsequently tested using an analogous transgenic strategy. Out of these gene candidates, the hp-PiGPB1 targeting the G protein β-subunit (PiGPB1) important for pathogenicity resulted in most restricted disease progress. Further, Illumina sequencing of inoculated transgenic potato leaves revealed sRNAs of 24/25 nt size homologous to the PiGPB1 gene in the transgenic plants indicating post-transcriptional silencing of the target gene. The work demonstrates that a host-induced gene-silencing approach is functional against P. infestans but is highly dependent on target gene for a successful outcome. This finding broadens the arsenal of control strategies to this important plant disease. PMID:25788734

  7. Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis.

    PubMed

    Mundodi, V; Kucknoor, A S; Klumpp, D J; Chang, T-H; Alderete, J F

    2004-08-01

    Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS-neo in sense and antisense orientations to generate plasmids pBS-neoS (S) and pBS-neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis. PMID:15306014

  8. Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis

    PubMed Central

    Mundodi, V.; Kucknoor, A. S.; Klumpp, D. J.; Chang, T.-H.; Alderete, J. F.

    2007-01-01

    Summary Host parasitism by Trichomonas vaginalis is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in T. vaginalis adherence, we silenced expression of ap65 using antisense RNA. The gene for ap65 was inserted into the vector pBS-neo in sense and antisense orientations to generate plasmids pBS-neoS (S) and pBS-neoAS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the neo gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of ap65 transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of ap65 was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of ap65. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in T. vaginalis to study the contribution of specific genes in pathogenesis. PMID:15306014

  9. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing

    NASA Astrophysics Data System (ADS)

    Mok, Hyejung; Lee, Soo Hyeon; Park, Ji Won; Park, Tae Gwan

    2010-03-01

    Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.

  10. Cell-free construction of disarmed Abutilon mosaic virus-based gene silencing vectors.

    PubMed

    Krenz, Björn; Wege, Christina; Jeske, Holger

    2010-10-01

    The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics. PMID:20638413

  11. Dietary and genetic effects on age-related loss of gene silencing reveal epigenetic plasticity of chromatin repression during aging.

    PubMed

    Jiang, Nan; Du, Guyu; Tobias, Ethan; Wood, Jason G; Whitaker, Rachel; Neretti, Nicola; Helfand, Stephen L

    2013-11-01

    During aging, changes in chromatin state that alter gene transcription have been postulated to result in expression of genes that are normally silenced, leading to deleterious age-related effects on cellular physiology. Despite the prevalence of this hypothesis, it is primarily in yeast that loss of gene silencing with age has been well documented. We use a novel position effect variegation (PEV) reporter in Drosophila melanogaster to show that age-related loss of repressive heterochromatin is associated with loss of gene silencing in metazoans and is affected by Sir2, as it is in yeast. The life span-extending intervention, calorie restriction (CR), delays the age-related loss of gene silencing, indicating that loss of gene silencing is a component of normal aging. Diet switch experiments show that such flies undergo a rapid change in their level of gene silencing, demonstrating the epigenetic plasticity of chromatin during aging and highlighting the potential role of diet and metabolism in chromatin maintenance, Thus, diet and related interventions may be of therapeutic importance for age-related diseases, such as cancer.

  12. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

    PubMed

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  13. Selective repression of gene expression in neuropathic pain by the neuron-restrictive silencing factor/repressor element-1 silencing transcription (NRSF/REST).

    PubMed

    Willis, Dianna E; Wang, Meng; Brown, Elizabeth; Fones, Lilah; Cave, John W

    2016-06-20

    Neuropathic pain often develops following nerve injury as a result of maladaptive changes that occur in the injured nerve and along the nociceptive pathways of the peripheral and central nervous systems. Multiple cellular and molecular mechanisms likely account for these changes; however, the exact nature of these mechanisms remain largely unknown. A growing number of studies suggest that alteration in gene expression is an important step in the progression from acute to chronic pain states and epigenetic regulation has been proposed to drive this change in gene expression. In this review, we discuss recent evidence that the DNA-binding protein neuron-restrictive silencing factor/repressor element-1 silencing transcription factor (NRSF/REST) is an important component in the development and maintenance of neuropathic pain through its role as a transcriptional regulator for a select subset of genes that it normally represses during development. PMID:26679228

  14. Gene silencing in Medicago truncatula roots using RNAi.

    PubMed

    Floss, Daniela S; Schmitz, Alexa M; Starker, Colby G; Gantt, J Stephen; Harrison, Maria J

    2013-01-01

    Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.

  15. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

    PubMed

    Mandegar, Mohammad A; Huebsch, Nathaniel; Frolov, Ekaterina B; Shin, Edward; Truong, Annie; Olvera, Michael P; Chan, Amanda H; Miyaoka, Yuichiro; Holmes, Kristin; Spencer, C Ian; Judge, Luke M; Gordon, David E; Eskildsen, Tilde V; Villalta, Jacqueline E; Horlbeck, Max A; Gilbert, Luke A; Krogan, Nevan J; Sheikh, Søren P; Weissman, Jonathan S; Qi, Lei S; So, Po-Lin; Conklin, Bruce R

    2016-04-01

    Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease. PMID:26971820

  16. Structure And Gene Silencing Activities of Monovalent And Pentavalent Cationic Lipid Vectors Complexed With Sirna

    SciTech Connect

    Bouxsein, N.F.; McAllister, C.S.; Ewert, K.K.; Samuel, C.E.; Safinya, C.R.; /UC, Santa Barbara

    2007-07-03

    Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (chg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given chg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and chg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.

  17. Post-transcriptional gene silencing in controlling viruses of the Tomato yellow leaf curl virus complex.

    PubMed

    Abhary, M K; Anfoka, G H; Nakhla, M K; Maxwell, D P

    2006-12-01

    Tomato yellow leaf curl disease (TYLCD) is caused by a group of geminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complex and are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causes great yield losses in many countries throughout the Mediterranean region and the Middle East. In this study, the efficacy of post-transcriptional gene silencing (PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curl virus-mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curl Malaga virus, and tomato yellow leaf curl Sardinia virus-Spain [2] were selected and used to design a construct that can trigger broad resistance against different viruses that cause tomato yellow leaf curl disease. The silencing construct was cloned into an Agrobacterium-binary vector in sense and antisense orientation and used in transient assay to infiltrate tomato and Nicotiana benthamiana plants. A high level of resistance was obtained when plants were agro-infiltrated with an infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or challenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16-20 days post infiltration with the silencing construct. Results of the polymerase chain reaction showed that the resistance was effective against all three viruses. Furthermore, dot blot hybridization and PCR failed to detect viral DNA in symptomless, silenced plants. A positive correlation between resistance and the accumulation of TYLCV-specific siRNAs was observed in silenced plants. Together, these data provide compelling evidence that PTGS can be used to engineer geminivirus-resistant plants. PMID:16862387

  18. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.

    PubMed

    Shen, Jianliang; Xu, Rong; Mai, Junhua; Kim, Han-Cheon; Guo, Xiaojing; Qin, Guoting; Yang, Yong; Wolfram, Joy; Mu, Chaofeng; Xia, Xiaojun; Gu, Jianhua; Liu, Xuewu; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2013-11-26

    Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

  19. Epigenetic gene silencing underlies C-fiber dysfunctions in neuropathic pain.

    PubMed

    Uchida, Hitoshi; Ma, Lin; Ueda, Hiroshi

    2010-03-31

    Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Na(v)1.8 sodium channel and mu-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Na(v)1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Na(v)1.8 gene expressions, C-fiber hypoesthesia, and the losses of peripheral morphine analgesia and Na(v)1.8-selective blocker-induced hypoesthesia. Together, these data suggest that NRSF causes pathological and pharmacological dysfunction of C-fibers, which underlies the negative symptoms in neuropathic pain. PMID:20357131

  20. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing

    PubMed Central

    Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production. PMID:27078685

  1. The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

    PubMed

    Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

    2013-12-23

    Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

  2. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

    PubMed

    Fukuma, Miki; Ganmyo, Yuto; Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

  3. Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

    PubMed

    Fukuma, Miki; Ganmyo, Yuto; Miura, Osamu; Ohyama, Takashi; Shimizu, Noriaki

    2016-01-01

    Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production. PMID:27078685

  4. Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing.

    PubMed

    Del Olmo, Iván; López, Juan A; Vázquez, Jesús; Raynaud, Cécile; Piñeiro, Manuel; Jarillo, José A

    2016-07-01

    Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells.

  5. Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing

    PubMed Central

    del Olmo, Iván; López, Juan A.; Vázquez, Jesús; Raynaud, Cécile; Piñeiro, Manuel; Jarillo, José A.

    2016-01-01

    Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1. We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells. PMID:26980282

  6. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells.

    PubMed

    Novobrantseva, Tatiana I; Borodovsky, Anna; Wong, Jamie; Klebanov, Boris; Zafari, Mohammad; Yucius, Kristina; Querbes, William; Ge, Pei; Ruda, Vera M; Milstein, Stuart; Speciner, Lauren; Duncan, Rick; Barros, Scott; Basha, Genc; Cullis, Pieter; Akinc, Akin; Donahoe, Jessica S; Narayanannair Jayaprakash, K; Jayaraman, Muthusamy; Bogorad, Roman L; Love, Kevin; Whitehead, Katie; Levins, Chris; Manoharan, Muthiah; Swirski, Filip K; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G; de Fougerolles, Antonin; Nahrendorf, Matthias; Koteliansky, Victor

    2012-01-01

    Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells. PMID:23344621

  7. Patterning of virus-infected Glycine max seed coat is associated with suppression of endogenous silencing of chalcone synthase genes.

    PubMed

    Senda, Mineo; Masuta, Chikara; Ohnishi, Shizen; Goto, Kazunori; Kasai, Atsushi; Sano, Teruo; Hong, Jin-Sung; MacFarlane, Stuart

    2004-04-01

    Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction. PMID:15037735

  8. Efficient transformation and artificial miRNA gene silencing in Lemna minor

    PubMed Central

    Cantó-Pastor, Alex; Mollá-Morales, Almudena; Ernst, Evan; Dahl, William; Zhai, Jixian; Yan, Yiheng; Meyers, Blake; Shanklin, John; Martienssen, Robert

    2015-01-01

    Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a Magnesium Chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic Lemna minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  9. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    PubMed Central

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  10. Strategies for altering plant traits using virus-induced gene silencing technologies.

    PubMed

    Lacomme, Christophe

    2015-01-01

    The rapid progress in genome sequencing and transcriptome analysis in model and crop plants has made possible the identification of a vast number of genes potentially associated with economically important complex traits. The ultimate goal is to assign functions to these genes by using forward and reverse genetic screens. Plant viruses have been developed for virus-induced gene silencing (VIGS) to generate rapid gene knockdown phenotypes in numerous plant species. To fulfill its potential for high-throughput phenomics, it is of prime importance to ensure that parameters conditioning the VIGS response, i.e., plant-virus interactions and associated loss-of-function screens, are "fit for purpose" and optimized to unequivocally conclude the role of a gene of interest in relation to a given trait. This chapter will review and discuss the different strategies used for the development of VIGS-based phenomics in model and crop species. PMID:25740354

  11. Strategies for altering plant traits using virus-induced gene silencing technologies.

    PubMed

    Lacomme, Christophe

    2015-01-01

    The rapid progress in genome sequencing and transcriptome analysis in model and crop plants has made possible the identification of a vast number of genes potentially associated with economically important complex traits. The ultimate goal is to assign functions to these genes by using forward and reverse genetic screens. Plant viruses have been developed for virus-induced gene silencing (VIGS) to generate rapid gene knockdown phenotypes in numerous plant species. To fulfill its potential for high-throughput phenomics, it is of prime importance to ensure that parameters conditioning the VIGS response, i.e., plant-virus interactions and associated loss-of-function screens, are "fit for purpose" and optimized to unequivocally conclude the role of a gene of interest in relation to a given trait. This chapter will review and discuss the different strategies used for the development of VIGS-based phenomics in model and crop species.

  12. Polycomb silencing of the Drosophila 4E-BP gene regulates imaginal disc cell growth.

    PubMed

    Mason-Suares, Heather; Tie, Feng; Yan, Christopher M; Harte, Peter J

    2013-08-01

    Polycomb group (PcG) proteins are best known for their role in maintaining stable, mitotically heritable silencing of the homeotic (HOX) genes during development. In addition to loss of homeotic gene silencing, some PcG mutants also have small imaginal discs. These include mutations in E(z), Su(z)12, esc and escl, which encode Polycomb repressive complex 2 (PRC2) subunits. The cause of this phenotype is not known, but the human homologs of PRC2 subunits have been shown to play a role in cell proliferation, are over-expressed in many tumors, and appear to be required for tumor proliferation. Here we show that the small imaginal disc phenotype arises, at least in part, from a cell growth defect. In homozygous E(z) mutants, imaginal disc cells are smaller than cells in normally proliferating discs. We show that the Thor gene, which encodes eIF4E-binding protein (4E-BP), the evolutionarily conserved inhibitor of cap-dependent translation and potent inhibitor of cell growth, is involved in the development of this phenotype. The Thor promoter region contains DNA binding motifs for transcription factors found in well-characterized Polycomb response elements (PREs), including PHO/PHOL, GAGA factor, and others, suggesting that Thor may be a direct target of Polycomb silencing. We present chromatin immunoprecipitation evidence that PcG proteins are bound to the Thor 5' region in vivo. The Thor gene is normally repressed in imaginal discs, but Thor mRNA and 4E-BP protein levels are elevated in imaginal discs of PRC2 subunit mutant larvae. Deletion of the Thor gene in E(z) mutants partially restores imaginal disc size toward wild-type and results in an increase in the fraction of larvae that pupariate. These results thus suggest that PcG proteins can directly modulate cell growth in Drosophila, in part by regulating Thor expression.

  13. Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.].

    PubMed

    Trusov, Yuri; Botella, José Ramón

    2006-01-01

    Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

  14. Silencing of grapevine pectate lyase-like genes VvPLL2 and VvPLL3 confers resistance against Erysiphe necator and differentially modulates gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Broad-spectrum resistance against powdery mildew (PM) has been reported by silencing susceptibility genes in the model plant Arabidopsis. Here we used artificial microRNA constructs in PM-susceptible Vitis vinifera cv. Chardonnay to stably silence two pectate lyase-like orthologs (VvPLL2 and VvPLL3)...

  15. RNAi Dynamics in Juvenile Fasciola spp. Liver Flukes Reveals the Persistence of Gene Silencing In Vitro

    PubMed Central

    McVeigh, Paul; McCammick, Erin M.; McCusker, Paul; Morphew, Russell M.; Mousley, Angela; Abidi, Abbas; Saifullah, Khalid M.; Muthusamy, Raman; Gopalakrishnan, Ravikumar; Spithill, Terry W.; Dalton, John P.; Brophy, Peter M.; Marks, Nikki J.; Maule, Aaron G.

    2014-01-01

    Background Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST). Methodology/Principal Findings Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively. Conclusions/Significance In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the

  16. Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.

    PubMed

    Tamura, Masato; Kawano, Mitsuoki; Sato, Mari; Nashimoto, Masayuki

    2015-10-01

    tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl‑β‑cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis‑inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin‑, caveolae‑ and raft‑independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis‑inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA‑mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically‑targeted and potent sgRNA drugs.

  17. Silencing of meiosis-critical genes for engineering male sterility in plants.

    PubMed

    Wang, Xiping; Singer, Stacy D; Liu, Zongrang

    2012-04-01

    The potential for pollen-mediated transgene flow into wild or closely related species has provoked unease in terms of transgenic modification of agricultural plant species. One approach to remedy this situation in species whose seeds and fruits are not of particular value is to engineer male sterility into the transgenic lines. In this study, three meiosis-critical genes, namely AHP2, AtRAD51C and SWITCH1 (SWI), were chosen as silencing targets to test the feasibility of incorporating sterility into plants using an RNAi-based approach. Our results indicated that the silencing of each of these genes via hairpin RNA (termed AHPi, RAD51Ci and SWIi lines) in Arabidopsis thaliana yielded a proportion of transgenic plants exhibiting a similar 'partially sterile' phenotype in which less than 50% of pollen was viable. In addition, a 'sterile' phenotype was also evident in a minority of RAD51Ci and SWIi, but not AHPi, lines in which plants yielded no seeds and either produced inviable pollen (RAD51Ci lines) or displayed a complete absence of pollen (SWIi lines). This suggests that AtRAD51C and SWI may function at distinct stages of meiosis. Further analyses of SWIi lines demonstrated that the 'sterile' phenotype was associated with a substantial reduction in the level of targeted gene transcript in floral tissues and resulted from sterility of the male, but not female gametes. This work demonstrates that generating male sterility through the silencing of key genes involved in the regulation of meiosis is feasible, and its advantages and potential applications for transgene containment are discussed. PMID:22120011

  18. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

    PubMed Central

    2010-01-01

    Background High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. Results Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). Conclusion We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable. PMID:20626919

  19. A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L. cv. Bukang).

    PubMed

    Chung, Eunsook; Seong, Eunsoo; Kim, Yeoung-Cheol; Chung, Eun Joo; Oh, Sang-Keun; Lee, Sanghyeob; Park, Jeong Mee; Joung, Young Hee; Choi, Doil

    2004-04-30

    Using a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system, expression of phytogene desaturase (PDS) and ribulose-1,5-bisphosphate carboxylase small-subuit (rbcS) genes was suppressed in Nicotiana benthamiana and pepper plants (Capsicum annuum L. cv. Bukang). The silenced phenotypes of pale yellow (rbcS), and photobleached leaves (PDS), were invariably obvious 2 weeks after inoculation with the TRV-based vector. In a parallel experiment, the same set of genes was silenced in N. benthamiana and yielded identical phenotypes to pepper 1 week after inoculation. Northern blot analyses showed that the endogenous levels of CarbcS and CaPDS transcripts were dramatically reduced in the silenced leaf tissues. These observations confirm that the silenced phenotype is closely correlated with the pattern of tissue expression. To our knowledge, this is the first high frequency VIGS method in pepper plants. It should provide a tool for large-scale gene silencing studies in pepper functional genomics.

  20. eIF4AII is dispensable for miRNA-mediated gene silencing

    PubMed Central

    Galicia-Vázquez, Gabriela; Chu, Jennifer; Pelletier, Jerry

    2015-01-01

    MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through partial complementary base-pairing to the 3′ untranslated region (UTR) of target mRNAs. Inhibition of translation initiation has been identified as an early event of miRNA-mediated gene repression, but the underlying mechanistic details of this process are not well understood. Recently, eukaryotic initiation factor (eIF) 4AII was identified as a critical modulator of miRNA activity with depletion of this factor alleviating miRNA-mediated gene repression. Using the CRISPR/Cas9-editing system, we generated a novel cell line in which expression of eIF4AII was eliminated. The absence of eIF4AII does not affect cell viability, proliferation, or global mRNA translation. Importantly, we show that eIF4AII is dispensable for miRNA-mediated gene silencing. PMID:26286746

  1. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing.

    PubMed

    Kola, Vijaya Sudhakara Rao; Renuka, P; Madhav, Maganti Sheshu; Mangrauthia, Satendra K

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21-24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  2. In Vitro Gene Silencing of the Fish Microsporidian Heterosporis saurida by RNA Interference

    PubMed Central

    Kumar, Gokhlesh; Abdel-Baki, Abdel-Azeem; Dkhil, Mohamed A.; El-Matbouli, Mansour; Al-Quraishy, Saleh

    2016-01-01

    Heterosporis saurida, a microsporidian parasite of lizardfish, Saurida undosquamis, causes severe economic losses in marine aquaculture. Among the novel approaches being explored for treatment of parasitic infections in aquaculture is small interfering RNA molecules. The aim of the present study was to investigate the efficiency of using siRNA to knock down expression of specific genes of H. saurida in vitro. For this purpose, siRNAs specific for ATP/ADP antiporter 1 and methionine aminopeptidase II genes were designed and tested using a previously developed in vitro cultivation model. Silencing of H. saurida target genes was assessed and the efficacy of using siRNA for inhibition of gene expression was measured by quantitative real-time polymerase chain reaction (PCR). Silencing of ATP/ADP antiporter 1 or methionine aminopeptidase II by siRNA reduced H. saurida infection levels in EK-1 cells 40% and 60%, respectively, as measured by qRT-PCR and spore counts. Combined siRNA treatment of both ATP/ADP antiporter 1 and methionine aminopeptidase II siRNAs was more effective against H. saurida infection as seen by the 16S rRNA level and spore counts. Our study concluded that siRNA could be used to advance development of novel approaches to inhibit H. saurida and provide an alternative approach to combat microsporidia. PMID:27228357

  3. Key enzymes and proteins of crop insects as candidate for RNAi based gene silencing

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Madhav, Maganti Sheshu; Mangrauthia, Satendra K.

    2015-01-01

    RNA interference (RNAi) is a mechanism of homology dependent gene silencing present in plants and animals. It operates through 21–24 nucleotides small RNAs which are processed through a set of core enzymatic machinery that involves Dicer and Argonaute proteins. In recent past, the technology has been well appreciated toward the control of plant pathogens and insects through suppression of key genes/proteins of infecting organisms. The genes encoding key enzymes/proteins with the great potential for developing an effective insect control by RNAi approach are actylcholinesterase, cytochrome P450 enzymes, amino peptidase N, allatostatin, allatotropin, tryptophan oxygenase, arginine kinase, vacuolar ATPase, chitin synthase, glutathione-S-transferase, catalase, trehalose phosphate synthase, vitellogenin, hydroxy-3-methylglutaryl coenzyme A reductase, and hormone receptor genes. Through various studies, it is demonstrated that RNAi is a reliable molecular tool which offers great promises in meeting the challenges imposed by crop insects with careful selection of key enzymes/proteins. Utilization of RNAi tool to target some of these key proteins of crop insects through various approaches is described here. The major challenges of RNAi based insect control such as identifying potential targets, delivery methods of silencing trigger, off target effects, and complexity of insect biology are very well illustrated. Further, required efforts to address these challenges are also discussed. PMID:25954206

  4. Epigenetic silencing of neurofilament genes promotes an aggressive phenotype in breast cancer

    PubMed Central

    Calmon, Marilia Freitas; Jeschke, Jana; Zhang, Wei; Dhir, Mashaal; Siebenkäs, Cornelia; Herrera, Alexander; Tsai, Hsing-Chen; O'Hagan, Heather M; Pappou, Emmanouil P; Hooker, Craig M; Fu, Tao; Schuebel, Kornel E; Gabrielson, Edward; Rahal, Paula; Herman, James G; Baylin, Stephen B; Ahuja, Nita

    2015-01-01

    Neurofilament heavy polypeptide (NEFH) has recently been identified as a candidate DNA hypermethylated gene within the functional breast cancer hypermethylome. NEFH exists in a complex with neurofilament medium polypeptide (NEFM) and neurofilament light polypeptide (NEFL) to form neurofilaments, which are structural components of the cytoskeleton in mature neurons. Recent studies reported the deregulation of these proteins in several malignancies, suggesting that neurofilaments may have a role in other cell types as well. Using a comprehensive approach, we studied the epigenetic inactivation of neurofilament genes in breast cancer and the functional significance of this event. We report that DNA methylation-associated silencing of NEFH, NEFL, and NEFM in breast cancer is frequent, cancer-specific, and correlates with clinical features of disease progression. DNA methylation-mediated inactivation of these genes occurs also in multiple other cancer histologies including pancreas, gastric, and colon. Restoration of NEFH function, the major subunit of the neurofilament complex, reduces proliferation and growth of breast cancer cells and arrests them in Go/G1 phase of the cell cycle along with a reduction in migration and invasion. These findings suggest that DNA methylation-mediated silencing of the neurofilament genes NEFH, NEFM, and NEFL are frequent events that may contribute to the progression of breast cancer and possibly other malignancies. PMID:25985363

  5. Epigenetic silencing of neurofilament genes promotes an aggressive phenotype in breast cancer.

    PubMed

    Calmon, Marilia Freitas; Jeschke, Jana; Zhang, Wei; Dhir, Mashaal; Siebenkäs, Cornelia; Herrera, Alexander; Tsai, Hsing-Chen; O'Hagan, Heather M; Pappou, Emmanouil P; Hooker, Craig M; Fu, Tao; Schuebel, Kornel E; Gabrielson, Edward; Rahal, Paula; Herman, James G; Baylin, Stephen B; Ahuja, Nita

    2015-01-01

    Neurofilament heavy polypeptide (NEFH) has recently been identified as a candidate DNA hypermethylated gene within the functional breast cancer hypermethylome. NEFH exists in a complex with neurofilament medium polypeptide (NEFM) and neurofilament light polypeptide (NEFL) to form neurofilaments, which are structural components of the cytoskeleton in mature neurons. Recent studies reported the deregulation of these proteins in several malignancies, suggesting that neurofilaments may have a role in other cell types as well. Using a comprehensive approach, we studied the epigenetic inactivation of neurofilament genes in breast cancer and the functional significance of this event. We report that DNA methylation-associated silencing of NEFH, NEFL, and NEFM in breast cancer is frequent, cancer-specific, and correlates with clinical features of disease progression. DNA methylation-mediated inactivation of these genes occurs also in multiple other cancer histologies including pancreas, gastric, and colon. Restoration of NEFH function, the major subunit of the neurofilament complex, reduces proliferation and growth of breast cancer cells and arrests them in Go/G1 phase of the cell cycle along with a reduction in migration and invasion. These findings suggest that DNA methylation-mediated silencing of the neurofilament genes NEFH, NEFM, and NEFL are frequent events that may contribute to the progression of breast cancer and possibly other malignancies.

  6. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

    PubMed Central

    Craven, R J; Petes, T D

    2001-01-01

    Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p. PMID:11333225

  7. Efficient Gene Silencing by Self-Assembled Complexes of siRNA and Symmetrical Fatty Acid Amides of Spermine

    PubMed Central

    Metwally, Abdelkader A.; Pourzand, Charareh; Blagbrough, Ian S.

    2011-01-01

    Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing. PMID:24310492

  8. Functional genomics tool: Gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA

    PubMed Central

    2010-01-01

    Background Ticks are blood-sucking arthropods responsible for transmitting a wide variety of disease-causing agents, and constitute important public health threats globally. Ixodes scapularis is the primary vector of the Lyme disease agent in the eastern and central U.S. RNAi is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. Here, we describe an optimized protocol for effectively suppressing gene expression in the egg and nymphal stages of I. scapularis by electroporation. Results The genes encoding the putative Phospholipase A2 (PLA2), cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin were targeted by delivering the dsRNA encoding the specific gene coding regions in the unfed nymphs. Silencing was measured using real time qRT-PCR. Electroporation as a mode of dsRNA delivery appears to be substantially efficient and less traumatic to the tick than dsRNA microinjection in the unfed nymphs. Using Cy3-labeled dsRNA to monitor the movement, electroporated dsRNA entered the nymphs and spread to salivary glands and other tissues. The significant disruption of β-actin and cytoplasmic Cystatin transcripts in tick eggs demonstrate the applicability of this technique. The PLA2, cytoplasmic Cystatin, Syntaxin-5, β-Actin and Calreticulin genes were also significantly silenced, suggesting that this method has the potential to introduce dsRNA in eggs and unfed nymphs. Conclusions Our study demonstrates that electroporation can be used as a simple dsRNA delivery tool in assessing the functional role of tick genes in the vector-host interactions. This technique represents a novel approach for specific gene suppression in immature stages of ticks. PMID:20074328

  9. Gene silencing and sex determination by programmed DNA elimination in parasitic nematodes.

    PubMed

    Streit, Adrian; Wang, Jianbin; Kang, Yuanyuan; Davis, Richard E

    2016-08-01

    Maintenance of genome integrity is essential. However, programmed DNA elimination removes specific DNA sequences from the genome during development. DNA elimination occurs in unicellular ciliates and diverse metazoa ranging from nematodes to vertebrates. Two distinct groups of nematodes use DNA elimination to silence germline-expressed genes in the soma (ascarids) or for sex determination (Strongyloides spp.). Data suggest that DNA elimination likely evolved independently in these nematodes. Recent studies indicate that differential CENP-A deposition within chromosomes defines which sequences are retained and lost during Ascaris DNA elimination. Additional studies are needed to determine the distribution, functions, and mechanisms of DNA elimination in nematodes. PMID:27315434

  10. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    SciTech Connect

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  11. Nanogyroids incorporating multivalent lipids: enhanced membrane charge density and pore forming ability for gene silencing.

    PubMed

    Leal, Cecília; Ewert, Kai K; Shirazi, Rahau S; Bouxsein, Nathan F; Safinya, Cyrus R

    2011-06-21

    The self-assembly of a custom-synthesized pentavalent cationic lipid (MVL5) and glycerol monooleate (GMO) with small interfering RNA (siRNA) results in the formation of a double-gyroid bicontinuous inverted cubic phase with colocalized lipid/siRNA domains as shown by synchrotron X-ray scattering and fluorescence microscopy. The high charge density (due to MVL5) and positive Gaussian modulus of the GMO-containing membranes confer optimal electrostatic and elastic properties for endosomal escape, enabling efficient siRNA delivery and effective, specific gene silencing.

  12. Nanogyroids Incorporating Multivalent Lipids: Enhanced Membrane Charge Density and Pore Forming Ability for Gene Silencing

    PubMed Central

    Leal, Cecília; Ewert, Kai K.; Shirazi, Rahau S.; Bouxsein, Nathan F.; Safinya, Cyrus R.

    2011-01-01

    The self-assembly of a custom-synthesized pentavalent cationic lipid (MVL5) and glycerol monooleate (GMO) with small interfering RNA (siRNA) results in the formation of a double-gyroid bicontinuous inverted cubic phase with co-localized lipid/siRNA domains as shown by synchrotron X-ray scattering and fluorescence microscopy. The high charge density (due to MVL5) and positive Gaussian modulus of the GMO-containing membranes confer optimal electrostatic and elastic properties for endosomal escape, enabling efficient siRNA delivery and effective, specific gene silencing. PMID:21612245

  13. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments

    PubMed Central

    Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Győrffy, Balázs

    2016-01-01

    No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency. PMID:27673562

  14. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments.

    PubMed

    Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Győrffy, Balázs

    2016-01-01

    No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal-Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E-06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E-04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency. PMID:27673562

  15. Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids.

    PubMed

    Chen, Z J; Comai, L; Pikaard, C S

    1998-12-01

    Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

  16. New Aspects of Gene-Silencing for the Treatment of Cardiovascular Diseases

    PubMed Central

    Koenig, Olivia; Walker, Tobias; Perle, Nadja; Zech, Almuth; Neumann, Bernd; Schlensak, Christian; Wendel, Hans-Peter; Nolte, Andrea

    2013-01-01

    Coronary heart disease (CHD), mainly caused by atherosclerosis, represents the single leading cause of death in industrialized countries. Besides the classical interventional therapies new applications for treatment of vascular wall pathologies are appearing on the horizon. RNA interference (RNAi) represents a novel therapeutic strategy due to sequence-specific gene-silencing through the use of small interfering RNA (siRNA). The modulation of gene expression by short RNAs provides a powerful tool to theoretically silence any disease-related or disease-promoting gene of interest. In this review we outline the RNAi mechanisms, the currently used delivery systems and their possible applications to the cardiovascular system. Especially, the optimization of the targeting and transfection procedures could enhance the efficiency of siRNA delivery drastically and might open the way to clinical applicability. The new findings of the last years may show the techniques to new innovative therapies and could probably play an important role in treating CHD in the future. PMID:24276320

  17. An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept

    PubMed Central

    Smith, Nikaïa; Vidalain, Pierre-Olivier; Nisole, Sébastien; Herbeuval, Jean-Philippe

    2016-01-01

    Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol. PMID:27412723

  18. Widespread disruption of repressor element-1 silencing transcription factor/neuron-restrictive silencer factor occupancy at its target genes in Huntington's disease.

    PubMed

    Zuccato, Chiara; Belyaev, Nikolai; Conforti, Paola; Ooi, Lezanne; Tartari, Marzia; Papadimou, Evangelia; MacDonald, Marcy; Fossale, Elisa; Zeitlin, Scott; Buckley, Noel; Cattaneo, Elena

    2007-06-27

    Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD. PMID:17596446

  19. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  20. Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing.

    PubMed

    Rodríguez-Negrete, Edgar; Lozano-Durán, Rosa; Piedra-Aguilera, Alvaro; Cruzado, Lucia; Bejarano, Eduardo R; Castillo, Araceli G

    2013-07-01

    Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defence against transposons and invading DNA viruses. Previous data showed that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Here, we describe a novel counter-defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases, METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3), in both locally and systemically infected tissues. We demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widespread among geminivirus species. Additionally, we identified Rep (Replication associated protein) as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 down-regulation. The presence of Rep suppressed TGS of an Arabidopsis thaliana transgene and of host loci whose expression was strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for replication, displays TGS suppressor activity through a mechanism distinct from that thus far described for geminiviruses. PMID:23614786

  1. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  2. DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica.

    PubMed

    Omotezako, Tatsuya; Onuma, Takeshi A; Nishida, Hiroki

    2015-05-22

    RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5'-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa.

  3. DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica

    PubMed Central

    Omotezako, Tatsuya; Onuma, Takeshi A.; Nishida, Hiroki

    2015-01-01

    RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5′-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa. PMID:25904672

  4. A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing*

    PubMed Central

    Cordeiro, Tiago N.; García, Jesús; Bernadó, Pau; Millet, Oscar; Pons, Miquel

    2015-01-01

    The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related. PMID:26085102

  5. DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica.

    PubMed

    Omotezako, Tatsuya; Onuma, Takeshi A; Nishida, Hiroki

    2015-05-22

    RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5'-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa. PMID:25904672

  6. Viral/Nonviral Chimeric Nanoparticles To Synergistically Suppress Leukemia Proliferation via Simultaneous Gene Transduction and Silencing.

    PubMed

    Hong, Cheol Am; Cho, Soo Kyung; Edson, Julius A; Kim, Jane; Ingato, Dominique; Pham, Bryan; Chuang, Anthony; Fruman, David A; Kwon, Young Jik

    2016-09-27

    Single modal cancer therapy that targets one pathological pathway often turns out to be inefficient. For example, relapse of chronic myelogenous leukemia (CML) after inhibiting BCR-ABL fusion protein using tyrosine kinase inhibitors (TKI) (e.g., Imatinib) is of significant clinical concern. This study developed a dual modal gene therapy that simultaneously tackles two key BCR-ABL-linked pathways using viral/nonviral chimeric nanoparticles (ChNPs). Consisting of an adeno-associated virus (AAV) core and an acid-degradable polymeric shell, the ChNPs were designed to simultaneously induce pro-apoptotic BIM expression by the AAV core and silence pro-survival MCL-1 by the small interfering RNA (siRNA) encapsulated in the shell. The resulting BIM/MCL-1 ChNPs were able to efficiently suppress the proliferation of BCR-ABL+ K562 and FL5.12/p190 cells in vitro and in vivo via simultaneously expressing BIM and silencing MCL-1. Interestingly, the synergistic antileukemic effects generated by BIM/MCL-1 ChNPs were specific to BCR-ABL+ cells and independent of a proliferative cytokine, IL-3. The AAV core of ChNPs was efficiently shielded from inactivation by anti-AAV serum and avoided the generation of anti-AAV serum, without acute toxicity. This study demonstrates the development of a synergistically efficient, specific, and safe therapy for leukemia using gene carriers that simultaneously manipulate multiple and interlinked pathological pathways. PMID:27472284

  7. HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

    PubMed Central

    2011-01-01

    Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent). Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were

  8. Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

    PubMed

    Eftekhariyan Ghamsari, Mohammad Reza; Karimi, Farah; Mousavi Gargari, Seyed Latif; Hosseini Tafreshi, Seyed Ali; Salami, Seyed Alireza

    2014-12-01

    Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium. PMID:25070062

  9. Effect of RNAi-mediated silencing of Livin gene on biological properties of colon cancer cell line LoVo.

    PubMed

    Zou, A M; Wang, H F; Zhu, W F; Wang, F X; Shen, J J

    2014-05-16

    This study aimed to investigate the effect of RNAi-mediated silencing of the Livin gene on biological properties of the colon cancer cell line LoVo. Interference vectors pSilencer4.1-Ll and pSilencer4.1-L2 targeting the Livin gene were constructed and transfected into LoVo cells. The expression of the Livin gene was determined by RT-PCR and Western blotting. The apoptosis, cell cycle, colony formation, proliferation of LoVo cells, as well as their sensitivity to cisplatin, were detected by flow cytometry, colony formation assay and MTT. Livin mRNA and protein expression in LoVo cells could be effectively silenced by pSilencer4.1-Ll but not pSilencer4.1-L2. In the pSilencer4.1-Ll transfection group, the apoptosis rate of LoVo cells was significantly higher than in the control group (24.2 ± 3.2 vs 8.1 ± 1.4%, P < 0.01), and after 72 h, cell proliferation was clearly decreased (about 70% inhibition). Compared with the control group, the colony formation rate in pSilencer4.1-Ll transfection group was obviously decreased (15 ± 4.6 vs 85 ± 5.8%, P < 0.01), with increased proportion of S phase cells (45.7 ± 4.9 vs 28.0 ± 3.0%, P < 0.01), decreased proportion of G1 phase cells (43.0 ± 5.2 vs 62.8 ± 5.1%, P < 0.01), and increased sensitivity to cisplatin (apoptosis rate increased from 43.4 ± 6.9 to 65.3 ± 6.2%, P < 0.01). pSilencer4.1-Ll can effectively silence Livin gene expression in LoVo colon cancer cells, inhibit cell proliferation and colony formation, induce apoptosis, and enhance sensitivity to cisplatin.

  10. Development of self-compatible B. rapa by RNAi-mediated S locus gene silencing.

    PubMed

    Jung, Hee-Jeong; Jung, Hyo-Jin; Ahmed, Nasar Uddin; Park, Jong-In; Kang, Kwon-Kyoo; Hur, Yoonkang; Lim, Yong-Pyo; Nou, Ill-Sup

    2012-01-01

    The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the S-locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S locus Cysteine-rich (SP11/SCR) protein. Here, we examined the function of S(60), one SP11/SCR allele of B. rapa cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in B. rapa.

  11. Heterochromatin-mediated gene silencing facilitates the diversification of olfactory neurons

    PubMed Central

    Lyons, David B.; Magklara, Angeliki; Goh, Tracie; Sampath, Srihari; Schaefer, Anne; Schotta, Gunnar; Lomvardas, Stavros

    2014-01-01

    SUMMARY An astounding property of the nervous system is its cellular diversity. This diversity, which was initially realized by morphological and electrophysiological differences, is ultimately produced by variations in gene expression programs. In most cases these variations are determined by external cues. However, a growing number of neuronal types have been identified in which inductive signals cannot explain the few but decisive transcriptional differences that cause cell diversification. Here, we show that heterochromatic silencing, which we find is governed by histone methyltransferases G9a (KMT1C) and GLP (KMT1D), is essential for stochastic and singular OR expression. Deletion of G9a and GLP dramatically reduces the complexity of the OR transcriptome, resulting in transcriptional domination by a few ORs and loss of singularity in OR expression. Thus, in addition to its previously known functions, our data suggest that heterochromatin creates an epigenetic platform that affords stochastic, mutually exclusive gene choices and promotes cellular diversity. PMID:25437545

  12. Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2

    PubMed Central

    Oravecz, Attila; Apostolov, Apostol; Polak, Katarzyna; Jost, Bernard; Le Gras, Stéphanie; Chan, Susan; Kastner, Philippe

    2015-01-01

    T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4−CD8− thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4−CD8− cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells. PMID:26549758

  13. Development of Self-Compatible B. rapa by RNAi-Mediated S Locus Gene Silencing

    PubMed Central

    Jung, Hee-Jeong; Jung, Hyo-Jin; Ahmed, Nasar Uddin; Park, Jong-In; Kang, Kwon-Kyoo; Hur, Yoonkang; Lim, Yong-Pyo; Nou, Ill-Sup

    2012-01-01

    The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the S-locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S locus Cysteine-rich (SP11/SCR) protein. Here, we examined the function of S60, one SP11/SCR allele of B. rapa cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in B. rapa. PMID:23145180

  14. RNAi-mediated silencing of fungal acuD gene attenuates the virulence of Penicillium marneffei.

    PubMed

    Sun, Jiufeng; Li, Xiqing; Feng, Peiying; Zhang, Junmin; Xie, Zhi; Song, Erwei; Xi, Liyan

    2014-02-01

    A number of pathogens, most of them intracellular, employ the glyoxylate cycle in order to ingest fatty acids as carbon sources as a way of coping with nutrient deprivation during the infection process. Isocitrate lyase, which is encoded by the pathogen's acuD gene, plays a pivotal role in the glyoxylate cycle, which has been implicated in fungal pathogenesis. In this study, the acuD gene of Penicillium marneffei was knocked down using siRNA expressed by a filamentous fungi expression system. The acuD siRNA reduced the acuD gene's mRNA and protein expression by 21.5 fold and 3.5 fold, respectively. When macrophages were infected with different transformants of P. marneffei, the knockdown of acuD expression with RNA interference was lethal to the pathogens. In addition, the secretion of tumor necrosis factor-alpha and interferon-gamma from the infected macrophages was reduced. Moreover, the RNAi-mediated silencing of acuD expression reduced the fungal burden in the nude mice infected with P. marneffei; inhibited the inflammatory response in the lungs, livers, and spleens during the chronic phase instead of the acute phase of infection; and thus prolonged survival of the infected animals. Collectively, our data indicate that the RNAi-mediated silencing of acuD expression could attenuate virulence of P. marneffei. The endogenous expression of the delivered siRNA vector could be used to evaluate the role of functional genes by continuous and stable expression of siRNA.

  15. Effect of RhoA gene silencing on proliferation and migration of gastric MGC-803 cells

    PubMed Central

    Duan, Ju-Tao; Wang, Xi-Mo; Zhang, Shu-Quan; Zhao, Guan-Jie

    2015-01-01

    In this study, the expression of silencing RhoA gene in gastric MGC-803 Cells was investigated, in order to discuss the effect of RhoA gene on cell proliferation, cell cycles and tumor migration. SiRNA sequence of RhoA gene was designed and synthesized; MGC-803 cells were transfected by LipofectamineTM2000. The expression of RhoA gene in mRNA and protein after interference was detected by RT-PCR and Western blot; flow cytometry was used to detect the cell cycle; cell proliferation was detected by CCK-8 assay and cell migration was detected by scratch healing assay. RhoA expression in mRNA and protein of the experimental group was significantly lower than that of the control group and blank group, and the difference was statistically significant (P < 0.05). The growth rate significantly slowed down in experimental group; the cell cycle was arrested in the G0/G1 phase and the number of cells in S-phase reduced; there was a statistically significant difference (P < 0.05). Scratch healing assay showed that cell migration of the experimental group was significantly decreased, with a statistically significant difference (P < 0.05). Specific interference on RhoA gene expression could inhibit the proliferation and migration of MGC-803 cells; therefore, siRNA sequences of RhoA gene may be an effective target for the treatment of gastric cancer. PMID:26550428

  16. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Facchini, Peter J

    2012-10-01

    Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3'-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3'-hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3'-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine.

  17. Integrated Analysis of Dysregulated miRNA-gene Expression in HMGA2-silenced Retinoblastoma Cells

    PubMed Central

    Venkatesan, Nalini; Deepa, PR; Vasudevan, Madavan; Khetan, Vikas; Reddy, Ashwin M; Krishnakumar, Subramanian

    2014-01-01

    Retinoblastoma (RB) is a primary childhood eye cancer. HMGA2 shows promise as a molecule for targeted therapy. The involvement of miRNAs in genome-level molecular dys-regulation in HMGA2-silenced RB cells is poorly understood. Through miRNA expression microarray profiling, and an integrated array analysis of the HMGA2-silenced RB cells, the dysregulated miRNAs and the miRNA-target relationships were modelled. Loop network analysis revealed a regulatory association between the transcription factor (SOX5) and the deregulated miRNAs (miR-29a, miR-9*, miR-9-3). Silencing of HMGA2 deregulated the vital oncomirs (miR-7, miR-331, miR-26a, miR-221, miR-17~92 and miR-106b∼25) in RB cells. From this list, the role of the miR-106b∼25 cluster was examined further for its expression in primary RB tumor tissues (n = 20). The regulatory targets of miR-106b∼25 cluster namely p21 (cyclin-dependent kinase inhibitor) and BIM (pro-apoptotic gene) were elevated, and apoptotic cell death was observed, in RB tumor cells treated with the specific antagomirs of the miR-106b∼25 cluster. Thus, suppression of miR-106b∼25 cluster controls RB tumor growth. Taken together, HMGA2 mediated anti-tumor effect present in RB is, in part, mediated through the miR-106b∼25 cluster. PMID:25232279

  18. Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

    PubMed Central

    Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

    2013-01-01

    Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis. PMID:23956416

  19. Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.

    PubMed

    Brunkard, Jacob O; Burch-Smith, Tessa M; Runkel, Anne M; Zambryski, Patricia

    2015-01-01

    Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

  20. 5'-C-Malonyl RNA: Small Interfering RNAs Modified with 5'-Monophosphate Bioisostere Demonstrate Gene Silencing Activity.

    PubMed

    Zlatev, Ivan; Foster, Donald J; Liu, Jingxuan; Charisse, Klaus; Brigham, Benjamin; Parmar, Rubina G; Jadhav, Vasant; Maier, Martin A; Rajeev, Kallanthottathil G; Egli, Martin; Manoharan, Muthiah

    2016-04-15

    5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.

  1. Carrier-free Gene Silencing by Amphiphilic Nucleic Acid Conjugates in Differentiated Intestinal Cells.

    PubMed

    Moroz, Elena; Lee, Soo Hyeon; Yamada, Ken; Halloy, François; Martínez-Montero, Saúl; Jahns, Hartmut; Hall, Jonathan; Damha, Masad J; Castagner, Bastien; Leroux, Jean-Christophe

    2016-01-01

    Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2'-deoxy-2'-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets. PMID:27648924

  2. Efficient transformation and artificial miRNA gene silencing in Lemna minor.

    PubMed

    Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

    2015-01-01

    Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae.

  3. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis

    PubMed Central

    Sánchez-Rangel, Diana; Chávez-Martínez, Ana I.; Rodríguez-Hernández, Aída A.; Maruri-López, Israel; Urano, Kaoru; Shinozaki, Kazuo; Jiménez-Bremont, Juan F.

    2016-01-01

    Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes. PMID:27014322

  4. Efficient transformation and artificial miRNA gene silencing in Lemna minor.

    PubMed

    Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

    2015-01-01

    Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  5. Carrier-free Gene Silencing by Amphiphilic Nucleic Acid Conjugates in Differentiated Intestinal Cells

    PubMed Central

    Moroz, Elena; Lee, Soo Hyeon; Yamada, Ken; Halloy, François; Martínez-Montero, Saúl; Jahns, Hartmut; Hall, Jonathan; Damha, Masad J; Castagner, Bastien; Leroux, Jean-Christophe

    2016-01-01

    Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2′-deoxy-2′-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets. PMID:27648924

  6. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis.

    PubMed

    Sánchez-Rangel, Diana; Chávez-Martínez, Ana I; Rodríguez-Hernández, Aída A; Maruri-López, Israel; Urano, Kaoru; Shinozaki, Kazuo; Jiménez-Bremont, Juan F

    2016-01-01

    Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes. PMID:27014322

  7. Carrier-free Gene Silencing by Amphiphilic Nucleic Acid Conjugates in Differentiated Intestinal Cells.

    PubMed

    Moroz, Elena; Lee, Soo Hyeon; Yamada, Ken; Halloy, François; Martínez-Montero, Saúl; Jahns, Hartmut; Hall, Jonathan; Damha, Masad J; Castagner, Bastien; Leroux, Jean-Christophe

    2016-01-01

    Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2'-deoxy-2'-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets.

  8. Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis.

    PubMed

    Sánchez-Rangel, Diana; Chávez-Martínez, Ana I; Rodríguez-Hernández, Aída A; Maruri-López, Israel; Urano, Kaoru; Shinozaki, Kazuo; Jiménez-Bremont, Juan F

    2016-01-01

    Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes.

  9. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.

    PubMed

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases. PMID:26699507

  10. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.

    PubMed

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

  11. MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response

    PubMed Central

    Pothof, Joris; Verkaik, Nicole S; van IJcken, Wilfred; Wiemer, Erik A C; Ta, Van T B; van der Horst, Gijsbertus T J; Jaspers, Nicolaas G J; van Gent, Dik C; Hoeijmakers, Jan H J; Persengiev, Stephan P

    2009-01-01

    DNA damage provokes DNA repair, cell-cycle regulation and apoptosis. This DNA-damage response encompasses gene-expression regulation at the transcriptional and post-translational levels. We show that cellular responses to UV-induced DNA damage are also regulated at the post-transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA-mediated gene-silencing inhibition by knocking down essential components of the microRNA-processing pathway (Dicer and Ago2). UV damage triggered a cell-cycle-dependent relocalization of Ago2 into stress granules and various microRNA-expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV-responsive microRNA expression was only partially ATM/ATR independent. Both microRNA-expression changes and stress-granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA-mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV-inducible miR-16 that downregulates checkpoint-gene CDC25a and regulates cell proliferation. We conclude that microRNA-mediated gene regulation adds a new dimension to the DNA-damage response. PMID:19536137

  12. Robust Hepatic Gene Silencing for Functional Studies Using Helper-Dependent Adenoviral Vectors

    PubMed Central

    Ruiz, Rafaela; Witting, Scott R.; Saxena, Romil

    2009-01-01

    Abstract RNA interference is currently envisioned as the basis of gene function and drug target validation studies. This novel technology has the advantage of providing a remarkably faster tool for gene silencing than traditional transgenic animal methodologies. In vivo administration of short interfering RNA (siRNA) typically results in reduced target gene expression for approximately 1 week. Viral vectors offer the possibility to express constitutive levels of short hairpin RNA (shRNA) so that the effects of knocking down the target gene can be studied for a few weeks, rather than a few days. Helper-dependent vectors have a significant advantage over previous generations of adenoviral vectors because of their much higher cloning capacity, potential for long-term transgene expression, and enhanced safety profiles on administration in vivo. Therefore, this advanced type of vector is an excellent tool to carry out in vivo studies directed at constitutive expression of shRNA. Here we show it is possible to obtain more than 90% target gene knockdown in an animal model of type 2 diabetes for several weeks, thereby consolidating this technology as an alternative to generating liver-specific knockout animals. PMID:18828727

  13. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

    PubMed Central

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light–oxygen–voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na+-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na+ currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases. PMID:26699507

  14. Silencing of Cited2 and Akap12 genes in radiation-induced rat osteosarcomas

    SciTech Connect

    Daino, Kazuhiro

    2009-12-18

    We have previously studied genomic copy number changes and global gene expression patterns in rat osteosarcomas (OS) induced by the bone-seeking alpha emitter {sup 238}Pu by comparative genomic hybridization (CGH) and oligonucleotide microarray analyses, respectively. Among the previously identified genes that were down-regulated in radiation-induced rat OS tumors, Cited2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2) and Akap12 (a kinase anchoring protein, also known as src-suppressed C-kinase substrate, SSeCKS) genes mapped to the most frequently lost regions on chromosome 1p. In the present study, relative copy number losses of Cited2 and Akap12 genes were observed in 8 of 15 (53%) and 10 of 15 (67%) tumors by quantitative PCR analysis. Loss of Cited2 and Akap12 in the tumors was confirmed at the levels of mRNA and protein expression by quantitative RT-PCR and immunoblot analyses, respectively. These results indicate that Cited2 and Akap12 are silenced in radiation-induced OS, and therefore are novel candidate tumor-suppressor genes of this tumor.

  15. Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells

    PubMed Central

    Mitsuda, Sho-hei; Shimizu, Noriaki

    2016-01-01

    A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model. PMID:27525955

  16. Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells.

    PubMed

    Mitsuda, Sho-Hei; Shimizu, Noriaki

    2016-01-01

    A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a "DNA methylation-core and chromatin-spread" model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model. PMID:27525955

  17. The human involucrin gene is transcriptionally repressed through a tissue-specific silencer element recognized by Oct-2.

    PubMed

    Azuara-Liceaga, Elisa; Sandoval, Marisol; Corona, Matilde; Gariglio, Patricio; López-Bayghen, Esther

    2004-05-28

    Involucrin is an important marker of epithelial differentiation which expression is upregulated just after basal cells are pushed into the suprabasal layer in stratified epithelia. Several transcription factors and regulatory elements had been described as responsible for turning on the gene. However, it is evident that in basal cell layer, additional mechanisms are involved in keeping the gene silent before the differentiation process starts. In this work, we located a potential transcriptional silencer in a 52bp sequence whose integrity is necessary for silencing the proximal enhancer promoter element (PEP) in multiplying keratinocytes. Octamer-binding sites were noticed in this fragment and the specific binding of Oct-2 transcription factor was detected. Oct-2 appears to be implicated in an epithelial-specific repression activity recorded only in keratinocytes and C33-A cell line. Overexpression of Oct-2 repressed the involucrin promoter activity in epithelial cells and in the presence of the silencer element.

  18. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum).

    PubMed

    Fragkostefanakis, Sotirios; Sedeek, Khalid E M; Raad, Maya; Zaki, Marwa Samir; Kalaitzis, Panagiotis

    2014-07-01

    Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.

  19. Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum

    PubMed Central

    Lu, Yanhui; Park, Yoonseong; Gao, Xiwu; Zhang, Xin; Yao, Jianxiu; Pang, Yuan-Ping; Jiang, Haobo; Zhu, Kun Yan

    2012-01-01

    We compared biological functions of two acetylcholinesterase genes (TcAce1 and TcAce2) in Tribolium castaneum, a globally distributed major pest of stored grain products and an emerging model organism, by using RNA interference. Although both genes expressed at all developmental stages and mainly in the brain, the transcript level of TcAce1 was 1.2- to 8.7-fold higher than that of TcAce2, depending on developmental stages. Silencing TcAce1 in 20-day larvae led to 100% mortality within two weeks after eclosion and increased larval susceptibilities to anticholinesterase insecticides. In contrast, silencing TcAce2 did not show insect mortality and significantly affect insecticide susceptibility, but delayed insect development and reduced female egg-laying and egg hatching. These results demonstrate for the first time that TcAce1 plays a major role in cholinergic functions and is the target of anticholinesterase insecticides, whereas TcAce2 plays an important, non-cholinergic role in female reproduction, embryo development, and growth of offspring. PMID:22371826

  20. Construction and identification of multiple genes Co silence of plasmid shRNA.

    PubMed

    Sun, Jin; Wang, Liang; Dong, Ming-Min; Cao, Hua; Tian, Xiu-Fen

    2015-01-01

    Objective To construct and identify the eukaryotic vector expressing shRNA (Plasmid-1), which expressed the VEGF, C-myc, Survivin and hTERT gene at the same time. To detect its interference effects on the nasopharyngeal carcinoma cell line (CNE-2Z) compared with single gene plasmid VEFG (Plasmid-2). Methods According to the sequence of VEGF, C-myc, Survivin and hTERT gene, we designed 2 oligonucleotide sequences and synthesized a complementary DNA chain, then inserted it into the eukaryotic vector expressing pGenesil 1. The cell proliferation activity was detected by MTT method. The interference efficacy on human nasopharyngeal carcinoma cell line (CNE-2Z) in the level of mRNA and protein were detected by RT-PCR and Western-bolt. The inhibitory effect of plasmid on tumor in nude mice was also observed in vivo. Results The restriction enzyme digestion and sequencing technologies confirmed the construction of recombinant eukaryotic vector expressing was correct. The plasmid was transfected into CNE-2Z cells, green fluorescence can be seen clearly in the single gene and multi gene transfected cells under fluorescent microscope. MTT showed that the proliferation of cell was inhibited, the invasive ability was decreased in vitro, and the inhibition effects of single gene plasmid on the growth and proliferation of cells were lower than multi gene. Real-time-PCR and Western-bolt confirmed that the expression of target gene was decreased in the level of mRNA and protein, and the interference effect of multi gene was better than the single gene. The nude mice experiment showed that the interference effect of shRNA plasmid on the growth of tumor cell was better than single gene plasmid Conclusion We constructed a shRNA plasmid encoded four different genes successfully. After transfected with nasopharyngeal carcinoma cells, it can interfere the expression of VEGF, C-myc, Survivin and hTERT gene at the same time. And the interference effect was better than silence VEGF alone

  1. Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis

    PubMed Central

    Matvienko, Marta; Piskurewicz, Urszula; Xu, Huaqin; Martineau, Belinda; Wong, Joan; Govindarajulu, Manjula; Kozik, Alexander; Michelmore, Richard W.

    2014-01-01

    The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. PMID:25344399

  2. Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators.

    PubMed

    Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

    2014-01-01

    Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators.

  3. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    SciTech Connect

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  4. Illuminating the gateway of gene silencing: perspective of RNA interference technology in clinical therapeutics.

    PubMed

    Sindhu, Annu; Arora, Pooja; Chaudhury, Ashok

    2012-07-01

    A novel laboratory revolution for disease therapy, the RNA interference (RNAi) technology, has adopted a new era of molecular research as the next generation "Gene-targeted prophylaxis." In this review, we have focused on the chief technological challenges associated with the efforts to develop RNAi-based therapeutics that may guide the biomedical researchers. Many non-curable maladies, like neurodegenerative diseases and cancers have effectively been cured using this technology. Rapid advances are still in progress for the development of RNAi-based technologies that will be having a major impact on medical research. We have highlighted the recent discoveries associated with the phenomenon of RNAi, expression of silencing molecules in mammals along with the vector systems used for disease therapeutics.

  5. Therapeutic potentials of gene silencing by RNA interference: principles, challenges, and new strategies.

    PubMed

    Deng, Yan; Wang, Chi Chiu; Choy, Kwong Wai; Du, Quan; Chen, Jiao; Wang, Qin; Li, Lu; Chung, Tony Kwok Hung; Tang, Tao

    2014-04-01

    During recent decades there have been remarkable advances in biology, in which one of the most important discoveries is RNA interference (RNAi). RNAi is a specific post-transcriptional regulatory pathway that can result in silencing gene functions. Efforts have been done to translate this new discovery into clinical applications for disease treatment. However, technical difficulties restrict the development of RNAi, including stability, off-target effects, immunostimulation and delivery problems. Researchers have attempted to surmount these barriers and improve the bioavailability and safety of RNAi-based therapeutics by optimizing the chemistry and structure of these molecules. This paper aimed to describe the principles of RNA interference, review the therapeutic potential in various diseases and discuss the new strategies for in vivo delivery of RNAi to overcome the challenges.

  6. HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing1[OPEN

    PubMed Central

    Hristova, Emilija; Fal, Kateryna; Klemme, Laurin; Windels, David; Bucher, Etienne

    2015-01-01

    To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides. PMID:25918117

  7. Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system.

    PubMed

    Takemoto, Hiroyasu; Ishii, Atsushi; Miyata, Kanjiro; Nakanishi, Masataka; Oba, Makoto; Ishii, Takehiko; Yamasaki, Yuichi; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2010-11-01

    An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery.

  8. Disruption of Spodoptera exigua larval development by silencing chitin synthase gene A with RNA interference.

    PubMed

    Chen, X; Tian, H; Zou, L; Tang, B; Hu, J; Zhang, W

    2008-12-01

    RNA interference (RNAi) is a powerful tool for rapidly analyzing gene functions. However, little is known about the possible use of dsRNA/siRNA as a pest control method. Here, we demonstrate that dsRNA/siRNA can induce the silence of chitin synthase gene A (CHSA), which is an important gene for the growth and development of cuticles and trachea in beet armyworm, Spodoptera exigua. Based on the in vitro RNAi experiments in an insect cell line (Trichoplusia ni High 5), in vivo RNAi was performed by injecting synthesized dsRNA/siRNA into the 4th instar larvae of S. exigua. Significantly lower levels of CHSA transcripts were detected. In addition, the cuticle of these insects was disordered and the epithelial walls of larval trachea did not expand uniformly in injected individuals. Moreover, Injections significantly increased abnormalities relative to control larvae. These results highlighted the possibility of dsRNA/siRNA for gene function studies in lepidopteran insects and future pest control. PMID:18662430

  9. Arabidopsis RPT2a, 19S proteasome subunit, regulates gene silencing via DNA methylation.

    PubMed

    Sako, Kaori; Maki, Yuko; Kanai, Tomoyuki; Kato, Eriko; Maekawa, Shugo; Yasuda, Shigetaka; Sato, Takeo; Watahiki, Masaaki K; Yamaguchi, Junji

    2012-01-01

    The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation. PMID:22615900

  10. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

    PubMed Central

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

    2016-01-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  11. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants.

    PubMed

    Li, Jian-Feng; Chung, Hoo Sun; Niu, Yajie; Bush, Jenifer; McCormack, Matthew; Sheen, Jen

    2013-05-01

    Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

  12. Polyethyleneimine (PEI) Mediated siRNA Gene Silencing in the Schistosoma mansoni Snail Host, Biomphalaria glabrata

    PubMed Central

    Knight, Matty; Miller, Andre; Liu, Yijia; Scaria, Puthupparampil; Woodle, Martin; Ittiprasert, Wannaporn

    2011-01-01

    An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a ‘mock’ fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease. PMID:21765961

  13. Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes.

    PubMed Central

    Zwicky, Roman; Müntener, Kathrin; Goldring, Mary B; Baici, Antonio

    2002-01-01

    Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies. PMID:12086583

  14. Muscle cell atrophy induced by HSP gene silencing was counteracted by HSP overexpression

    NASA Astrophysics Data System (ADS)

    Choi, Inho; Lee, Joo-Hee; Nikawa, Takeshi; Gwag, Taesik; Park, Kyoungsook; Park, Junsoo

    Heat shock proteins (HSP), as molecular chaperones, are known to assist protein quality control under various stresses. Although overexpression of HSP70 was found to contribute to muscle size retention under an unloading condition, it remains largely unclarified whether muscle atrophy is induced by active suppression of HSP expression. In this study, we pre-treated Hsp70 siRNA to rat L6 cells for the HSP gene silencing, and determined myotube diameter, HSP72 expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (CEL), the HSP70 inducer. Relative to a negative control (NC), muscle cell diameter was reduced 0.89-fold in the siRNA-treated group, increased 1.2-fold in the CEL-treated group and retained at the size of NC in the siRNA+CEL group. HSP72 expression was decreased 0.35-fold by siRNA whereas the level was increased 6- to 8-fold in the CEL and siRNA+CEL groups. Expression of FoxO3 and atrogin-1 was increased 1.8- to 4.8-fold by siRNA, which was abolished by CEL treatment. Finally, phosphorylation of Akt1, S6K and ERK1/2 was not affected by siRNA, but was elevated 2- to 6-fold in the CEL and siRNA+CEL groups. Taken together, HSP downregulation by Hsp gene silencing led to muscle cell atrophy principally via increases in catabolic activities and that such anti-atrophic effect was counteracted by HSP overexpression.

  15. Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing

    PubMed Central

    Davuluri, Ganga Rao; van Tuinen, Ageeth; Mustilli, Anna Chiara; Manfredonia, Alessandro; Newman, Robert; Burgess, Diane; Brummell, David A.; King, Stephen R.; Palys, Joe; Uhlig, John; Pennings, Henk M. J.; Bowler, Chris

    2013-01-01

    Summary The tomato HIGH PIGMENT-2 gene encodes an orthologue of the Arabidopsis nuclear protein DE-ETIOLATED 1 (DET1). From genetic analyses it has been proposed that DET1 is a negative regulator of light signal transduction, and recent results indicate that it may control light-regulated gene expression at the level of chromatin remodelling. To gain further understanding about the function of DET1 during plant development, we generated a range of overexpression constructs and introduced them into tomato. Unexpectedly, we only observed phenotypes characteristic of DET1 inactivation, i.e. hyper-responsiveness to light. Molecular analysis indicated in all cases that these phenotypes were a result of suppression of endogenous DET1 expression, due to post-transcriptional gene silencing. DET1 silencing was often lethal when it occurred at relatively early stages of plant development, whereas light hyper-responsive phenotypes were obtained when silencing occurred later on. The appearance of phenotypes correlated with the generation of siRNAs but not DNA hypermethylation, and was most efficient when using constructs with mutations in the DET1 coding sequence or with constructs containing only the 3′-terminal portion of the gene. These results indicate an important function for DET1 throughout plant development and demonstrate that silencing of DET1 in fruits results in increased carotenoids, which may have biotechnological potential. PMID:15469492

  16. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  17. Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

    PubMed Central

    Earley, Keith; Lawrence, Richard J.; Pontes, Olga; Reuther, Rachel; Enciso, Angel J.; Silva, Manuela; Neves, Nuno; Gross, Michael; Viegas, Wanda; Pikaard, Craig S.

    2006-01-01

    Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications. PMID:16648464

  18. Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes

    PubMed Central

    Matsuoka, Yuji; Bando, Tetsuya; Watanabe, Takahito; Ishimaru, Yoshiyasu; Noji, Sumihare; Popadić, Aleksandar; Mito, Taro

    2015-01-01

    In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. PMID:25948756

  19. Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.

    PubMed

    Matsuoka, Yuji; Bando, Tetsuya; Watanabe, Takahito; Ishimaru, Yoshiyasu; Noji, Sumihare; Popadić, Aleksandar; Mito, Taro

    2015-01-01

    In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. PMID:25948756

  20. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Facchini, Peter J

    2012-10-01

    Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3'-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3'-hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3'-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine. PMID:22725256

  1. Position-dependent silencing of germline Vß segments on TCRß alleles containing preassembled VßDJßCß1 genes.

    PubMed

    Brady, Brenna L; Oropallo, Michael A; Yang-Iott, Katherine S; Serwold, Thomas; Hochedlinger, Konrad; Jaenisch, Rudolf; Weissman, Irving L; Bassing, Craig H

    2010-09-15

    The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

  2. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

    PubMed

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  3. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

    PubMed Central

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  4. Rescue of Metabolic Alterations in AR113Q Skeletal Muscle by Peripheral Androgen Receptor Gene Silencing.

    PubMed

    Giorgetti, Elisa; Yu, Zhigang; Chua, Jason P; Shimamura, Ryosuke; Zhao, Lili; Zhu, Fan; Venneti, Sriram; Pennuto, Maria; Guan, Yuanfang; Hung, Gene; Lieberman, Andrew P

    2016-09-27

    Spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder, is caused by a CAG/glutamine expansion in the androgen receptor (polyQ AR). Recent studies demonstrate that skeletal muscle is an important site of toxicity that contributes to the SBMA phenotype. Here, we sought to identify critical pathways altered in muscle that underlie disease manifestations in AR113Q mice. This led to the unanticipated identification of gene expression changes affecting regulators of carbohydrate metabolism, similar to those triggered by denervation. AR113Q muscle exhibits diminished glycolysis, altered mitochondria, and an impaired response to exercise. Strikingly, the expression of genes regulating muscle energy metabolism is rescued following peripheral polyQ AR gene silencing by antisense oligonucleotides (ASO), a therapeutic strategy that alleviates disease. Our data establish the occurrence of a metabolic imbalance in SBMA muscle triggered by peripheral expression of the polyQ AR and indicate that alterations in energy utilization contribute to non-neuronal disease manifestations. PMID:27681426

  5. Applications of RNA interference-based gene silencing in animal agriculture.

    PubMed

    Long, Charles R; Tessanne, Kimberly J; Golding, Michael C

    2010-01-01

    Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.

  6. Rescue of Metabolic Alterations in AR113Q Skeletal Muscle by Peripheral Androgen Receptor Gene Silencing.

    PubMed

    Giorgetti, Elisa; Yu, Zhigang; Chua, Jason P; Shimamura, Ryosuke; Zhao, Lili; Zhu, Fan; Venneti, Sriram; Pennuto, Maria; Guan, Yuanfang; Hung, Gene; Lieberman, Andrew P

    2016-09-27

    Spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder, is caused by a CAG/glutamine expansion in the androgen receptor (polyQ AR). Recent studies demonstrate that skeletal muscle is an important site of toxicity that contributes to the SBMA phenotype. Here, we sought to identify critical pathways altered in muscle that underlie disease manifestations in AR113Q mice. This led to the unanticipated identification of gene expression changes affecting regulators of carbohydrate metabolism, similar to those triggered by denervation. AR113Q muscle exhibits diminished glycolysis, altered mitochondria, and an impaired response to exercise. Strikingly, the expression of genes regulating muscle energy metabolism is rescued following peripheral polyQ AR gene silencing by antisense oligonucleotides (ASO), a therapeutic strategy that alleviates disease. Our data establish the occurrence of a metabolic imbalance in SBMA muscle triggered by peripheral expression of the polyQ AR and indicate that alterations in energy utilization contribute to non-neuronal disease manifestations.

  7. Transformation of the US bread wheat 'Butte 86' and silencing of omega-5 gliadin genes.

    PubMed

    Altenbach, Susan B; Allen, Paul V

    2011-01-01

    Complex groups of proteins determine the unique functional properties of wheat flour and are sometimes responsible for food intolerances and allergies in individuals that consume wheat products. Transgenic approaches can be used to explore the functions of different flour proteins, but are limited to the few wheat cultivars that can be transformed and also by the lack of detailed information about genes and proteins expressed in grain from those cultivars. The US bread wheat Butte 86 has been extensively characterized and a comprehensive proteome map was developed in which flour proteins were distinguished by mass spectrometry and associated with specific gene sequences. Here, this information has been used to design an RNA interference construct to silence the expression of genes encoding omega gliadins that trigger the food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA). The construct was introduced into immature embryos from Butte 86 using biolistics and bialaphos-resistant plants were regenerated. Stable transformation and inheritance of the transgene were confirmed by PCR. Analysis of proteins in grain from transgenic plants demonstrated that the omega-5 gliadins were either absent or substantially reduced relative to non-transformed controls. The ability to genetically transform Butte 86 makes it possible to alter flour composition in a targeted manner in a commercial US wheat cultivar and should accelerate future research on flour quality and immunogenic potential. PMID:21844700

  8. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae.

    PubMed

    Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W; Zhu, Kun Yan; Duman-Scheel, Molly

    2015-03-25

    Vector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

  9. Demethylation and re-expression of epigenetically silenced tumor suppressor genes: sensitization of cancer cells by combination therapy.

    PubMed

    Sarkar, Sibaji; Goldgar, Sarah; Byler, Shannon; Rosenthal, Shoshana; Heerboth, Sarah

    2013-02-01

    Epigenetic regulation in eukaryotic and mammalian systems is a complex and emerging field of study. While histone modifications create an open chromatin conformation allowing for gene transcription, CpG methylation adds a further dimension to the expression of specific genes in developmental pathways and carcinogenesis. In this review, we will highlight DNA methylation as one of the distinct mechanisms for gene silencing and try to provide insight into the role of epigenetics in cancer progenitor cell formation and carcinogenesis. We will also introduce the concept of a dynamic methylation-demethylation system and the potential for the existence of a demethylating enzyme in this process. Finally, we will explain how re-expression of epigenetically silenced tumor suppressor genes could be exploited to develop effective drug therapies. In particular, we will consider how a combination therapy that includes epigenetic drugs could possibly kill cancer progenitor cells and reduce the chance of relapse following chemotherapy. PMID:23414323

  10. V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants.

    PubMed

    Wang, Bi; Li, Fangfang; Huang, Changjun; Yang, Xiuling; Qian, Yajuan; Xie, Yan; Zhou, Xueping

    2014-01-01

    Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity. PMID:24187017

  11. Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus.

    PubMed

    de Ronde, Dryas; Butterbach, Patrick; Lohuis, Dick; Hedil, Marcio; van Lent, Jan W M; Kormelink, Richard

    2013-05-01

    As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs(RI) protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N(RI) (/) (RB) protein, or when NSs(RB) was expressed. Although NSs(RI) was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs(RB) had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.

  12. An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

    PubMed

    Frøkjær-Jensen, Christian; Jain, Nimit; Hansen, Loren; Davis, M Wayne; Li, Yongbin; Zhao, Di; Rebora, Karine; Millet, Jonathan R M; Liu, Xiao; Kim, Stuart K; Dupuy, Denis; Jorgensen, Erik M; Fire, Andrew Z

    2016-07-14

    Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure. PMID:27374334

  13. Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

    PubMed

    Peralta-Zaragoza, Oscar; De-la-O-Gómez, Faustino; Deas, Jessica; Fernández-Tilapa, Gloria; Fierros-Zárate, Geny Del Socorro; Gómez-Cerón, Claudia; Burguete-García, Ana; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Madrid-Marina, Vicente

    2015-01-01

    RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells. PMID:25348304

  14. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

    PubMed

    Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. PMID:26079224

  15. Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

    PubMed

    Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

    2016-02-01

    Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes.

  16. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  17. Gene silencing of 4-1BB by RNA interference inhibits acute rejection in rats with liver transplantation.

    PubMed

    Shi, Yang; Hu, Shuqun; Song, Qingwei; Yu, Shengcai; Zhou, Xiaojun; Yin, Jun; Qin, Lei; Qian, Haixin

    2013-01-01

    The 4-1BB signal pathway plays a key role in organ transplantation tolerance. In this study, we have investigated the effect of gene silencing of 4-1BB by RNA interference (RNAi) on the acute rejection in rats with liver transplantation. The recombination vector of lentivirus that contains shRNA targeting the 4-1BB gene (LV-sh4-1BB) was constructed. The liver transplantation was performed using the two-cuff technique. Brown-Norway (BN) recipient rats were infected by the recombinant LVs. The results showed that gene silencing of 4-1BB by RNAi downregulated the 4-1BB gene expression of the splenic lymphocytes in vitro, and the splenic lymphocytes isolated from the rats with liver transplantation. LV-sh4-1BB decreased the plasma levels of liver injury markers including AST, ALT, and BIL and also decreased the level of plasma IL-2 and IFN- γ in recipient rats with liver transplantation. Lentivirus-mediated delivery of shRNA targeting 4-1BB gene prolonged the survival time of recipient and alleviated the injury of liver morphology in recipient rats with liver transplantation. In conclusion, our results demonstrate that gene silencing of 4-1BB by RNA interference inhibits the acute rejection in rats with liver transplantation.

  18. Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

    PubMed

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

    2009-12-31

    phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

  19. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

    PubMed

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

    2016-06-28

    Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.

  20. Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga.

    PubMed

    Rodriguez, Fernando; Arkhipova, Irina R

    2016-05-01

    RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25-31 nucleotides in length and have a strong 5'-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3'-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome.

  1. Multitasking of the piRNA Silencing Machinery: Targeting Transposable Elements and Foreign Genes in the Bdelloid Rotifer Adineta vaga.

    PubMed

    Rodriguez, Fernando; Arkhipova, Irina R

    2016-05-01

    RNA-mediated silencing processes play a key role in silencing of transposable elements, especially in the germ line, where piwi-interacting RNAs (piRNAs) are responsible for suppressing transposon mobility and maintaining genome integrity. We previously reported that the genome of Adineta vaga, the first sequenced representative of the phylum Rotifera (class Bdelloidea), is characterized by massive levels of horizontal gene transfer, by unusually low transposon content, and by highly diversified RNA-mediated silencing machinery. Here, we investigate genome-wide distribution of pi-like small RNAs, which in A. vaga are 25-31 nucleotides in length and have a strong 5'-uridine bias, while lacking ping-pong amplification signatures. In agreement with expectations, 71% of mapped reads corresponded to annotated transposons, with 93% of these reads being in the antisense orientation. Unexpectedly, a significant fraction of piRNAs originate from predicted coding regions corresponding to genes of putatively foreign origin. The distribution of piRNAs across foreign genes is not biased toward 3'-UTRs, instead resembling transposons in uniform distribution pattern throughout the gene body, and in predominantly antisense orientation. We also find that genes with small RNA coverage, including a number of genes of metazoan origin, are characterized by higher occurrence of telomeric repeats in the surrounding genomic regions, and by higher density of transposons in the vicinity, which have the potential to promote antisense transcription. Our findings highlight the complex interplay between RNA-based silencing processes and acquisition of genes at the genome periphery, which can result either in their loss or eventual domestication and integration into the host genome. PMID:27017627

  2. Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    SciTech Connect

    Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo

    2013-04-05

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

  3. siRNA Versus miRNA as Therapeutics for Gene Silencing.

    PubMed

    Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

    2015-09-15

    Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed.

  4. siRNA Versus miRNA as Therapeutics for Gene Silencing

    PubMed Central

    Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

    2015-01-01

    Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed. PMID:26372022

  5. Nuclear factor 1 regulates the distal silencer of the human PIT1/GHF1 gene.

    PubMed Central

    Rajas, F; Delhase, M; De La Hoya, M; Verdood, P; Castrillo, J L; Hooghe-Peters, E L

    1998-01-01

    Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2. 3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region. PMID:9639565

  6. Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

    PubMed

    Wan, Jian-Xin; Fukuda, Noboru; Ueno, Takahiro; Watanabe, Takayoshi; Matsuda, Hiroyuki; Saito, Kosuke; Nagase, Hiroki; Matsumoto, Yoshiaki; Matsumoto, Koichi

    2011-01-01

    Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-β1 (TGF-β1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-β1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-β1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.

  7. Stability and immunogenicity properties of the gene-silencing polypurine reverse Hoogsteen hairpins.

    PubMed

    Villalobos, Xenia; Rodríguez, Laura; Prévot, Jeanne; Oleaga, Carlota; Ciudad, Carlos J; Noé, Véronique

    2014-01-01

    Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.

  8. Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis.

    PubMed

    van der Linde, Karina; Kastner, Christine; Kumlehn, Jochen; Kahmann, Regine; Doehlemann, Gunther

    2011-01-01

    Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.

  9. Enteral siRNA delivery technique for therapeutic gene silencing in the liver via the lymphatic route.

    PubMed

    Murakami, Masahiro; Nishina, Kazutaka; Watanabe, Chie; Yoshida-Tanaka, Kie; Piao, Wenying; Kuwahara, Hiroya; Horikiri, Yuji; Miyata, Kanjiro; Nishiyama, Nobuhiro; Kataoka, Kazunori; Yoshida, Masayuki; Mizusawa, Hidehiro; Yokota, Takanori

    2015-11-23

    An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver.

  10. Enteral siRNA delivery technique for therapeutic gene silencing in the liver via the lymphatic route

    PubMed Central

    Murakami, Masahiro; Nishina, Kazutaka; Watanabe, Chie; Yoshida-Tanaka, Kie; Piao, Wenying; Kuwahara, Hiroya; Horikiri, Yuji; Miyata, Kanjiro; Nishiyama, Nobuhiro; Kataoka, Kazunori; Yoshida, Masayuki; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver. PMID:26593819

  11. Evaluation of two gene-silencing constructs for resistance to tomato yellow leaf curl viruses in Nicotiana benthamiana plants.

    PubMed

    Gharsallah Chouchane, S; Gorsane, F; Nakhla, M K; Salus, M; Martin, C T; Maxwell, D P; Marrakchi, M; Fakhfakh, H

    2008-01-01

    Infiltration of Agrobacterium tumefaciens into intact plant leaves of N. benthamiana was used to test the efficiency of two virus-based silencing constructs conferring resistance to the closely related begomoviruses. The constructs contained the most conserved sequences of the coat protein (CP) gene and replication-associated protein (Rep) gene of Tomato yellow leaf curl Sardinia virus (Sicily strain) (TYLCSV-[Sic]). Both constructs formed a hairpin structure that enhanced the post-transcriptional gene-silencing mechanism. When agro-infiltrated plants were challenged separately with infectious viruses TYLCSV-[Sic] and Tomato yellow leaf curl virus (TYLCV), the plants showed resistance to TYLCSV-[Sic], but not to the related TYLCV. PMID:18999888

  12. Use of Posttranscription Gene Silencing in Squash to Induce Resistance against the Egyptian Isolate of the Squash Leaf Curl Virus.

    PubMed

    Taha, Omnia; Farouk, Inas; Abdallah, Abdelhadi; Abdallah, Naglaa A

    2016-01-01

    Squash leaf curl virus (SqLCV) is a bipartite begomovirus affecting squash plants. It is transmitted by whitefly Bemisia tabaci biotype B causing severe leaf curling, vein banding, and molting ending by stunting. In this study full-length genomic clone of SqLCV Egyptian isolated and posttranscriptional gene silencing (PTGS) has been induced to develop virus resistance. The Noubaria SqLCV has more than 95% homology with Jordon, Israel, Lebanon, Palestine, and Cairo isolates. Two genes fragment from SqLCV introduced in sense and antisense orientations using pFGC5049 vector to be expressed as hairpin RNA. The first fragment was 348 bp from replication associated protein gene (Rep). The second fragment was 879 bp representing the full sequence of the movement protein gene (BC1). Using real-time PCR, a silencing record of 97% has been recorded to Rep/TrAP construct; as a result it has prevented the appearance of viral symptoms in most tested plants up to two months after infection, while construct containing the BC1 gene scored a reduction in the accumulation of viral genome expression as appearing in real-time PCR results 4.6-fold giving a silencing of 79%, which had a positive effect on symptoms development in most tested plants. PMID:27034922

  13. Use of Posttranscription Gene Silencing in Squash to Induce Resistance against the Egyptian Isolate of the Squash Leaf Curl Virus

    PubMed Central

    Taha, Omnia; Farouk, Inas; Abdallah, Abdelhadi

    2016-01-01

    Squash leaf curl virus (SqLCV) is a bipartite begomovirus affecting squash plants. It is transmitted by whitefly Bemisia tabaci biotype B causing severe leaf curling, vein banding, and molting ending by stunting. In this study full-length genomic clone of SqLCV Egyptian isolated and posttranscriptional gene silencing (PTGS) has been induced to develop virus resistance. The Noubaria SqLCV has more than 95% homology with Jordon, Israel, Lebanon, Palestine, and Cairo isolates. Two genes fragment from SqLCV introduced in sense and antisense orientations using pFGC5049 vector to be expressed as hairpin RNA. The first fragment was 348 bp from replication associated protein gene (Rep). The second fragment was 879 bp representing the full sequence of the movement protein gene (BC1). Using real-time PCR, a silencing record of 97% has been recorded to Rep/TrAP construct; as a result it has prevented the appearance of viral symptoms in most tested plants up to two months after infection, while construct containing the BC1 gene scored a reduction in the accumulation of viral genome expression as appearing in real-time PCR results 4.6-fold giving a silencing of 79%, which had a positive effect on symptoms development in most tested plants. PMID:27034922

  14. A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

    PubMed Central

    Rosen, Ohad; Manor, Rivka; Weil, Simy; Gafni, Ohad; Linial, Assaf; Aflalo, Eliahu D.; Ventura, Tomer; Sagi, Amir

    2010-01-01

    In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins. PMID:21151555

  15. The gene silencing transcription factor REST represses miR-132 expression in hippocampal neurons destined to die.

    PubMed

    Hwang, Jee-Yeon; Kaneko, Naoki; Noh, Kyung-Min; Pontarelli, Fabrizio; Zukin, R Suzanne

    2014-10-01

    The gene silencing transcription factor REST [repressor element 1 silencing transcription factor]/NRSF (neuron-restrictive silencer factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of repressor element 1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke. PMID:25108103

  16. Methods for effective real-time RT-PCR analysis of virus-induced gene silencing.

    PubMed

    Rotenberg, Dorith; Thompson, Thea S; German, Thomas L; Willis, David K

    2006-12-01

    We applied real-time RT-PCR to the analysis of Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in Nicotiana benthamiana and tomato. Using a combination of direct measurement and mathematical assessment, we evaluated three plant genes, ubiquitin (ubi3), elongation factor-1 alpha (EF-1), and actin, for use as internal reference transcripts and found that EF-1 and ubi3 were least variable under our experimental conditions. Primer sets designed to amplify the 5' or 3' regions of endogenous PDS transcripts in tomato yielded similar reductions in transcript levels indicating a uniform VIGS-mediated degradation of target RNA. By measuring the ratio of the abundance of the PDS insert transcript to the TRV coat protein RNA, we established that the PDS insert within TRV was stable in both hosts. VIGS in N. benthamiana resulted in complete photo-bleaching of all foliar tissue compared to chimeric bleaching in tomato. PDS transcript levels were decreased eleven- and seven-fold in photobleached leaves of N. benthamiana and tomato, respectively, while sampling tomato leaflets on the basis of age rather than visible bleaching resulted in only a 17% reduction in PDS coupled with a large leaf-to-leaf variation. There was a significant inverse relationship (r2=76%, P=0.01) between the relative abundance of CP RNA and the amount of PDS transcript in rTRV::tPDS-infected tomato suggesting that virus spread and accumulation are required precursors for successful VIGS in this host. PMID:16959330

  17. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    SciTech Connect

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-06-15

    Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

  18. Gene silencing of nfa1 affects the in vitro cytotoxicity of Naegleria fowleri in murine macrophages.

    PubMed

    Jung, Suk-Yul; Kim, Jong-Hyun; Song, Kyoung-Ju; Lee, Yang-Jin; Kwon, Myung-Hee; Kim, Kyongmin; Park, Sun; Im, Kyung-il; Shin, Ho-Joon

    2009-05-01

    The gene nfa1 was isolated from the free-living pathogenic amoeba Naegleria fowleri. The protein Nfa1 is located in pseudopodia and specifically in food-cups. It is also involved in cytotoxicity. In this study, we used synthetic small interfering RNAs (siRNA) to examine the effects of nfa1 down-regulation. We observed the expression of nfa1 mRNA and Nfa1 protein using Northern and Western blots. We also examined the effects of nfa1 down-regulation on the in vitro cytotoxicity of N. fowleri. Four synthetic siRNAs were constructed, and of those, sinfa1-1 showed the highest down-regulation of an nfa1 mRNA and Nfa1 protein by 70 and 43%, respectively. In order to achieve long-lasting silencing of the transfected genes, we constructed two vectors which were pAct/SAGAH and pAct/asnfa1AGAH cloned with the sinfa1-1 and an antisense RNA to the nfa1 gene. In N. fowleri transfected with pAct/SAGAH, FACS revealed a 60 and 57% reduction in nfa1 mRNA and Nfa1 protein levels, respectively. To determine whether the Nfa1 proteins were related with in vitro cytotoxicity, LDH assays were used and showed that the cytotoxicity of these transfectants to macrophages was reduced by 26.4 and 36.2% at 17 and 24h, respectively. Moreover, after transfection with pAct/asnfa1AGAH, amoebic cytotoxicity decreased by 8.2 and 10% at 17 and at 24h, respectively. This is the first report to show the RNA interference in N. folweri trophozoites and also demonstrate the Nfa1 function in vitro for its cytotoxicity.

  19. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

    PubMed

    Ding, Xin Shun; Schneider, William L; Chaluvadi, Srinivasa Rao; Mian, M A Rouf; Nelson, Richard S

    2006-11-01

    Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

  20. Virus-induced gene silencing (VIGS) in Cysticapnos vesicaria, a zygomorphic-flowered Papaveraceae (Ranunculales, basal eudicots)

    PubMed Central

    Hidalgo, Oriane; Bartholmes, Conny; Gleissberg, Stefan

    2012-01-01

    Background and Aims Studies of evolutionary diversification in the basal eudicot family Papaveraceae, such as the transition from actinomorphy to zygomorphy, are hampered by the lack of comparative functional studies. So far, gene silencing methods are only available in the actinomorphic species Eschscholzia californica and Papaver somniferum. This study addresses the amenability of Cysticapnos vesicaria, a derived fumitory with zygomorphic flowers, to virus-induced gene silencing (VIGS), and describes vegetative and reproductive traits in this species. Methods VIGS-mediated downregulation of the C. vesicaria PHYTOENE DESATURASE gene (CvPDS) and of the FLORICAULA gene CvFLO was carried out using Agrobacterium tumefaciens transfer of Tobacco rattle virus (TRV)-based vectors. Wild-type and vector-treated plants were characterized using reverse transcription–PCR (RT–PCR), in situ hybridization, and macroscopic and scanning electron microscopic imaging. Key Results Cysticapnos vesicaria germinates rapidly, can be grown at high density, has a short life cycle and is self-compatible. Inoculation of C. vesicaria with a CvPDS-VIGS vector resulted in strong photobleaching of green parts and reduction of endogenous CvPDS transcript levels. Gene silencing persisted during inflorescence development until fruit set. Inoculation of plants with CvFLO-VIGS affected floral phyllotaxis, symmetry and floral organ identities. Conclusions The high penetrance, severity and stability of pTRV-mediated silencing, including the induction of meristem-related phenotypes, make C. vesicaria a very promising new focus species for evolutionary–developmental (evo–devo) studies in the Papaveraceae. This now enables comparative studies of flower symmetry, inflorescence determinacy and other traits that diversified in the Papaveraceae. PMID:22307568

  1. Epitope-tagged protein-based artificial microRNA (ETPamir) screens for optimized gene silencing in plants

    PubMed Central

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-01-01

    Artificial microRNA (amiRNA) technology offers highly specific and versatile gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple target genes encoding epitope-tagged proteins are constitutively or inducibly co-expressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with a commercial tag antibody, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 days. The ETPamir screens circumvent the widespread shortage of plant antibodies and the complexity of plant amiRNA silencing at target mRNA or/and protein levels. This method can be extended to verify predicted endogenous target genes for plant natural miRNAs. PMID:24675734

  2. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants.

    PubMed

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-04-01

    Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.

  3. A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica

    PubMed Central

    2014-01-01

    Background Striga hermonthica is a hemiparasitic weed that infects cereals in Sub Sahara Africa (SSA) resulting in up to 100% grain yield loss. This significant loss in grain yields is a major contributor to food insecurity and poverty in the region. Current strategies to control the parasite are costly, unavailable and remain unpracticed by small-scale farmers, underscoring the need for more economical and sustainable control strategies. Development of resistant germplasm is the most sustainable strategy in the control of S. hermonthica, but is constrained by paucity of resistance genes for introduction into crop germplasm. RNA interference (RNAi) has potential for developing host-derived resistance against S. hermonthica by transformation of host crops with RNAi sequences targeted at critical Striga genes. The application of RNAi in management of S. hermonthica is however constrained by lack of efficient high throughput screening protocols for the candidate genes for silencing, as well as sub optimal delivery of siRNAs into the parasite. In comparison to stable transformation, viral induced gene silencing (VIGS) is a rapid and powerful tool for plant functional genomics and provides an easy and effective strategy in screening for putative candidate genes to target through RNAi. In addition, VIGS allows for a secondary amplification of the RNAi signal increasing the siRNA threshold and facilitates siRNA transport through viral movement proteins. We tested the efficiency of the Tobacco rattle virus (TRV1 and TRV2) VIGS vectors in silencing S. hermonthica phytoene desaturase (PDS) gene through agrodrench and agro-infiltration. Results We report the validation of VIGS in S. hermonthica using a silencing cassette generated from TRV with a PDS gene insert. Agro-infiltrated and agro-drenched S. hermonthica leaves showed photo-bleaching phenotypes typical for PDS silencing within 7 and 14 days post infection respectively. In both cases S. hermonthica plants recovered

  4. Dentin Sialophosphoprotein (DSPP) Gene-Silencing Inhibits Key Tumorigenic Activities in Human Oral Cancer Cell Line, OSC2

    PubMed Central

    Joshi, Rajeshree; Tawfik, Amany; Edeh, Nneka; McCloud, Veronica; Looney, Stephen; Lewis, Jill; Hsu, Stephen; Ogbureke, Kalu U. E.

    2010-01-01

    Background We determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells. Methodology/Principal Findings Multiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y = 0.850x, p<0.001) (y = 1.156x, p<0.001)}, MMP-3 {(y = 0.994x, p<0.001) (y = 1.324x, p = 0.004)}, and MMP-9 {(y = 1.248x, p = 0.005, y = 0.809, p = 0.013)}. Conclusions/Significance DSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The

  5. RNAi revised - target mRNA-dependent enhancement of gene silencing

    PubMed Central

    Dornseifer, Simon; Willkomm, Sarah; Far, Rosel Kretschmer-Kazemi; Liebschwager, Janine; Beltsiou, Foteini; Frank, Kirsten; Laufer, Sandra D.; Martinetz, Thomas; Sczakiel, Georg; Claussen, Jens Christian; Restle, Tobias

    2015-01-01

    The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery. PMID:26578554

  6. Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana

    PubMed Central

    Hale, Christopher J.; Potok, Magdalena E.; Lopez, Jennifer; Do, Truman; Liu, Ao; Michaels, Scott D.; Jacobsen, Steven E.

    2016-01-01

    Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin. PMID:27253878

  7. Silencing of stat4 gene inhibits cell proliferation and invasion of colorectal cancer cells.

    PubMed

    Cheng, J M; Yao, M R; Zhu, Q; Wu, X Y; Zhou, J; Tan, W L; Zhan, S H

    2015-01-01

    Signal transducers and activators of transcription (STAT) play critical roles in development, proliferation, and immune defense. However the consequences of STAT hyperactivity can predispose to diseases, including colorectal cancer. In the present study, we aimed to evaluate the function of STAT4 in human colorectal cancer (CRC). The expression of STAT4 was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was carried out to investigate the effects of lentivirus-mediated STAT4 shRNA (Lv-shSTAT4) on cell proliferation and invasive potential indicated by MTT and Transwell assays in CRC cell lines (SW480 and Caco2). As a consequence, it was found that the expression of STAT4 protein was significantly increased in CRC tissues compared with that in adjacent non-cancerous tissues (ANCT) (71.1% vs 44.4%, P=0.015), and was related with the Duke’s staging and depth of invasion in CRC patients (P=0.022; P=0.001). Silencing of STAT4 gene suppressed cell proliferation and invasion of CRC cells. Taken together, these findings demonstrate that increased expression of STAT4 is positively correlated with the depth of invasion in CRC patients, and inhibition of STAT4 expression represses the growth and invasion of CRC cells, suggesting that STAT4 may be a promising therapeutic target for the treatment of CRC.

  8. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

    PubMed Central

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-01-01

    Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

  9. RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii.

    PubMed

    Godman, James E; Molnár, Attila; Baulcombe, David C; Balk, Janneke

    2010-11-01

    The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

  10. Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in glycine max seed coats.

    PubMed

    Tuteja, Jigyasa H; Zabala, Gracia; Varala, Kranthi; Hudson, Matthew; Vodkin, Lila O

    2009-10-01

    Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.

  11. RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis.

    PubMed

    Safavian, Darya; Zayed, Yara; Indriolo, Emily; Chapman, Laura; Ahmed, Abdalla; Goring, Daphne R

    2015-12-01

    Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance.

  12. RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis.

    PubMed

    Safavian, Darya; Zayed, Yara; Indriolo, Emily; Chapman, Laura; Ahmed, Abdalla; Goring, Daphne R

    2015-12-01

    Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance. PMID:26443677

  13. Quantifying the sequence–function relation in gene silencing by bacterial small RNAs

    PubMed Central

    Hao, Yue; Zhang, Zhongge J.; Erickson, David W.; Huang, Min; Huang, Yingwu; Li, Junbai; Hwa, Terence; Shi, Hualin

    2011-01-01

    Sequence–function relations for small RNA (sRNA)-mediated gene silencing were quantified for the sRNA RyhB and some of its mRNA targets in Escherichia coli. Numerous mutants of RyhB and its targets were generated and their in vivo functions characterized at various levels of target and RyhB expression. Although a core complementary region is required for repression by RyhB, variations in the complementary sequences of the core region gave rise to a continuum of repression strengths, correlated exponentially with the computed free energy of RyhB-target duplex formation. Moreover, sequence variations in the linker region known to interact with the RNA chaperone Hfq also gave rise to a continuum of repression strengths, correlated exponentially with the computed energy cost of keeping the linker region open. These results support the applicability of the thermodynamic model in predicting sRNA–mRNA interaction and suggest that sequences at these locations may be used to fine-tune the degree of repression. Surprisingly, a truncated RyhB without the Hfq-binding region is found to repress multiple targets of the wild-type RyhB effectively, both in the presence and absence of Hfq, even though the former is required for the activity of wild-type RyhB itself. These findings challenge the commonly accepted model concerning the function of Hfq in gene silencing—both in providing stability to the sRNAs and in catalyzing the target mRNAs to take on active conformations—and raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences. PMID:21742981

  14. Transcriptional Gene Silencing Mediated by a Plastid Inner Envelope Phosphoenolpyruvate/Phosphate Translocator CUE1 in Arabidopsis1[OA

    PubMed Central

    Shen, Jie; Ren, Xiaozhi; Cao, Rui; Liu, Jun; Gong, Zhizhong

    2009-01-01

    Mutations in REPRESSOR OF SILENCING1 (ROS1) lead to the transcriptional gene silencing (TGS) of ProRD29A:LUC (LUCIFERASE) and Pro35S:NPTII (Neomycin Phosphotransferase II) reporter genes. We performed a genetic screen to find suppressors of ros1 that identified two mutant alleles in the Arabidopsis (Arabidopsis thaliana) CHLOROPHYLL A/B BINDING PROTEIN UNDEREXPRESSED1 (CUE1) gene, which encodes a plastid inner envelope phosphoenolpyruvate/phosphate translocator. The cue1 mutations released the TGS of Pro35S:NPTII and the transcriptionally silent endogenous locus TRANSCRIPTIONAL SILENCING INFORMATION in a manner that was independent of DNA methylation but dependent on chromatin modification. The cue1 mutations did not affect the TGS of ProRD29A:LUC in ros1, which was dependent on RNA-directed DNA methylation. It is possible that signals from chloroplasts help to regulate the epigenetic status of a subset of genomic loci in the nucleus. PMID:19515789

  15. Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles.

    PubMed

    Ohnishi, Yusuke; Tokunaga, Katsushi; Kaneko, Kiyotoshi; Hohjoh, Hirohiko

    2006-02-28

    Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASPRNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer's disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3'-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid beta 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

  16. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

    PubMed Central

    Foda, Bardees M.; Singh, Upinder

    2015-01-01

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  17. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

  18. RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae1

    PubMed Central

    Subramanian, Senthil; Graham, Madge Y.; Yu, Oliver; Graham, Terrence L.

    2005-01-01

    Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing. PMID:15778457

  19. Silencing of the TGF-β1 Gene Increases the Immunogenicity of Cells from Human Ovarian Carcinoma

    PubMed Central

    Wei, Huafeng; Liu, Pu; Swisher, Elizabeth; Yip, Yuen Yee; Tse, Jee Hang; Agnew, Kathy; Hellström, Karl Erik; Hellström, Ingegerd

    2012-01-01

    Cells from many tumors produce transforming growth factor (TGF)-βwhich facilitates their escape from control by the immune system. We previously reported that non-immunogenic cells from either of two transplantable mouse tumors became effective as therapeutic tumor vaccines after lentivirus-mediated shRNA interference to ‘silence’the TGF-β1 gene. We now show that cells from in vitro cultured human ovarian carcinomas (OvC) make large amounts of TGF-β1 and that this can be prevented by ‘silencing’ the TGF-β1 gene. We further show that in vitro sensitization of peripheral blood mononuclear cells (PBMC) in the presence of either mitomycin-treated OvC cells whose TGF-β1 gene was silenced or in vitro matured dendritic cells (DC) which had been pulsed with homogenates from OvC cells with silenced TGF-β1 generated a stronger Th1/Tc1 immune response to the respective WT OvC and also to the OvC antigens mesothelin and HE4 as measured by ELIspot assays. The percentage of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing CD4+ and CD8+ T cells increased while there were fewer cells expressing markers characteristic for regulatory T cells or myeloid derived suppressor cells. Similar results were obtained when PBMC from a patient with OvC were sensitized to DC pulsed with homogenate from autologous TGF-β1-silenced tumor cells, and a cytolytic lymphocyte response was generated to autologous OvC cells. Our results support clinical evaluation of TGF-β1-silenced tumor vaccines for immunotherapy of OvC. PMID:22421944

  20. Analysis of the siRNA-mediated gene silencing process targeting three homologous genes controlling soybean seed oil quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the discovery of RNA silencing in the nineties, the implication and potential application of this new technology have been recognized. In the past decades, RNA silencing has gained significant attention because its success in genomic scale research and also in the genetic improvement of crop p...

  1. Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer

    PubMed Central

    Lando, Malin; Fjeldbo, Christina S; Wilting, Saskia M; Snoek, Barbara C; Aarnes, Eva-Katrine; Forsberg, Malin F; Kristensen, Gunnar B; Steenbergen, Renske DM; Lyng, Heidi

    2015-01-01

    Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved

  2. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    SciTech Connect

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-04-10

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  3. Fine-tuning silencing.

    PubMed

    Panning, Barbara

    2010-01-01

    Polycomb Repressive Complex 2 (PRC2) modifies chromatin to silence many embryonic patterning genes, restricting their expression to the appropriate cell populations. Two reports in Cell by Peng et al. (2009) and Shen et al. (2009) identify Jarid2/Jumonji, a new component of PRC2, which inhibits PRC2 enzymatic activity to fine-tune silencing.

  4. Reverse Genetics of Floral Scent: Application of Tobacco Rattle Virus-Based Gene Silencing in Petunia1[OA

    PubMed Central

    Spitzer, Ben; Zvi, Michal Moyal Ben; Ovadis, Marianna; Marhevka, Elena; Barkai, Oren; Edelbaum, Orit; Marton, Ira; Masci, Tania; Alon, Michal; Morin, Shai; Rogachev, Ilana; Aharoni, Asaph; Vainstein, Alexander

    2007-01-01

    Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes. PMID:17720754

  5. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

    PubMed

    Campos, Laura; Granell, Pablo; Tárraga, Susana; López-Gresa, Pilar; Conejero, Vicente; Bellés, José María; Rodrigo, Ismael; Lisón, Purificación

    2014-04-01

    We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

  6. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

    PubMed Central

    Kim, Kil Hyun; Lim, Seungmo; Kang, Yang Jae; Yoon, Min Young; Nam, Moon; Jun, Tae Hwan; Seo, Min-Jung; Baek, Seong-Bum; Lee, Jeom-Ho; Moon, Jung-Kyung; Lee, Suk-Ha; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun; Park, Chang-Hwan

    2016-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments. PMID:27147931

  7. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans.

    PubMed

    Kim, Kil Hyun; Lim, Seungmo; Kang, Yang Jae; Yoon, Min Young; Nam, Moon; Jun, Tae Hwan; Seo, Min-Jung; Baek, Seong-Bum; Lee, Jeom-Ho; Moon, Jung-Kyung; Lee, Suk-Ha; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun; Park, Chang-Hwan

    2016-04-01

    Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments. PMID:27147931

  8. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans.

    PubMed

    Kim, Kil Hyun; Lim, Seungmo; Kang, Yang Jae; Yoon, Min Young; Nam, Moon; Jun, Tae Hwan; Seo, Min-Jung; Baek, Seong-Bum; Lee, Jeom-Ho; Moon, Jung-Kyung; Lee, Suk-Ha; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun; Park, Chang-Hwan

    2016-04-01

    Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

  9. MagRET Nanoparticles: An Iron Oxide Nanocomposite Platform for Gene Silencing from MicroRNAs to Long Noncoding RNAs.

    PubMed

    Lellouche, E; Israel, L L; Bechor, M; Attal, S; Kurlander, E; Asher, V A; Dolitzky, A; Shaham, L; Izraeli, S; Lellouche, J-P; Michaeli, S

    2015-08-19

    Silencing of RNA to knock down genes is currently one of the top priorities in gene therapies for cancer. However, to become practical the obstacle of RNA delivery needs to be solved. In this study, we used innovative maghemite (γ-Fe2O3) nanoparticles, termed magnetic reagent for efficient transfection (MagRET), which are composed of a maghemite core that is surface-doped by lanthanide Ce(3/4+) cations using sonochemistry. Thereafter, a polycationic polyethylenimine (PEI) polymer phase is bound to the maghemite core via coordinative chemistry enabled by the [CeL(n)](3/4+)cations/complex. PEI oxidation was used to mitigate the in vivo toxicity. Using this approach, silencing of 80-100% was observed for mRNAs, microRNAs, and lncRNA in a variety of cancer cells. MagRET NPs are advantageous in hard to transfect leukemias. This versatile nanoscale carrier can silence all known types of RNAs and these MagRET NPs with oxidized PEI are not lethal upon injection, thus holding promise for therapeutic applications, as a theranostic tool. PMID:26056709

  10. ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

    PubMed

    Liu, Jing; Yu, Dongbo; Aiba, Yuichiro; Pendergraff, Hannah; Swayze, Eric E; Lima, Walt F; Hu, Jiaxin; Prakash, Thazha P; Corey, David R

    2013-11-01

    Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

  11. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  12. Silencing of Abcd1 and Abcd2 genes sensitizes astrocytes for inflammation: implication for X-adrenoleukodystrophy*

    PubMed Central

    Singh, Jaspreet; Khan, Mushfiquddin; Singh, Inderjit

    2009-01-01

    X-linked adrenoleukodystrophy is a metabolic disorder arising from a mutation/deletion in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the oxidation of very long chain fatty acids (VLCFAs). Thus, these VLCFAs accumulate. In a cerebral form of ALD (cALD), VLCFA accumulation induces neuroinflammation that leads to loss of oligodendrocytes and myelin, which ultimately shortens the lifespan. To establish a relationship between the metabolic disease and inflammatory disease induction, we document that small interfering RNA (siRNA)-mediated silencing of Abcd1 (ALDP) and Abcd2 [adrenoleukodystrophy-related protein (ALDRP)] genes in mice primary astrocyte cultures resulted in accumulation of VLCFA and induction of an inflammatory response characteristic of human cALD. Correction of the metabolic defect using monoenoic FAs in Abcd1/Abcd2-silenced cultured astrocytes decreased inducible nitric oxide synthase and inflammatory cytokine expression, suggesting a link between VLCFA accumulation and inflammation. The inflammatory response was found to be mediated by transcription factors NF-κB, AP-1, and C/EBP in Abcd1/Abcd2-silenced mouse primary astrocytes. Although mechanisms of VLCFA-mediated induction of the inflammatory response have been investigated here in vitro, the in vivo mediators remain elusive. Our data represent the first study to suggest a direct link between the accumulation of VLCFA and the induction of inflammatory mediators. PMID:18723473

  13. An Alpha Motif at Tas3C Terminus Mediates RITS Cis Spreading and Promotes Heterochromatic Gene Silencing

    SciTech Connect

    Li, H.; Motamedi, M; Yip, C; Wang, Z; Walz, T; Patel, D; Moazed, D

    2009-01-01

    RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 {alpha}-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.

  14. Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis

    PubMed Central

    Karki, Anju; Humphrey, Sean E.; Steele, Rebecca E.; Hess, David A.; Taparowsky, Elizabeth J.; Konieczny, Stephen F.

    2015-01-01

    Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis—events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer. PMID:26717480

  15. The Treatment of Fibrosis of Joint Synovium and Frozen Shoulder by Smad4 Gene Silencing in Rats

    PubMed Central

    Xue, MingFeng; Gong, SuiLiang; Dai, JiaPing; Chen, Gang; Hu, JunYu

    2016-01-01

    Soft tissue fibrosis at the joint induced by inflammation is the pathological basis of frozen shoulder. In the present study, we utilized a lentiviral approach to silence the Smad4 gene in an in vitro fibrosis model of fibroblasts and an in vivo frozen shoulder model. We observed the change in the fibrosis process and the biological indicators of frozen shoulder. The in vitro fibrosis models (Rat myoblasts L6, Rat synovial cell RSC-364 and Rat chondrocytes RCs) were established using TGF-β1 induction, and the effect of Smad4 gene silencing on fibrosis was analyzed. The method of Kanno A was employed to establish a rat model of frozen shoulder, and Smad4 in the relevant part was knocked down with the lentiviral approach. We then examined the abduction and rotation angles and the length of synovial intima and measured the inflammatory factors in effusion and the fibrotic markers of tissues. We found that Smad4 knockdown suppressed the proliferation and expression of fibrotic markers in L6, RSC-364 and RCs cells induced by TGF-β1. MMP activity measurements showed that Smad4 knockdown significantly reversed the decrease in MMP activity in these three cell lines that were induced by TGF-β1. Furthermore, using lentivirus in the rat frozen shoulder model, we found that Smad4 silencing attenuated the inflammatory response and fibrosis. It significantly inhibited the increase of the Vimentin, α-SMA, collagen I and III, Lama1 and Timp1 proteins in synovial tissue as well as the inflammatory factors of TNF-a, IL-1α/β, IL-6 and IL-10 in effusion. MMP acidity assays revealed that Smad4 silencing inhibited MMP activity in the synovial, cartilage and ligament tissues in the model animals. The assessment of the phosphorylated Smad2/3 in the nuclei isolated from the synovial tissues showed that Smad4 silencing significantly inhibited the phosphorylation and subsequent nuclear translocation of Smad2/3 proteins. Moreover, Smad4-shRNA lentivirus inhibited the decrease in both

  16. Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

    PubMed Central

    Koch, Aline; Kumar, Neelendra; Weber, Lennart; Keller, Harald; Imani, Jafargholi; Kogel, Karl-Heinz

    2013-01-01

    Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases. PMID:24218613

  17. Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy).

    PubMed

    Hileman, Lena C; Drea, Sinéad; Martino, Gemma; Litt, Amy; Irish, Vivian F

    2005-10-01

    Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are resistant to conventional genetic approaches. However, VIGS has been shown to be effective in only a few, closely related plant species. Tobacco rattle virus (TRV), a bipartite RNA virus, has a wide host range and so in principle could serve as an efficient vector for VIGS in a diverse array of plant species. Here we show that a vector based on TRV sequences is effective at silencing the endogenous phytoene desaturase (PapsPDS) gene in Papaver somniferum (opium poppy). We show that this vector does not compromise the growth or reproduction of poppy and the plants did not display viral symptoms. The silencing of PapsPDS resulted in a significant reduction in PapsPDS mRNA and a concomitant photobleached phenotype. The ability to rapidly assay gene function in P. somniferum will be valuable in manipulation of the opiate pathway in this pharmaceutically important species. We suggest that our vacuum infiltration method used to deliver TRV-based vectors into poppy is a promising approach for expanding VIGS to diverse angiosperm species in which traditional delivery methods fail to induce VIGS. Furthermore, these studies demonstrate the utility of TRV-VIGS for probing gene function in a basal eudicot species that is phylogenetically distant from model plant species.

  18. Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

    PubMed Central

    Lynch, Catherine A; Tycko, Benjamin; Bestor, Timothy H; Walsh, Colum P

    2002-01-01

    Background The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels. Results Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors. Conclusion These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma. PMID:12234381

  19. Simultaneous gene silencing of KRAS and anti-apoptotic genes as a multitarget therapy.

    PubMed

    Werner, Kristin; Lademann, Franziska; Thepkaysone, May-Linn; Jahnke, Beatrix; Aust, Daniela E; Kahlert, Christoph; Weber, Georg; Weitz, Jürgen; Grützmann, Robert; Pilarsky, Christian

    2016-01-26

    Pancreatic cancer is one of the most lethal tumor types worldwide and an effective therapy is still elusive. Targeted therapy focused against a specific alteration is by definition unable to attack broad pathway signaling modification. Tumor heterogeneity will render targeted therapies ineffective based on the regrowth of cancer cell sub-clones. Therefore multimodal therapy strategies, targeting signaling pathways simultaneously should improve treatment.SiRNAs against KRAS and the apoptosis associated genes BCLXL, FLIP, MCL1L, SURVIVIN and XIAP were transfected into human and murine pancreatic cancer cell lines. Induction of apoptosis was measured by Caspase 3/7 activation, subG1 FACS analysis and PARP cleavage. The therapeutic approach was tested in a subcutaneous allograft model with a murine cancer cell line.By using siRNAs as a systematic approach to remodel signal transduction in pancreatic cancer the results showed increasing inhibition of proliferation and apoptosis induction in vitro and in vivo. Thus, siRNAs are suitable to model multimodal therapy against signaling pathways in pancreatic cancer. Improvements in in vivo delivery of siRNAs against a multitude of targets might therefore be a potential therapeutic approach.

  20. Simultaneous gene silencing of KRAS and anti-apoptotic genes as a multitarget therapy

    PubMed Central

    Werner, Kristin; Lademann, Franziska; Thepkaysone, May-Linn; Jahnke, Beatrix; Aust, Daniela E.; Kahlert, Christoph; Weber, Georg; Weitz, Jürgen; Grützmann, Robert; Pilarsky, Christian

    2016-01-01

    Pancreatic cancer is one of the most lethal tumor types worldwide and an effective therapy is still elusive. Targeted therapy focused against a specific alteration is by definition unable to attack broad pathway signaling modification. Tumor heterogeneity will render targeted therapies ineffective based on the regrowth of cancer cell sub-clones. Therefore multimodal therapy strategies, targeting signaling pathways simultaneously should improve treatment. SiRNAs against KRAS and the apoptosis associated genes BCLXL, FLIP, MCL1L, SURVIVIN and XIAP were transfected into human and murine pancreatic cancer cell lines. Induction of apoptosis was measured by Caspase 3/7 activation, subG1 FACS analysis and PARP cleavage. The therapeutic approach was tested in a subcutaneous allograft model with a murine cancer cell line. By using siRNAs as a systematic approach to remodel signal transduction in pancreatic cancer the results showed increasing inhibition of proliferation and apoptosis induction in vitro and in vivo. Thus, siRNAs are suitable to model multimodal therapy against signaling pathways in pancreatic cancer. Improvements in in vivo delivery of siRNAs against a multitude of targets might therefore be a potential therapeutic approach. PMID:26716649

  1. Silencing of a metaphase I-specific gene results in a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene mutations.

    PubMed

    Bhullar, Ramanjot; Nagarajan, Ragupathi; Bennypaul, Harvinder; Sidhu, Gaganpreet K; Sidhu, Gaganjot; Rustgi, Sachin; von Wettstein, Diter; Gill, Kulvinder S

    2014-09-30

    Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I. PMID:25232038

  2. Identification of novel pepper genes involved in Bax- or INF1-mediated cell death responses by high-throughput virus-induced gene silencing.

    PubMed

    Lee, Jeong Hee; Kim, Young Cheol; Choi, Doil; Park, Jeong Mee

    2013-11-19

    Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.

  3. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles.

    PubMed

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye

    2015-09-11

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  4. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

    NASA Astrophysics Data System (ADS)

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

    2015-09-01

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

  5. RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles.

    PubMed

    Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Chuan, Tjin Swee; Yong, Ken-Tye

    2015-09-11

    RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications. PMID:26291710

  6. Corepressor for element-1-silencing transcription factor preferentially mediates gene networks underlying neural stem cell fate decisions.

    PubMed

    Abrajano, Joseph J; Qureshi, Irfan A; Gokhan, Solen; Molero, Aldrin E; Zheng, Deyou; Bergman, Aviv; Mehler, Mark F

    2010-09-21

    The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) silences neuronal genes in neural stem cells (NSCs) and nonneuronal cells through its role as a dynamic modular platform for recruitment of transcriptional and epigenetic regulatory cofactors to RE1-containing promoters. In embryonic stem cells, the REST regulatory network is highly integrated with the transcriptional circuitry governing self-renewal and pluripotency, although its exact functional role is unclear. The C-terminal cofactor for REST, CoREST, also acts as a modular scaffold, but its cell type-specific roles have not been elucidated. We used chromatin immunoprecipitation-on-chip to examine CoREST and REST binding sites in NSCs and their proximate progenitor species. In NSCs, we identified a larger number of CoREST (1,820) compared with REST (322) target genes. The majority of these CoREST targets do not contain known RE1 motifs. Notably, these CoREST target genes do play important roles in pluripotency networks, in modulating NSC identity and fate decisions and in epigenetic processes previously associated with both REST and CoREST. Moreover, we found that NSC-mediated developmental transitions were associated primarily with liberation of CoREST from promoters with transcriptional repression favored in less lineage-restricted radial glia and transcriptional activation favored in more lineage-restricted neuronal-oligodendrocyte precursors. Clonal NSC REST and CoREST gene manipulation paradigms further revealed that CoREST has largely independent and previously uncharacterized roles in promoting NSC multilineage potential and modulating early neural fate decisions. PMID:20823235

  7. Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

    PubMed Central

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

    2013-01-01

    The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  8. Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation.

    PubMed Central

    Mottus, R; Sobel, R E; Grigliatti, T A

    2000-01-01

    For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster. We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. We propose that the missense mutations reported here are acting as antimorphic mutations that "poison" the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus. PMID:10655219

  9. Inter-genomic DNA Exchanges and Homeologous Gene Silencing Shaped the Nascent Allopolyploid Coffee Genome (Coffea arabica L.)

    PubMed Central

    Lashermes, Philippe; Hueber, Yann; Combes, Marie-Christine; Severac, Dany; Dereeper, Alexis

    2016-01-01

    Allopolyploidization is a biological process that has played a major role in plant speciation and evolution. Genomic changes are common consequences of polyploidization, but their dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen to study genetic changes that accompany allopolyploid formation. Both RNA-seq and DNA-seq data were generated from two genetically distant C. arabica accessions. Genomic structural variation was investigated using C. canephora, one of its diploid progenitors, as reference genome. The fate of 9047 duplicate homeologous genes was inferred and compared between the accessions. The pattern of SNP density along the reference genome was consistent with the allopolyploid structure. Large genomic duplications or deletions were not detected. Two homeologous copies were retained and expressed in 96% of the genes analyzed. Nevertheless, duplicated genes were found to be affected by various genomic changes leading to homeolog loss or silencing. Genetic and epigenetic changes were evidenced that could have played a major role in the stabilization of the unique ancestral allotetraploid and its subsequent diversification. While the early evolution of C. arabica mainly involved homeologous crossover exchanges, the later stage appears to have relied on more gradual evolution involving gene conversion and homeolog silencing. PMID:27440920

  10. Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.

    PubMed

    MacFarlane, Ryan C; Singh, Upinder

    2008-06-01

    The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of "RNAi" negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem. PMID:18346737

  11. Conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing

    PubMed Central

    Chalamcharla, Venkata R.; Folco, H. Diego; Dhakshnamoorthy, Jothy; Grewal, Shiv I. S.

    2015-01-01

    Cotranscriptional RNA processing and surveillance factors mediate heterochromatin formation in diverse eukaryotes. In fission yeast, RNAi machinery and RNA elimination factors including the Mtl1–Red1 core and the exosome are involved in facultative heterochromatin assembly; however, the exact mechanisms remain unclear. Here we show that RNA elimination factors cooperate with the conserved exoribonuclease Dhp1/Rat1/Xrn2, which couples pre-mRNA 3′-end processing to transcription termination, to promote premature termination and facultative heterochromatin formation at meiotic genes. We also find that Dhp1 is critical for RNAi-mediated heterochromatin assembly at retroelements and regulated gene loci and facilitates the formation of constitutive heterochromatin at centromeric and mating-type loci. Remarkably, our results reveal that Dhp1 interacts with the Clr4/Suv39h methyltransferase complex and acts directly to nucleate heterochromatin. Our work uncovers a previously unidentified role for 3′-end processing and transcription termination machinery in gene silencing through premature termination and suggests that noncanonical transcription termination by Dhp1 and RNA elimination factors is linked to heterochromatin assembly. These findings have important implications for understanding silencing mechanisms targeting genes and repeat elements in higher eukaryotes. PMID:26631744

  12. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    PubMed

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  13. Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus.

    PubMed

    Eybishtz, Assaf; Peretz, Yuval; Sade, Dagan; Akad, Fouad; Czosnek, Henryk

    2009-09-01

    A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program-one susceptible (S), one resistant (R-that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance. PMID:19533378

  14. Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich’s Ataxia

    PubMed Central

    Soragni, Elisabetta; Chou, C. James; Rusche, James R.; Gottesfeld, Joel M.

    2015-01-01

    The genetic defect in Friedreich’s ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we

  15. Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded by Ageratum yellow vein virus, a monopartite begomovirus.

    PubMed

    Sharma, P; Ikegami, M; Kon, T

    2010-04-01

    Ageratum yellow vein disease (AYVD) is caused by the association of a Tomato leaf curl Java betasatellite [Indonesia:Indonesia 1:2003] (ToLCJB-[ID:ID1:03]) with a begomovirus component. Our previous results demonstrated that ToLCJB-[ID:ID:03] is essential for induction of leaf curl symptoms in plants and transgene expression of its betaC1 gene in Nicotiana benthamiana plants induces virus-like symptoms. Here we show that Ageratum yellow vein virus-Indonesia [Indonesia: Tomato] (AYVV-ID[ID:Tom]) alone could systemically infect the plants and induced upward leaf curl symptoms. ToLCJB-[ID:ID1:03] was required, in addition to AYVV-ID[ID:Tom], for induction of severe downward leaf curl disease in N. benthamiana plants. However, DNAbeta01fsbetaC1, which encompasses a frameshift mutation, did not induce severe symptoms in N. benthamiana when co-inoculated with AYVV-ID[ID:Tom]. The infectivity analysis of AYVV-ID[ID:Tom] and its associated betasatellite encoded genes using Potato virus X (PVX) vector were carried out in N. benthamiana, indicate that the V2 and betaC1 genes are symptom determinants. We have identified the DNA encoded V2 and its betasatellite, ToLCJB-[ID:ID1:03], encoded betaC1 proteins as efficient silencing suppressors of posttranscriptional gene silencing (PTGS) by using an Agrobacterium co-infiltration or heterologous PVX vector assays. However, the results also showed weak suppression of gene silencing activities for C2 and C4 induced by GFP and mRNA associated with GFP was detected. Furthermore, confocal imaging analysis of ToLCJB-[ID:ID1:03] betaC1 in the epidermal cells of N. benthamiana shows that this protein is accumulated towards the periphery of the cell and around the nucleus, however, V2 accumulated in the cell cytoplasm, C4 associated with plasma membrane and C2 exclusively targeted into nucleus. In this study, we identified as many as four distinct suppressors of RNA silencing encoded by AYVV-ID[ID:Tom] and its cognate betasatellite in the

  16. The Effect of Silencing HIF-1α Gene in BxPC-3 Cell Line on Glycolysis-Related Gene Expression, Cell Growth, Invasion, and Apoptosis.

    PubMed

    Jiang, Yi; Wu, Guo-Hao; He, Guo-Dong; Zhuang, Qiu-Lin; Xi, Qiu-Lei; Zhang, Bo; Han, Yu-Song; Fang, Jing

    2015-01-01

    Hypoxia has been proved to be a typical character of solid tumors. Tumor cells prefer to use glucose through the glycolysis pathway instead of aerobic respiration. However, the precise molecular mechanism underlying this so-called Warburg effect remains elusive. In the current study, siRNA was synthesized and transfected into BxPC-3 cell line to silence the expression of HIF-1α gene. It was found that hypoxia induced hypoxia-inducible factor 1α (HIF-1α) overexpression in BxPC-3 cells, enhanced the expression of pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A, thus facilitating glycolysis and making tumor cells more tolerant to hypoxic stress. The silencing of HIF-1α gene significantly attenuated glycolysis under hypoxic conditions, inhibited the growth and invasion ability of BxPC-3 cells, and enhanced hypoxia-induced cell apoptosis.

  17. MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma.

    PubMed

    Melaiu, Ombretta; Stebbing, Justin; Lombardo, Ylenia; Bracci, Elisa; Uehara, Norihisa; Bonotti, Alessandra; Cristaudo, Alfonso; Foddis, Rudy; Mutti, Luciano; Barale, Roberto; Gemignani, Federica; Giamas, Georgios; Landi, Stefano

    2014-01-01

    Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

  18. Silencing of the host factor eIF(iso)4E gene confers plum pox virus resistance in plum.

    PubMed

    Wang, Xinhua; Kohalmi, Susanne E; Svircev, Antonet; Wang, Aiming; Sanfaçon, Hélène; Tian, Lining

    2013-01-01

    Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.

  19. G9a Is Essential for Epigenetic Silencing of K+ Channel Genes in Acute-to-Chronic Pain Transition

    PubMed Central

    Laumet, Geoffroy; Garriga, Judit; Chen, Shao-Rui; Zhang, Yuhao; Li, De-Pei; Smith, Trevor M.; Dong, Yingchun; Jelinek, Jaroslav; Cesaroni, Matteo; Issa, Jean-Pierre; Pan, Hui-Lin

    2015-01-01

    Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K+ channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. Here we show that nerve injury increased H3K9me2 occupancy at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect DNA methylation levels of these genes in DRGs. Nerve injury increased activity of G9a, histone deacetylases and EZH2, but only G9a inhibition consistently restored K+ channel expression. Selective G9a knockout in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced K+ channel genes but also normalized 638 genes down- or up-regulated by nerve injury. Thus G9a plays a dominant role in transcriptional repression of K+ channels and in acute-to-chronic pain transition after nerve injury. PMID:26551542

  20. Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction.

    PubMed

    Singh, N K; Eliash, N; Stein, I; Kamer, Y; Ilia, Z; Rafaeli, A; Soroker, V

    2016-04-01

    The ectoparasitic mite Varroa destructor is one of the major threats to apiculture. Using a behavioural choice bioassay, we determined that phoretic mites were more successful in reaching a bee than reproductive mites, suggesting an energy trade-off between reproduction and host selection. We used both chemo-ecological and molecular strategies to identify the regulation of the olfactory machinery of Varroa and its association with reproduction. We focused on transcription regulation. Using primers designed to the conserved DNA binding region of transcription factors, we identified a gene transcript in V. destructor homologous to the pheromone receptor transcription factor (PRTF) gene of Pediculus humanus corporis. Quantitative PCR (qPCR) revealed that this PRTF-like gene transcript is expressed in the forelegs at higher levels than in the body devoid of forelegs. Subsequent comparative qPCR analysis showed that transcript expression was significantly higher in the phoretic as compared to the reproductive stage. Electrophysiological and behavioural studies revealed a reduction in the sensitivity of PRTF RNA interference-silenced mites to bee headspace, consistent with a reduction in the mites' ability to reach a host. In addition, vitellogenin expression was stimulated in PRTF-silenced mites to similar levels as found in reproductive mites. These data shed light upon the regulatory mechanism of host chemosensing in V. destructor. PMID:26801167

  1. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Al-Taher, Abdullah; Nakashima, Remi; Sakaguchi, Tomoya; Kandeel, Ali; Nagaya, Yuki; Kitamura, Yoshiaki; Kitade, Yukio

    2014-01-01

    Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

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

    PubMed Central

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

    2013-01-01

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

  3. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Al-Taher, Abdullah; Nakashima, Remi; Sakaguchi, Tomoya; Kandeel, Ali; Nagaya, Yuki; Kitamura, Yoshiaki; Kitade, Yukio

    2014-01-01

    Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain. PMID:24788663

  4. Silencing of the major family of NBS-LRR-encoding genes in lettuce results in the loss of multiple resistance specificities.

    PubMed

    Wroblewski, Tadeusz; Piskurewicz, Urszula; Tomczak, Anna; Ochoa, Oswaldo; Michelmore, Richard W

    2007-09-01

    The RGC2 gene cluster in lettuce (Lactuca sativa) is one of the largest known families of genes encoding nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins. One of its members, RGC2B, encodes Dm3 which determines resistance to downy mildew caused by the oomycete Bremia lactucae carrying the cognate avirulence gene, Avr3. We developed an efficient strategy for analysis of this large family of low expressed genes using post-transcriptional gene silencing (PTGS). We transformed lettuce cv. Diana (carrying Dm3) using chimeric gene constructs designed to simultaneously silence RGC2B and the GUS reporter gene via the production of interfering hairpin RNA (ihpRNA). Transient assays of GUS expression in leaves accurately predicted silencing of both genes and were subsequently used to assay silencing in transgenic T(1) plants and their offspring. Levels of mRNA were reduced not only for RGC2B but also for all seven diverse RGC2 family members tested. We then used the same strategy to show that the resistance specificity encoded by the genetically defined Dm18 locus in lettuce cv. Mariska is the result of two resistance specificities, only one of which was silenced by ihpRNA derived from RGC2B. Analysis of progeny from crosses between transgenic, silenced tester stocks and lettuce accessions carrying other resistance genes previously mapped to the RGC2 locus indicated that two additional resistance specificities to B. lactucae, Dm14 and Dm16, as well as resistance to lettuce root aphid (Pemphigus bursarius L.), Ra, are encoded by RGC2 family members. PMID:17587302

  5. Expression of cancer stem markers could be influenced by silencing of p16 gene in HeLa cervical carcinoma cells.

    PubMed

    Wu, H; Zhang, J; Shi, H

    2016-01-01

    Effect of the tumor suppression gene p16 on the biological characteristics of HeLa cervical carcinoma cells was explored. The expression of p16 protein was increased in HeLa tumor sphere cells, and no significant difference in tumor spheres from the first to the fourth passages. Compared with those of parental HeLa cells, the proportion of CD44+/CD24- and ABCG2+ cells increased significantly in tumor spheres. However after the cells were silenced by the p16-sh289 vector, expression of P16 protein and the cell number of CD44+/CD24- and ABCG2+ decreased. Moreover, HeLa cells with p16 gene silencing showed decreased abilities of sphere formation and matrigel invasion. More HeLa cells with p16 gene silence were needed for tumor formation in nude mice. Tumor size and weight in mouse model established with p16 gene silenced HeLa cells were less than those with HeLa parental cell model. The present results indicate that silencing of the p16 gene inhibits expression of cancer stem cell markers and tumorigenic ability of HeLa cells.

  6. High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing1

    PubMed Central

    Liu, Qing; Singh, Surinder P.; Green, Allan G.

    2002-01-01

    We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications. PMID:12177486

  7. Gene expression profiling and silencing reveal that monolignol biosynthesis plays a critical role in penetration defence in wheat against powdery mildew invasion

    PubMed Central

    Bhuiyan, Nazmul H.; Selvaraj, Gopalan; Wei, Yangdou; King, John

    2009-01-01

    Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat. PMID:19039100

  8. Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway.

    PubMed

    Scofield, Steven R; Huang, Li; Brandt, Amanda S; Gill, Bikram S

    2005-08-01

    Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.

  9. Silencing defense pathways in Arabidopsis by heterologous gene sequences from Brassica oleracea enhances the performance of a specialist and a generalist herbivorous insect.

    PubMed

    Zheng, Si-Jun; Zhang, Peng-Jun; van Loon, Joop J A; Dicke, Marcel

    2011-08-01

    The jasmonic acid (JA) signaling pathway and defensive secondary metabolites such as glucosinolates are generally considered to play central roles in the defense of brassicaceous plants against herbivorous insects. To determine the function of specific plant genes in plant-insect interactions, signaling or biosynthetic mutants are needed. However, mutants are not yet available for brassicaceous plants other than Arabidopsis thaliana, e.g., cabbage (Brassica oleracea). We employed virus-induced gene silencing (VIGS) by using tobacco rattle virus (TRV) to knock down the endogenous expression of lipoxygenase (LOX), an upstream enzyme of the JA pathway and thioglucoside glucohydrolase: myrosinase (TGG1/TGG2), a hydrolytic enzyme that catalyzes the release of defensive volatile products originating from glucosinolates, in Arabidopsis thaliana. This was done by using the heterologous gene sequences from B. oleracea. Silencing these genes in A. thaliana plants is efficient and specific. Only 18 nucleotides with 100% identity between the trigger (BoMYR) and the target (AtTGG1/2) sequence are sufficient to achieve gene silencing. LOX-silenced plants showed significantly reduced AtLOX2 transcript accumulation after Pieris rapae larval feeding. TGG-silenced plants exhibited significantly lower TGG1/TGG2 transcript levels only after shorter larval feeding. The inhibition of TGG1/TGG2 transcript accumulation via gene silencing may be overruled by longer larval feeding. Specialist P. rapae larvae developed significantly better on both types of silenced plants than on empty vector (EV) control plants, while generalist Mamestra brassicae larvae developed significantly better on the TGG1/TGG2 silenced plants than on EV control plants. This shows that not only the generalist herbivore but also the Brassicaceae-specialist P. rapae is negatively affected by the ability of brassicaceous plants to produce their specific secondary metabolites, i.e., glucosinolates. Our results

  10. Molecular mechanisms of nickel carcinogenesis: gene silencing by nickel delivery to the nucleus and gene activation/inactivation by nickel-induced cell signaling.

    PubMed

    Costa, Max; Yan, Yan; Zhao, Daoji; Salnikow, Konstantin

    2003-04-01

    We have summarized the molecular and cellular events involved in nickel (Ni) compound induced carcinogenesis. The major hypothesis for nickel carcinogenic action has involved the ability of the Ni compound to deliver high concentrations of Ni intracellularly, enter the nucleus and interact with chromatin. Ni has been found to selectively damage heterochromatin, and a major action of Ni is its ability to silence the expression of genes located near heterochromatin by inducing a loss of histone H4 and H3 acetylation and DNA hypermethylation. When Ni silences critical genes, such as tumor suppressor genes, the cell is altered to a greater state of neoplastic transformation. The carcinogenic hazard of Ni compounds has been directly related to the ability of that Ni compound to raise the intracellular Ni ions. The mechanisms of Ni-induced gene silencing will be discussed. However, recently it has been found that soluble Ni ions can interact with the cell surface receptors and activate cell signaling resulting in the induction of a variety of cellular genes. In particular, the Ca and hypoxia inducible factor pathway is activated in all cells exposed to soluble Ni ions. In the case of HIF-1 induction, a cell is now equipped with the expression of a variety of genes that will allow the cell to survive the lack of oxygen and thus should enable a previously initiated cancer cell to progress into a full malignant state and metastasize. These new findings support the view that soluble Ni ions exhibit carcinogenic potential by activating cell promotion and lend strength to the epidemiological data showing soluble Ni to be associated with cancer risk in Ni refinery workers. PMID:12729258

  11. ATOX1 gene silencing increases susceptibility to anticancer therapy based on copper ionophores or chelating drugs.

    PubMed

    Barresi, Vincenza; Spampinato, Giorgia; Musso, Nicolò; Trovato Salinaro, Angela; Rizzarelli, Enrico; Condorelli, Daniele Filippo

    2016-03-01

    Copper is a catalytic cofactor required for the normal function of many enzymes involved in fundamental biological processes but highly cytotoxic when in excess. Therefore its homeostasis and distribution is strictly regulated by a network of transporters and intracellular chaperones. ATOX1 (antioxidant protein 1) is a copper chaperone that plays a role in copper homeostasis by binding and transporting cytosolic copper to ATPase proteins in the trans-Golgi network. In the present study the Caco-2 cell line, a colon carcinoma cell line, was used as an in vitro model to evaluate if ATOX1 deficiency could affect sensitivity to experimentally induced copper dyshomeostasis. Silencing of ATOX1 increased toxicity of a short treatment with a high concentration of Cu(2+). Copper ionophores, such as 5-chloro-8-hydroxyquinoline, induced a copper-dependent cell toxicity which was significantly potentiated after ATOX1 silencing. The copper chelator TPEN (N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine) produced a form of cell toxicity that was reversed by the addition of Cu(2+). ATOX1 silencing increased Caco-2 cell sensitivity to TPEN toxicity. Our results suggest the possibility of a therapy with copper-chelating or ionophore drugs in subtypes of tumors showing specific alterations in ATOX1 expression. PMID:26784148

  12. Quantitative Silencing of EGFP Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol

    PubMed Central

    2012-01-01

    Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N4,N9-diacyl spermine conjugate, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N4,N9-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1

  13. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

    PubMed

    Moreno, Ana Beatriz; Martínez de Alba, Angel Emilio; Bardou, Florian; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis; Mallory, Allison C

    2013-04-01

    Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways. PMID:23482394

  14. The Chp1–Tas3 core is a multifunctional platform critical for gene silencing by RITS

    SciTech Connect

    Schalch, Thomas; Job, Godwin; Shanker, Sreenath; Partridge, Janet F.; Joshua-Tor, Leemor

    2011-11-13

    RNA interference (RNAi) is critical for the assembly of heterochromatin at Schizosaccharomyces pombe centromeres. Central to this process is the RNA-induced initiation of transcriptional gene silencing (RITS) complex, which physically anchors small noncoding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which forms a bridge between Chp1 and Ago1. Chp1 is a large protein with no recognizable domains, apart from its chromodomain. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing the tight association of Chp1 and Tas3. The structure also shows a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1–Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.

  15. TLR agonist–Stat3 siRNA conjugates: cell-specific gene silencing and enhanced antitumor immune responses

    PubMed Central

    Kortylewski, Marcin; Swiderski, Piotr; Herrmann, Andreas; Wang, Lin; Kowolik, Claudia; Kujawski, Maciej; Lee, Heehyoung; Scuto, Anna; Liu, Yong; Yang, Chunmei; Deng, Jiehui; Soifer, Harris S.; Raubitschek, Andrew; Forman, Stephen; Rossi, John J.; Pardoll, Drew M.; Jove, Richard; Yu, Hua

    2010-01-01

    Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment. PMID:19749770

  16. pH-Sensitive siRNA nanovector for targeted gene silencing and cytotoxic effect in cancer cells.

    PubMed

    Mok, Hyejung; Veiseh, Omid; Fang, Chen; Kievit, Forrest M; Wang, Freddy Y; Park, James O; Zhang, Miqin

    2010-12-01

    A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.

  17. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

    PubMed Central

    Lee, Y W; Klein, C B; Kargacin, B; Salnikow, K; Kitahara, J; Dowjat, K; Zhitkovich, A; Christie, N T; Costa, M

    1995-01-01

    A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms. PMID:7537850

  18. Trans-silencing by P elements inserted in subtelomeric heterochromatin involves the Drosophila Polycomb group gene, Enhancer of zeste.

    PubMed Central

    Roche, S E; Rio, D C

    1998-01-01

    Drosophila P-element transposition is regulated by a maternally inherited state known as P cytotype. An important aspect of P cytotype is transcriptional repression of the P-element promoter. P cytotype can also repress non-P-element promoters within P-element ends, suggesting that P cytotype repression might involve chromatin-based transcriptional silencing. To learn more about the role of chromatin in P cytotype repression, we have been studying the P strain Lk-P(1A). This strain contains two full-length P elements inserted in the heterochromatic telomere-associated sequences (TAS elements) at cytological location 1A. Mutations in the Polycomb group gene (Pc-G gene), Enhancer of zeste (E(z)), whose protein product binds at 1A, resulted in a loss of Lk-P(1A) cytotype control. E(z) mutations also affected the trans-silencing of heterologous promoters between P-element termini by P-element transgenes inserted in the TAS repeats. These data suggest that pairing interactions between P elements, resulting in exchange of chromatin structures, may be a mechanism for controlling the expression and activity of P elements. PMID:9691041

  19. Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas

    PubMed Central

    Kola, Vijaya Sudhakara Rao; Renuka, P.; Padmakumari, Ayyagari Phani; Mangrauthia, Satendra K.; Balachandran, Sena M.; Ravindra Babu, V.; Madhav, Maganti S.

    2016-01-01

    RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5′ FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12–15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach. PMID:26903874

  20. Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas.

    PubMed

    Kola, Vijaya Sudhakara Rao; Renuka, P; Padmakumari, Ayyagari Phani; Mangrauthia, Satendra K; Balachandran, Sena M; Ravindra Babu, V; Madhav, Maganti S

    2016-01-01

    RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5' FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12-15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

  1. Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ-aminobutyric acid metabolic pathway.

    PubMed

    Bao, Hexigeduleng; Chen, Xianyang; Lv, Sulian; Jiang, Ping; Feng, Juanjuan; Fan, Pengxiang; Nie, Lingling; Li, Yinxin

    2015-03-01

    γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress.

  2. Evidence That the Transcriptional Regulators Sin3 and Rpd3, and a Novel Gene (Sds3) with Similar Functions, Are Involved in Transcriptional Silencing in S. Cerevisiae

    PubMed Central

    Vannier, D.; Balderes, D.; Shore, D.

    1996-01-01

    In a screen for extragenic suppressors of a silencing defective rap1(s) hmrΔA strain, recessive mutations in 21 different genes were found that restored repression to HMR. We describe the characterization of three of these SDS (suppressors of defective silencing) genes. SDS16 and SDS6 are known transcriptional modifiers, SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively, while the third is a novel gene, SDS3. SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in haploid cells and is necessary for sporulation in diploid cells. However, sds3 mutations differ from sin3 and rpd3 mutations in that they do not derepress TRK2. These sds mutations suppress a variety of cis- and trans-defects, which impair the establishment of silencing at HMR. Any one of the sds mutations slightly increases telomere position effect while a striking synergistic increase in repression is observed in a rap1(s) background. Epistasis studies suggest that SDS3 works in a different pathway from RPD3 and SIN3 to affect silencing at HMR. Together these results show that defects in certain general transcriptional modifiers can have a pronounced influence on position-effect gene silencing in yeast. Mechanisms for this increase in postion effect are discussed. PMID:8978024

  3. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis

    PubMed Central

    Groten, Karin; Pahari, Nabin T.; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T.

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  4. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    PubMed

    Groten, Karin; Pahari, Nabin T; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  5. Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.

    PubMed

    Kolniak, Tiffany A; Sullivan, Jack M

    2011-05-01

    Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding

  6. Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents.

    PubMed

    Kolniak, Tiffany A; Sullivan, Jack M

    2011-05-01

    Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular co-localization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding

  7. Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

    PubMed Central

    Khongnomnan, Kritsada; Makkoch, Jarika; Poomipak, Witthaya; Poovorawan, Yong

    2015-01-01

    MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3′-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3′-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future. PMID:26080461

  8. Identification of a class of human cancer germline genes with transcriptional silencing refractory to the hypomethylating drug 5-aza-2′-deoxycytidine.

    PubMed Central

    Almatrafi, Ahmed; Feichtinger, Julia; Vernon, Ellen G.; Escobar, Natalia Gomez; Wakeman, Jane A.; Larcombe, Lee D.; McFarlane, Ramsay J.

    2014-01-01

    Bona fide germline genes have expression restricted to the germ cells of the gonads. Testis-specific germline development-associated genes can become activated in cancer cells and can potentially drive the oncogenic process and serve as therapeutic/biomarker targets; such germline genes are referred to as cancer/testis genes. Many cancer/testis genes are silenced via hypermethylation of CpG islands in their associated transcriptional control regions and become activated upon treatment with DNA hypomethylating agents; such hypomethylation-induced activation of cancer/testis genes provides a potential combination approach to augment immunotherapeutics. Thus, understanding cancer/testis gene regulation is of increasing clinical importance. Previously studied cancer/testis gene activation has focused on X chromosome encoded cancer/testis genes. Here we find that a sub-set of non-X encoded cancer/testis genes are silenced in non-germline cells via a mechanism that is refractory to epigenetic dysregulation, including treatment with the hypomethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor tricostatin A. These findings formally indicate that there is a sub-group of the clinically important cancer/testis genes that are unlikely to be activated in clinical therapeutic approaches using hypomethylating agents and it indicates a unique transcriptional silencing mechanism for germline genes in non-germline cells that might provide a target mechanism for new clinical therapies. PMID:25594001

  9. Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

    PubMed

    Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Kwon, Suk-Yoon; Moon, Jae Sun

    2016-05-01

    We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR.

  10. Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

    PubMed

    Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Kwon, Suk-Yoon; Moon, Jae Sun

    2016-05-01

    We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR. PMID:26950504

  11. Requirement for flap endonuclease 1 (FEN1) to maintain genomic stability and transcriptional gene silencing in Arabidopsis.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Zhu, Jian-Kang; Gong, Zhizhong

    2016-09-01

    As a central component in the maturation of Okazaki fragments, flap endonuclease 1 (FEN1) removes the 5'-flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. The Arabidopsis fen1-1 mutant is hypersensitive to methyl methanesulfonate and shows reduced telomere length. Interestingly, genome-wide chromatin immunoprecipitation and RNA sequencing results demonstrate that FEN1 mutation leads to a decrease in the level of H3K27me3 and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS as well as maintaining genome stability in Arabidopsis.

  12. Screening for trans-acting factors and other factors involved in the activating or silencing of the gamma-globin gene during human ontogeny.

    PubMed

    Ma, Yan-Ni; Zhang, Xin; Zhang, Jun-Wu; Zhang, Xin-Hua; Wang, Rong-Xin

    2007-06-01

    Researchers hope to increase gamma-globin expression by controlling potential trans-acting factors that specifically activate the gamma-globin gene in fetuses or silence this gene in adults to potentially treat sickle cell disease and beta-thalassemias. To characterize genes encoding such factors, we analyzed the differential expression of mRNAs in erythroid induction cultures of CD34+ cells derived from normal adult bone marrow, umbilical cord blood, and bone marrow from a patient with heterocellular hereditary persistence of fetal hemoglobin. Using differential-display - reverse-transcription PCR analysis, we identified a number of genes with differential expression in the above-mentioned cells. The differential expression of some genes was also confirmed by quantitative real-time PCR. Our data provide important clues for identifying and validating trans-activators that activate the gamma-globin gene in fetuses, and trans-acting factors involved in silencing the gamma-globin gene in adults.

  13. Double Strand Breaks Can Initiate Gene Silencing and SIRT1-Dependent Onset of DNA Methylation in an Exogenous Promoter CpG Island

    PubMed Central

    O'Hagan, Heather M.; Mohammad, Helai P.; Baylin, Stephen B.

    2008-01-01

    Chronic exposure to inducers of DNA base oxidation and single and double strand breaks contribute to tumorigenesis. In addition to the genetic changes caused by this DNA damage, such tumors often contain epigenetically silenced genes with aberrant promoter region CpG island DNA hypermethylation. We herein explore the relationships between such DNA damage and epigenetic gene silencing using an experimental model in which we induce a defined double strand break in an exogenous promoter construct of the E-cadherin CpG island, which is frequently aberrantly DNA hypermethylated in epithelial cancers. Following the onset of repair of the break, we observe recruitment to the site of damage of key proteins involved in establishing and maintaining transcriptional repression, namely SIRT1, EZH2, DNMT1, and DNMT3B, and the appearance of the silencing histone modifications, hypoacetyl H4K16, H3K9me2 and me3, and H3K27me3. Although in most cells selected after the break, DNA repair occurs faithfully with preservation of activity of the promoter, a small percentage of the plated cells demonstrate induction of heritable silencing. The chromatin around the break site in such a silent clone is enriched for most of the above silent chromatin proteins and histone marks, and the region harbors the appearance of increasing DNA methylation in the CpG island of the promoter. During the acute break, SIRT1 appears to be required for the transient recruitment of DNMT3B and subsequent methylation of the promoter in the silent clones. Taken together, our data suggest that normal repair of a DNA break can occasionally cause heritable silencing of a CpG island–containing promoter by recruitment of proteins involved in silencing. Furthermore, with contribution of the stress-related protein SIRT1, the break can lead to the onset of aberrant CpG island DNA methylation, which is frequently associated with tight gene silencing in cancer. PMID:18704159

  14. Potato Snakin-1 Gene Silencing Affects Cell Division, Primary Metabolism, and Cell Wall Composition1[W

    PubMed Central

    Nahirñak, Vanesa; Almasia, Natalia Inés; Fernandez, Paula Virginia; Hopp, Horacio Esteban; Estevez, José Manuel; Carrari, Fernando; Vazquez-Rovere, Cecilia

    2012-01-01

    Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from potato (Solanum tuberosum) that was classified as a member of the Snakin/Gibberellic Acid Stimulated in Arabidopsis protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1, potato lines did not show remarkable morphological differences from the wild type; SN1 silencing resulted in reduced height, which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of the adaxial epidermis of mature leaves revealed that silenced lines had 70% to 90% increases in mean cell size with respect to wild-type leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-green fluorescent protein fusion protein was localized in plasma membrane, and bimolecular fluorescence complementation assays revealed that SN1 self-interacted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry, Fourier transform infrared spectroscopy, and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in seven cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism, and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense. PMID:22080603

  15. Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes

    PubMed Central

    Mahadevan, Chidambareswaren; Jaleel, Abdul; Deb, Lokesh; Thomas, George; Sakuntala, Manjula

    2015-01-01

    Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS. PMID:25918840

  16. RNAi-induced silencing in floral tissues of Petunia hybrida by agroinfiltration: a rapid assay for chalcone isomerase gene function analysis.

    PubMed

    Keykha, F; Bagheri, A; Moshtaghi, N; Bahrami, A R; Sharifi, A

    2016-01-01

    Variegation in flower color is commonly observed in many plant species and also occurs on Petunia (Petunia hybrida) as an ornamental plant. Variegated plants are of highly valuable in the floricultural market. Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants. Transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for color modification assay in floral tissues of Petunia with different colors. chiRNAi construct was cloned into the pBI121 vector under the control of 35S promoter. Transient RNA silencing of chi in the floral tissues of Petunia was induced by delivering 530 bp chi hairpin RNAs (hpRNAs) into the petals of flowers using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the petals of four colors of Petunia. Silencing of the endogenous chi mRNAs was highly effective in reduction of chi gene and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in Petunia through chi gene suppression. PMID:27609470

  17. Utilizing virus-induced gene silencing for the functional characterization of maize genes during infection with the fungal pathogen Ustilago maydis.

    PubMed

    van der Linde, Karina; Doehlemann, Gunther

    2013-01-01

    While in dicotyledonous plants virus-induced gene silencing (VIGS) is well established to study plant-pathogen interaction, in monocots only few examples of efficient VIGS have been reported so far. One of the available systems is based on the brome mosaic virus (BMV) which allows gene silencing in different cereals including barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays).Infection of maize plants by the corn smut fungus Ustilago maydis leads to the formation of large tumors on stem, leaves, and inflorescences. During this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed comprehensive and stage-specific changes in host gene expression during disease progression.To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a VIGS system based on the Brome mosaic virus (BMV) to maize at conditions that allow successful U. maydis infection of BMV pre-infected maize plants. This setup enables quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (q(RT)-PCR)-based readout.

  18. Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs.

    PubMed

    Biragyn, Arya; Bodogai, Monica; Olkhanud, Purevdorj B; Denny-Brown, Sinan R; Puri, Nitin; Ayukawa, Koichi; Kanegasaki, Shiro; Hogaboam, Cory M; Wejksza, Katarzyna; Lee-Chang, Catalina

    2013-05-01

    Despite significant attractiveness of antisense oligonucleotide/RNAi technology, its clinical application has been precluded by a lack of methods for targeted delivery and transduction of primary immune cells in vivo. Here, we devised a chemokine CCL17-based strategy (TARC-arp) that transiently silences expression of genes in immune cells by delivering inhibitory oligonucleotides through their chemokine receptors. In modeling studies using mice with established 4T1.2 breast cancer, we show that IL10 produced by CCR4 cells, in particular FoxP3 regulatory T cells (Tregs), plays an important role in lung metastasis. As such, TARC-arp-mediated silencing of IL10 or FoxP3 in CCR4 Tregs is sufficient to block lung metastasis. Thus, we provide a simple solution that circumvents the problems of RNAi use in vivo, indicating that a disease outcome can be successfully controlled by delivering inhibitory oligonucleotides with chemokines to inactivate a selective subset of immune cells. PMID:23603860

  19. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.

    PubMed

    Sarot, Emeline; Payen-Groschêne, Geneviève; Bucheton, Alain; Pélisson, Alain

    2004-03-01

    In Drosophila melanogaster, the endogenous retrovirus gypsy is repressed by the functional alleles (restrictive) of an as-yet-uncloned heterochromatic gene called flamenco. Using gypsy-lacZ transcriptional fusions, we show here that this repression takes place not only in the follicle cells of restrictive ovaries, as was previously observed, but also in restrictive larval female gonads. Analyses of the role of gypsy cis-regulatory sequences in the control of gypsy expression are also presented. They rule out the hypothesis that gypsy would contain a single binding region for a putative Flamenco repressor. Indeed, the ovarian expression of a chimeric yp3-lacZ construct was shown to become sensitive to the Flamenco regulation when any of three different 5'-UTR gypsy sequences (ranging from 59 to 647 nucleotides) was incorporated into the heterologous yp3-lacZ transcript. The piwi mutation, which is known to affect RNA-mediated homology-dependent transgene silencing, was also shown to impede the repression of gypsy in restrictive female gonads. Finally, a RNA-silencing model is also supported by the finding in ovaries of short RNAs (25-27 nucleotides long) homologous to sequences from within the gypsy 5'-UTR. PMID:15082550

  20. Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

    PubMed

    Zheng, Wenjie; Sai, Wenli; Yao, Min; Gu, Hongbin; Yao, Yao; Qian, Qi; Yao, Dengfu

    2015-05-01

    Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells. PMID:25600802

  1. Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling.

    PubMed

    Spirin, P V; Lebedev, T D; Orlova, N N; Gornostaeva, A S; Prokofjeva, M M; Nikitenko, N A; Dmitriev, S E; Buzdin, A A; Borisov, N M; Aliper, A M; Garazha, A V; Rubtsov, P M; Stocking, C; Prassolov, V S

    2014-11-01

    The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

  2. Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.

    PubMed

    Ramesh Kumar, D; Saravana Kumar, P; Gandhi, M Rajiv; Al-Dhabi, Naif Abdullah; Paulraj, M Gabriel; Ignacimuthu, S

    2016-05-01

    RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control. PMID:26794313

  3. Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction

    PubMed Central

    Podlesek, Zdravko; Busby, Stephen J. W.; Žgur-Bertok, Darja; Butala, Matej

    2015-01-01

    Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has “chosen” highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids. PMID:26114960

  4. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    PubMed Central

    Zebec, Ziga; Zink, Isabelle Anna; Kerou, Melina; Schleper, Christa

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms. PMID:27507792

  5. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    SciTech Connect

    Kubo, Takanori; Yanagihara, Kazuyoshi; Takei, Yoshifumi; Mihara, Keichiro; Sato, Yuichiro; Seyama, Toshio

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  6. A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci

    PubMed Central

    Sivertsen, Audun; Pedersen, Torunn; Larssen, Kjersti Wik; Bergh, Kåre; Rønning, Torunn Gresdal; Radtke, Andreas

    2016-01-01

    We report an outbreak of vancomycin-variable vanA+ enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA+ Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised. PMID:27139479

  7. A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer

    PubMed Central

    Kumegawa, Kohei; Maruyama, Reo; Yamamoto, Eiichiro; Ashida, Masami; Kitajima, Hiroshi; Tsuyada, Akihiro; Niinuma, Takeshi; Kai, Masahiro; Yamano, Hiro-o; Sugai, Tamotsu; Tokino, Takashi; Shinomura, Yasuhisa; Imai, Kohzoh; Suzuki, Hiromu

    2016-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA. PMID:27215978

  8. Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex

    PubMed Central

    Clancey, Shelly Applen; Wang, Xuying; Heitman, Joseph

    2016-01-01

    RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein) and Qip1 (a homolog of N. crassa Qip) were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor) and Fzc28 (a transcription factor) are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory. PMID:26943821

  9. Silencing of Eag1 Gene Inhibits Osteosarcoma Proliferation and Migration by Targeting STAT3-VEGF Pathway

    PubMed Central

    Wu, Xinyu; Chen, Zhida; Zeng, Wengrong; Zhong, Yuanfu; Liu, Qingjun; Wu, Jin

    2015-01-01

    So far, the role of Ether à go-go 1 (Eag1) potassium channels in migration and invasion progression of cancers remains elusive. In the present study, the effects of Eag1 knockdown on osteosarcoma cell proliferation, growth, and apoptosis were examined. Then, we evaluated the effects of Eag1 silencing on osteosarcoma cell migration and invasion. In addition, we detected the expression of vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in osteosarcoma cell treated with Eag1 small interfering RNAs (siRNAs). Finally, STAT3 siRNA was employed to determine the influence of downregulation of STAT3 on cell proliferation and migration. The results showed that knockdown of Eag1 significantly suppressed osteosarcoma cell proliferation and osteosarcoma xenografts growth. However, Eag1 silencing had little effect on cell apoptosis. Additionally, osteosarcoma cell adhesion, migration, and invasion were also potently attenuated. Notably, the expression levels of VEGF decreased evidently upon Eag1 siRNAs treatment, paralleled with reductions in the expression levels of STAT3. Moreover, a similar pattern was observed in osteosarcoma cell proliferation and migration suppression between STAT3 siRNA and Eag1 siRNAs groups. Our data indicated that Eag1 promotes osteosarcoma proliferation and migration, at least in part, by targeting STAT3-VEGF pathway. PMID:26783521

  10. Polycomb repressor complex 1 promotes gene silencing through H2AK119 mono-ubiquitination in acinar-to-ductal metaplasia and pancreatic cancer cells

    PubMed Central

    Reinhard, Tobias; Popp, Anna; Schäffer, Isabell; Raulefs, Susanne; Kong, Bo; Esposito, Irene

    2016-01-01

    Acinar-to-ductal metaplasia (ADM) occurring in cerulein-mediated pancreatitis or in oncogenic Kras-driven pancreatic cancer development is accompanied by extensive changes in the transcriptional program. In this process, acinar cells shut down the expression of acinar specific differentiation genes and re-express genes usually found in embryonic pancreatic progenitor cells. Previous studies have demonstrated that a loss of acinar-specific transcription factors sensitizes the cells towards oncogenic transformation, ultimately resulting in cancer development. However, the mechanism behind the transcriptional silencing of acinar cell fate genes in ADM and pancreatic cancer is largely unknown. Here, we analyzed whether elevated levels of the polycomb repressor complex 1 (PRC1) components Bmi1 and Ring1b and their catalyzed histone modification H2AK119ub in ADMs and tumor cells, are responsible for the mediation of acinar gene silencing. Therefore, we performed chromatin-immunoprecipitation in in vitro generated ADMs and isolated murine tumor cells against the repressive histone modifications H3K27me3 and H2AK119ub. We established that the acinar transcription factor complex Ptf1-L is epigenetically silenced in ADMs as well as in pancreatic tumor cells. For the first time, this work presents a possible mechanism of acinar gene silencing, which is an important prerequisite in the initiation and maintenance of a dedifferentiated cell state in ADMs and tumor cells. PMID:26716510

  11. Silencing SlELP2L, a tomato Elongator complex protein 2-like gene, inhibits leaf growth, accelerates leaf, sepal senescence, and produces dark-green fruit.

    PubMed

    Zhu, Mingku; Li, Yali; Chen, Guoping; Ren, Lijun; Xie, Qiaoli; Zhao, Zhiping; Hu, Zongli

    2015-01-09

    The multi-subunit complex Elongator interacts with elongating RNA polymerase II (RNAPII) and is thought to facilitate transcription through histone acetylation. Elongator is highly conserved in eukaryotes, yet has multiple kingdom-specific functions in diverse organisms. Recent genetic studies performed in Arabidopsis have demonstrated that Elongator functions in plant growth and development, and in response to biotic and abiotic stress. However, little is known about its roles in other plant species. Here, we study the function of an Elongator complex protein 2-like gene in tomato, here designated as SlELP2L, through RNAi-mediated gene silencing. Silencing SlELP2L in tomato inhibits leaf growth, accelerates leaf and sepal senescence, and produces dark-green fruit with reduced GA and IAA contents in leaves, and increased chlorophyll accumulation in pericarps. Gene expression analysis indicated that SlELP2L-silenced plants had reduced transcript levels of ethylene- and ripening-related genes during fruit ripening with slightly decreased carotenoid content in fruits, while the expression of DNA methyltransferase genes was up-regulated, indicating that SlELP2L may modulate DNA methylation in tomato. Besides, silencing SlELP2L increases ABA sensitivity in inhibiting seedling growth. These results suggest that SlELP2L plays important roles in regulating plant growth and development, as well as in response to ABA in tomato.

  12. Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    PubMed Central

    Rajeswaran, Rajendran; Gubaeva, Ekaterina G.; Zvereva, Anna S.; Windels, David; Vazquez, Franck; Blevins, Todd; Farinelli, Laurent; Pooggin, Mikhail M.

    2012-01-01

    In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs. PMID:23028332

  13. Silencing of an aphid carboxylesterase gene by use of plant-mediated RNAi impairs Sitobion avenae tolerance of Phoxim insecticides.

    PubMed

    Xu, Lanjie; Duan, Xiaoliang; Lv, Yanhua; Zhang, Xiaohua; Nie, Zhansheng; Xie, Chaojie; Ni, Zhongfu; Liang, Rongqi

    2014-04-01

    RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA level. Its specificity is sequence-based and depends on the sequence of one strand of the dsRNA corresponding to part or all of a specific gene transcript. In this study we adopted plant-mediated RNAi technology that targets Sitobion avenae (S. avenae) to enable gene silencing in the aphid and to minimize handling of the insects during experiments. S. avenae was selected for this study because it causes serious economic losses to wheat throughout the world. The carboxylesterase (CbE E4) gene in S. avenae was homologously cloned, which increased synthesis of a protein known to be critical to the resistance (tolerance) this species has developed to a wide range of pesticides. A plant RNAi vector was constructed, and transgenic Triticum aestivum (dsCbE1-5 and dsCbE2-2 lines) expressing CbE E4 dsRNA were developed. S. avenae were fed on dsCbE1-5 and dsCbE2-2 lines stably producing the CbE E4 dsRNA. CbE E4 gene expression in S. avenae was reduced by up to 30-60%. The number of aphids raised on dsCbE1-5 and dsCbE2-2 was lower than the number raised on non-transgenic plants. A solution of CbE E4 enzyme from S. avenae fed on dsCbE1-5 and dsCbE2-2 plants hydrolyzed only up to 20-30% Phoxim solution within 40 min whereas a solution of the enzyme from CbE E4 fed on control plants hydrolyzed 60% of Phoxim solution within 40 min. CbE E4 gene silencing was achieved by our wheat-mediated RNAi approach. This plant-mediated RNAi approach for addressing degradation-based pesticide resistance mechanisms in aphids and may prove useful in pest management for diverse agro-ecosystems. PMID:24242160

  14. Silencing of an aphid carboxylesterase gene by use of plant-mediated RNAi impairs Sitobion avenae tolerance of Phoxim insecticides.

    PubMed

    Xu, Lanjie; Duan, Xiaoliang; Lv, Yanhua; Zhang, Xiaohua; Nie, Zhansheng; Xie, Chaojie; Ni, Zhongfu; Liang, Rongqi

    2014-04-01

    RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA level. Its specificity is sequence-based and depends on the sequence of one strand of the dsRNA corresponding to part or all of a specific gene transcript. In this study we adopted plant-mediated RNAi technology that targets Sitobion avenae (S. avenae) to enable gene silencing in the aphid and to minimize handling of the insects during experiments. S. avenae was selected for this study because it causes serious economic losses to wheat throughout the world. The carboxylesterase (CbE E4) gene in S. avenae was homologously cloned, which increased synthesis of a protein known to be critical to the resistance (tolerance) this species has developed to a wide range of pesticides. A plant RNAi vector was constructed, and transgenic Triticum aestivum (dsCbE1-5 and dsCbE2-2 lines) expressing CbE E4 dsRNA were developed. S. avenae were fed on dsCbE1-5 and dsCbE2-2 lines stably producing the CbE E4 dsRNA. CbE E4 gene expression in S. avenae was reduced by up to 30-60%. The number of aphids raised on dsCbE1-5 and dsCbE2-2 was lower than the number raised on non-transgenic plants. A solution of CbE E4 enzyme from S. avenae fed on dsCbE1-5 and dsCbE2-2 plants hydrolyzed only up to 20-30% Phoxim solution within 40 min whereas a solution of the enzyme from CbE E4 fed on control plants hydrolyzed 60% of Phoxim solution within 40 min. CbE E4 gene silencing was achieved by our wheat-mediated RNAi approach. This plant-mediated RNAi approach for addressing degradation-based pesticide resistance mechanisms in aphids and may prove useful in pest management for diverse agro-ecosystems.

  15. Mutational bias of Turnip Yellow Mosaic Virus in the context of host anti-viral gene silencing.

    PubMed

    Ma, Jinmin; Pallett, Denise; Jiang, Hui; Hou, Yong; Wang, Hui

    2015-12-01

    Plant Dicer-like (DCL) enzymes exhibit a GC-preference during anti-viral post-transcriptional gene silencing (PTGS), delivering an evolutionary selection pressure resulting in plant viruses with GC-poor genomes. However, some viruses, e.g. Turnip Yellow Mosaic Virus (TYMV, genus Tymovirus) have GC-rich genomes, raising the question as to whether or not DCL derived selection pressure affects these viruses. In this study we analyzed the virus-derived small interfering RNAs from TYMV-infected leaves of Brassica juncea showed that the TYMV population accumulated a mutational bias with AU replacing GC (GC-AU), demonstrating PTGS pressure. Interestingly, at the highly polymorphic sites the GC-AU bias was no longer observed. This suggests the presence of an unknown mechanism preventing mutational drift of the viral population and maintaining viral genome stability, despite the host PTGS pressure.

  16. Mutational bias of Turnip Yellow Mosaic Virus in the context of host anti-viral gene silencing.

    PubMed

    Ma, Jinmin; Pallett, Denise; Jiang, Hui; Hou, Yong; Wang, Hui

    2015-12-01

    Plant Dicer-like (DCL) enzymes exhibit a GC-preference during anti-viral post-transcriptional gene silencing (PTGS), delivering an evolutionary selection pressure resulting in plant viruses with GC-poor genomes. However, some viruses, e.g. Turnip Yellow Mosaic Virus (TYMV, genus Tymovirus) have GC-rich genomes, raising the question as to whether or not DCL derived selection pressure affects these viruses. In this study we analyzed the virus-derived small interfering RNAs from TYMV-infected leaves of Brassica juncea showed that the TYMV population accumulated a mutational bias with AU replacing GC (GC-AU), demonstrating PTGS pressure. Interestingly, at the highly polymorphic sites the GC-AU bias was no longer observed. This suggests the presence of an unknown mechanism preventing mutational drift of the viral population and maintaining viral genome stability, despite the host PTGS pressure. PMID:26379088

  17. Silencing of the CaCP gene delays salt- and osmotic-induced leaf senescence in Capsicum annuum L.

    PubMed

    Xiao, Huai-Juan; Yin, Yan-Xu; Chai, Wei-Guo; Gong, Zhen-Hui

    2014-05-12

    Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene, CaCP, was involved in leaf senescence in pepper (Capsicum annuum L.). The full-length CaCP cDNA is comprised of 1316 bp, contains 1044 nucleotides in open reading frame (ORF), and encodes a 347 amino acid protein. The deduced protein belongs to the papain-like cysteine proteases (CPs) superfamily, containing a highly conserved ERFNIN motif, a GCNGG motif and a conserved catalytic triad. This protein localized to the vacuole of plant cells. Real-time quantitative PCR analysis revealed that the expression level of CaCP gene was dramatically higher in leaves and flowers than that in roots, stems and fruits. Moreover, CaCP transcripts were induced upon during leaf senescence. CaCP expression was upregulated by plant hormones, especially salicylic acid. CaCP was also significantly induced by abiotic and biotic stress treatments, including high salinity, mannitol and Phytophthora capsici. Loss of function of CaCP using the virus-induced gene-silencing technique in pepper plants led to enhanced tolerance to salt- and osmotic-induced stress. Taken together, these results suggest that CaCP is a senescence-associated gene, which is involved in developmental senescence and regulates salt- and osmotic-induced leaf senescence in pepper.

  18. Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants[W

    PubMed Central

    Li, Jian-Feng; Chung, Hoo Sun; Niu, Yajie; Bush, Jenifer; McCormack, Matthew; Sheen, Jen

    2013-01-01

    Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5′ coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology. PMID:23645631

  19. Silencing of the CaCP Gene Delays Salt- and Osmotic-Induced Leaf Senescence in Capsicum annuum L.

    PubMed Central

    Xiao, Huai-Juan; Yin, Yan-Xu; Chai, Wei-Guo; Gong, Zhen-Hui

    2014-01-01

    Cysteine proteinases have been known to participate in developmental processes and in response to stress in plants. Our present research reported that a novel CP gene, CaCP, was involved in leaf senescence in pepper (Capsicum annuum L.). The full-length CaCP cDNA is comprised of 1316 bp, contains 1044 nucleotides in open reading frame (ORF), and encodes a 347 amino acid protein. The deduced protein belongs to the papain-like cysteine proteases (CPs) superfamily, containing a highly conserved ERFNIN motif, a GCNGG motif and a conserved catalytic triad. This protein localized to the vacuole of plant cells. Real-time quantitative PCR analysis revealed that the expression level of CaCP gene was dramatically higher in leaves and flowers than that in roots, stems and fruits. Moreover, CaCP transcripts were induced upon during leaf senescence. CaCP expression was upregulated by plant hormones, especially salicylic acid. CaCP was also significantly induced by abiotic and biotic stress treatments, including high salinity, mannitol and Phytophthora capsici. Loss of function of CaCP using the virus-induced gene-silencing technique in pepper plants led to enhanced tolerance to salt- and osmotic-induced stress. Taken together, these results suggest that CaCP is a senescence-associated gene, which is involved in developmental senescence and regulates salt- and osmotic-induced leaf senescence in pepper. PMID:24823878

  20. Silencing abnormal wing disc gene of the Asian citrus psyllid, Diaphorina citri disrupts adult wing development and increases nymph mortality.

    PubMed

    El-Shesheny, Ibrahim; Hajeri, Subhas; El-Hawary, Ibrahim; Gowda, Siddarame; Killiny, Nabil

    2013-01-01

    Huanglongbing (HLB) causes considerable economic losses to citrus industries worldwide. Its management depends on controlling of the Asian citrus Psyllid (ACP), the vector of the bacterium, Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB. Silencing genes by RNA interference (RNAi) is a promising tool to explore gene functions as well as control pests. In the current study, abnormal wing disc (awd) gene associated with wing development in insects is used to interfere with the flight of psyllids. Our study showed that transcription of awd is development-dependent and the highest level was found in the last instar (5(th)) of the nymphal stage. Micro-application (topical application) of dsRNA to 5(th) instar of nymphs caused significant nymphal mortality and adult wing-malformation. These adverse effects in ACP were positively correlated with the amounts of dsRNA used. A qRT-PCR analysis confirmed the dsRNA-mediated transcriptional down-regulation of the awd gene. Significant down-regulation was required to induce a wing-malformed phenotype. No effect was found when dsRNA-gfp was used, indicating the specific effect of dsRNA-awd. Our findings suggest a role for awd in ACP wing development and metamorphosis. awd could serve as a potential target for insect management either via direct application of dsRNA or by producing transgenic plants expressing dsRNA-awd. These strategies will help to mitigate HLB by controlling ACP.

  1. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    PubMed Central

    Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L.; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

    2013-01-01

    The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination. PMID:24705165

  2. Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    PubMed Central

    Zylicz, Jan J; Dietmann, Sabine; Günesdogan, Ufuk; Hackett, Jamie A; Cougot, Delphine; Lee, Caroline; Surani, M Azim

    2015-01-01

    Early mouse development is accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2), which is essential for embryonic development. Here we show that genome-wide accumulation of H3K9me2 is crucial for postimplantation development, and coincides with redistribution of enhancer of zeste homolog 2 (EZH2)-dependent histone H3 lysine 27 trimethylation (H3K27me3). Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in cell cycle regulation, germline development and embryogenesis. Notably, the H3K9me2 modification extends to active enhancer elements where it promotes developmentally-linked gene silencing and directly marks promoters and gene bodies. This epigenetic mechanism is important for priming gene regulatory networks for critical cell fate decisions in rapidly proliferating postimplantation epiblast cells. DOI: http://dx.doi.org/10.7554/eLife.09571.001 PMID:26551560

  3. Targeted silencing of BjMYB28 transcription factor gene directs development of low glucosinolate lines in oilseed Brassica juncea.

    PubMed

    Augustine, Rehna; Mukhopadhyay, Arundhati; Bisht, Naveen C

    2013-09-01

    Brassica juncea (Indian mustard), a globally important oilseed crop, contains relatively high amount of seed glucosinolates ranging from 80 to 120 μmol/g dry weight (DW). One of the major breeding objectives in oilseed Brassicas is to improve the seed-meal quality through the development of low-seed-glucosinolate lines (<30 μmol/g DW), as high amounts of certain seed glucosinolates are known to be anti-nutritional and reduce the meal palatability. Here, we report the development of transgenic B. juncea lines having seed glucosinolates as low as 11.26 μmol/g DW, through RNAi-based targeted suppression of BjMYB28, a R2R3-MYB transcription factor family gene involved in aliphatic glucosinolate biosynthesis. Targeted silencing of BjMYB28 homologs provided significant reduction in the anti-nutritional aliphatic glucosinolates fractions, without altering the desirable nonaliphatic glucosinolate pool, both in leaves and seeds of transgenic plants. Molecular characterization of single-copy, low glucosinolate homozygous lines confirmed significant down-regulation of BjMYB28 homologs vis-à-vis enhanced accumulation of BjMYB28-specific siRNA pool. Consequently, these low glucosinolate lines also showed significant suppression of genes involved in aliphatic glucosinolate biosynthesis. The low glucosinolate trait was stable in subsequent generations of the transgenic lines with no visible off-target effects on plant growth and development. Various seed quality parameters including fatty acid composition, oil content, protein content and seed weight of the low glucosinolate lines also remained unaltered, when tested under containment conditions in the field. Our results indicate that targeted silencing of a key glucosinolate transcriptional regulator MYB28 has huge potential for reducing the glucosinolates content and improving the seed-meal quality of oilseed Brassica crops. PMID:23721233

  4. In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation

    PubMed Central

    Hegde, Vikas; Hickerson, Robyn P.; Nainamalai, Sitheswaran; Campbell, Paul A.; Smith, Frances J.D.; McLean, W.H. Irwin; Leslie Pedrioli, Deena M.

    2014-01-01

    Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis — the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available “self-delivery” modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and “self-delivery” siRNAs. Remarkably, a sustained > 40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use. PMID:25449884

  5. In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation.

    PubMed

    Hegde, Vikas; Hickerson, Robyn P; Nainamalai, Sitheswaran; Campbell, Paul A; Smith, Frances J D; McLean, W H Irwin; Pedrioli, Deena M Leslie

    2014-12-28

    Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.

  6. Identification of Winter-Responsive Proteins in Bread Wheat Using Proteomics Analysis and Virus-Induced Gene Silencing (VIGS).

    PubMed

    Zhang, Ning; Huo, Wang; Zhang, Lingran; Chen, Feng; Cui, Dangqun

    2016-09-01

    Proteomic approaches were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from two cultivars (UC1110 and PI 610750) and their derivatives, as well as the F10 recombinant inbred line population differing in cold-tolerance, a total of 20 common protein spots representing 16 unique proteins were successfully identified using 2-DE method. Of these, 14 spots had significantly enhanced abundance in the cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with the cold-tolerant parental cultivar PI 610750 and its 20 descendant lines. Six protein spots with reduced abundance were also detected. The identified protein spots are involved in stress/defense, carbohydrate metabolism, protein metabolism, nitrogen metabolism, energy metabolism, and photosynthesis. The 20 differentially expressed protein spots were chosen for quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression changes at the RNA level. The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models. Among the three unknown proteins, Spot 20 (PAP6-like) showed high sequence similarities with PAP6. qRT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to freezing stress, these plants had more serious droop and wilt, an increased rate of relative electrolyte leakage, reduced relative water content (RWC) and decreased tocopherol levels when compared with viral control plants. However, the plants that were silenced for the other two unknown proteins had no significant differences in comparison to the BSMV0-inoculated plants under freezing conditions. These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat. PMID

  7. Cheminformatics Approach to Gene Silencing: Z Descriptors of Nucleotides and SVM Regression Afford Predictive Models for siRNA Potency.

    PubMed

    Ebalunode, Jerry O; Zheng, Weifan

    2010-12-17

    Short interfering RNA mediated gene silencing technology has been through tremendous development over the past decade, and has found broad applications in both basic biomedical research and pharmaceutical development. Critical to the effective use of this technology is the development of reliable algorithms to predict the potency and selectivity of siRNAs under study. Existing algorithms are mostly built upon sequence information of siRNAs and then employ statistical pattern recognition or machine learning techniques to derive rules or models. However, sequence-based features have limited ability to characterize siRNAs, especially chemically modified ones. In this study, we proposed a cheminformatics approach to describe siRNAs. Principal component scores (z1, z2, z3, z4) have been derived for each of the 5 nucleotides (A, U, G, C, T) from the descriptor matrix computed by the MOE program. Descriptors of a given siRNA sequence are simply the concatenation of the z values of its composing nucleotides. Thus, for each of the 2431 siRNA sequences in the Huesken dataset, 76 descriptors were generated for the 19-NT representation, and 84 descriptors were generated for the 21-NT representation of siRNAs. Support Vector Machine regression (SVMR) was employed to develop predictive models. In all cases, the models achieved Pearson correlation coefficient r and R about 0.84 and 0.65 for the training sets and test sets, respectively. A minimum of 25 % of the whole dataset was needed to obtain predictive models that could accurately predict 75 % of the remaining siRNAs. Thus, for the first time, a cheminformatics approach has been developed to successfully model the structure-potency relationship in siRNA-based gene silencing data, which has laid a solid foundation for quantitative modeling of chemically modified siRNAs.

  8. Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stress-responsive genes in crop plants

    PubMed Central

    Ramegowda, Venkategowda; Mysore, Kirankumar S.; Senthil-Kumar, Muthappa

    2014-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for gene function analysis in plants. Over the last decade, VIGS has been successfully used as both a forward and reverse genetics technique for gene function analysis in various model plants, as well as crop plants. With the increased identification of differentially expressed genes under various abiotic stresses through high-throughput transcript profiling, the application of VIGS is expected to be important in the future for functional characterization of a large number of genes. In the recent past, VIGS was proven to be an elegant tool for functional characterization of genes associated with abiotic stress responses. In this review, we provide an overview of how VIGS is used in different crop species to characterize genes associated with drought-, salt-, oxidative- and nutrient-deficiency-stresses. We describe the examples from studies where abiotic stress related genes are characterized using VIGS. In addition, we describe the major advantages of VIGS over other currently available functional genomics tools. We also summarize the recent improvements, limitations and future prospects of using VIGS as a tool for studying plant responses to abiotic stresses. PMID:25071806

  9. Effects of targeted silencing of FOXC1 gene on proliferation and in vitro migration of human non-small-cell lung carcinoma cells

    PubMed Central

    Chen, Sumei; Jiao, Shunchang; Jia, Youchao; Li, Yang

    2016-01-01

    Background: The aim of this study was to evaluate the effects of targeted silencing of forkhead box C1 (FOXC1) gene with small interfering RNA (siRNA) on the proliferation and in vitro migration of human non-small-cell lung carcinoma (NSCLC) A549 and NCIH460 cells, and to explore the molecular mechanism. Methods: These cells were divided into FOXC1 siRNA groups and negative control groups. Results: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that compared with normal cells and paracancerous tissues, FOXC1 mRNA expressions in NSCLC cells and tissues were significantly higher (P<0.05). qRT-PCR and Western blot showed that FOXC1 siRNA effectively silenced FOXC1 gene expression in NSCLC cells. EdU labeling assay revealed that the proliferative capacity significantly decreased compared with that of normal control group after FOXC1 silencing (P<0.05). Significantly fewer cells in the transfected group migrated than those in negative control group did. After FOXC1 silencing, NSCLC cells were arrested in the G0/G1 phase, which were significantly different from those in negative control group (P<0.05). Compared with negative control group, the expression of cyclin D1 decreased and that of E-cadherin increased. Meanwhile, vimentin and MMP-2 expressions significantly reduced (P<0.05). FOXC1 siRNA effectively silenced FOXC1 gene expressions in NSCLC cells, inhibited their proliferation and invasion, and arrested them in the G0/G1 phase, suggesting that FOXC1 affected proliferation probably by regulating the expression of cell cycle-related protein cyclin D1. Conclusion: Silencing FOXC1 may evidently inhibit the migration of these cells by reversing the EMT process through suppressing cadherin, being associated with the expressions of extracellular MMPs. PMID:27648121

  10. RNAi-directed post transcriptional gene silencing of an Arabidopsis Myb transgene in tobacco

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The AtMyb90 gene encodes the 'production of anthocyanin pigment 2' (PAP2) transcription factor of Arabidopsis thaliana and is able to induce a visible hyper-pigmented phenotype when expressed in tobacco. Based upon this phenotype, we have used the AtMyb90 gene as a reporter gene to examine RNAi-dire...

  11. A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance.

    PubMed

    Sade, Dagan; Eybishtz, Assaf; Gorovits, Rena; Sobol, Iris; Czosnek, Henryk

    2012-10-01

    To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40 days after sowing (20 days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus. PMID:22843056

  12. A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance.

    PubMed

    Sade, Dagan; Eybishtz, Assaf; Gorovits, Rena; Sobol, Iris; Czosnek, Henryk

    2012-10-01

    To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40 days after sowing (20 days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.

  13. Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

    PubMed Central

    Jung, Jae-Young; Lee, Seung Eun; Hwang, Eun Mi

    2016-01-01

    Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population. PMID:27358580

  14. Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System.

    PubMed

    Jung, Jae-Young; Lee, Seung Eun; Hwang, Eun Mi; Lee, C Justin

    2016-06-01

    Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population. PMID:27358580

  15. The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis

    PubMed Central

    Bednarek, Ilona; Sypniewski, Daniel; Gawlik, Natalia; Goraus, Karol

    2012-01-01

    Aim of the study Abnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells. Material and methods The study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA. Results In transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed. Conclusions Application of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells. PMID:23788901

  16. Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

    PubMed

    Zhu, Xinjiang; Liu, Jian; Xu, Xiaoyang; Zhang, Chundong; Dai, Dongqiu

    2015-05-01

    The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies. PMID:25738530

  17. Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

    PubMed

    Zhu, Xinjiang; Liu, Jian; Xu, Xiaoyang; Zhang, Chundong; Dai, Dongqiu

    2015-05-01

    The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies.

  18. The Code of Silence: Widespread Associations Between Synonymous Codon Biases and Gene Function.

    PubMed

    Supek, Fran

    2016-01-01

    Some mutations in gene coding regions exchange one synonymous codon for another, and thus do not alter the amino acid sequence of the encoded protein. Even though they are often called 'silent,' these mutations may exhibit a plethora of effects on the living cell. Therefore, they are often selected during evolution, causing synonymous codon usage biases in genomes. Comparative analyses of bacterial, archaeal, fungal, and human cancer genomes have found many links between a gene's biological role and the accrual of synonymous mutations during evolution. In particular, highly expressed genes in certain functional categories are enriched with optimal codons, which are decoded by the abundant tRNAs, thus enhancing the speed and accuracy of the translating ribosome. The set of genes exhibiting codon adaptation differs between genomes, and these differences show robust associations to organismal phenotypes. In addition to selection for translation efficiency, other distinct codon bias patterns have been found in: amino acid starvation genes, cyclically expressed genes, tissue-specific genes in animals and plants, oxidative stress response genes, cellular differentiation genes, and oncogenes. In addition, genomes of organisms harboring tRNA modifications exhibit particular codon preferences. The evolutionary trace of codon bias patterns across orthologous genes may be examined to learn about a gene's relevance to various phenotypes, or, more generally, its function in the cell. PMID:26538122

  19. Expression of genes involved in the salicylic acid pathway in type h1 thioredoxin transiently silenced pepper plants during a begomovirus compatible interaction.

    PubMed

    Luna-Rivero, Marianne S; Hernández-Zepeda, Cecilia; Villanueva-Alonzo, Hernán; Minero-García, Yereni; Castell-González, Salvador E; Moreno-Valenzuela, Oscar A

    2016-04-01

    The type-h thioredoxins (TRXs) play a fundamental role in oxidative stress tolerance and defense responses against pathogens. In pepper plants, type-h TRXs participate in the defense mechanism against Cucumber mosaic virus. The goal of this study was to analyze the role of the CaTRXh1-cicy gene in pepper plants during compatible interaction with a DNA virus, the Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The effects of a transient silencing of the CaTRXh1-cicy gene in pepper plants wëre evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants under different treatments. The accumulation of salicylic acid (SA) and the relative expression of the defense genes NPR1 and PR10 were also evaluated. Results showed that viral DNA accumulation was higher in transiently CaTRXh1-cicy silenced plants that were also infected with EuMV-YP. Symptoms in these plants were more severe compared to the non-silenced plants infected with EuMV-YP. The SA levels in the EuMV-YP-infected plants were rapidly induced at 1 h post infection (hpi) in comparison to the non-silenced plants inoculated with EuMV-YP. Additionally, in pepper plants infected with EuMV-YP, the expression of NPR1 decreased by up to 41 and 58 % at 28 days post infection (dpi) compared to the non-silenced pepper plants infected with only EuMV-YP and healthy non-inoculated pepper plants, respectively. PR10 gene expression decreased by up to 70 % at 28 dpi. Overall, the results indicate that the CaTRXh1-cicy gene participates in defense mechanisms during the compatible interaction of pepper plants with the EuMV-YP DNA virus.

  20. Expression of genes involved in the salicylic acid pathway in type h1 thioredoxin transiently silenced pepper plants during a begomovirus compatible interaction.

    PubMed

    Luna-Rivero, Marianne S; Hernández-Zepeda, Cecilia; Villanueva-Alonzo, Hernán; Minero-García, Yereni; Castell-González, Salvador E; Moreno-Valenzuela, Oscar A

    2016-04-01

    The type-h thioredoxins (TRXs) play a fundamental role in oxidative stress tolerance and defense responses against pathogens. In pepper plants, type-h TRXs participate in the defense mechanism against Cucumber mosaic virus. The goal of this study was to analyze the role of the CaTRXh1-cicy gene in pepper plants during compatible interaction with a DNA virus, the Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The effects of a transient silencing of the CaTRXh1-cicy gene in pepper plants wëre evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants under different treatments. The accumulation of salicylic acid (SA) and the relative expression of the defense genes NPR1 and PR10 were also evaluated. Results showed that viral DNA accumulation was higher in transiently CaTRXh1-cicy silenced plants that were also infected with EuMV-YP. Symptoms in these plants were more severe compared to the non-silenced plants infected with EuMV-YP. The SA levels in the EuMV-YP-infected plants were rapidly induced at 1 h post infection (hpi) in comparison to the non-silenced plants inoculated with EuMV-YP. Additionally, in pepper plants infected with EuMV-YP, the expression of NPR1 decreased by up to 41 and 58 % at 28 days post infection (dpi) compared to the non-silenced pepper plants infected with only EuMV-YP and healthy non-inoculated pepper plants, respectively. PR10 gene expression decreased by up to 70 % at 28 dpi. Overall, the results indicate that the CaTRXh1-cicy gene participates in defense mechanisms during the compatible interaction of pepper plants with the EuMV-YP DNA virus. PMID:26606929

  1. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  2. Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

    PubMed

    Liu, Zheng; Hirani, Arvind H; McVetty, Peter B E; Daayf, Fouad; Quiros, Carlos F; Li, Genyi

    2012-05-01

    The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

  3. [Silencing of Dominant Genes in Heterozygous Genotypes of Interspecific Hybrids Fagopyrum esculentum Moench. x C2026 F. homotropicum Ohnishi].

    PubMed

    Fesenko, N N; Fesenko, I N

    2016-04-01

    Fagopyrum homotropicum Ohnishi is a very polymorphic self-pollinating species with homostylous flowers, which morphologically different lineages are differ also in ability to hybridize with F. esculentum Moench. (closely related outcrosser with heterostyly). A lineage C2026 F. homotropicum diverged from F. esculentum with forming noticeable pre-zygotic and post-zygotic barriers: the most successful interspecific crossing F esculentum x C2026 resulted wrinkled hybrid seeds germinated in Petri dishes. These interspecific hybrids and backcrosses F. esculentum x F₁, being heterozygous at loci DET/det, SHT/sht and homostyly gene of F. homotropicum, in our experiments often formed phenotype like a recessive homozygote for at least one of these genes, i.e. dominant alleles were silenced. Apparently, these effects can be caused by disorders of epigenetic regulation associated with the divergence of hybridized species. Such disorders, especially those that occur at the stage of seed development, represent one of the main experimentally confirmed mechanisms of pre-zygotic isolation between species. Apparently, F. esculentum and the lineage C2026 of F. homotropicum represent an example of intermediate stage of post-zygotic isolation development process which based on epigenetic deregulation of gene expression in the hybrids. Sometimes it may be revealed not only at the stage of seed development, but also at later stages of ontogenesis. PMID:27529977

  4. Silencing of a gene adjacent to the breakpoint of a widespread Drosophila inversion by a transposon-induced antisense RNA.

    PubMed

    Puig, Marta; Cáceres, Mario; Ruiz, Alfredo

    2004-06-15

    Adaptive changes in nature occur by a variety of mechanisms, and Drosophila chromosomal inversions was one of the first studied examples. However, the precise genetic causes of the adaptive value of inversions remain uncertain. Here we investigate the impact of the widespread inversion 2j of Drosophila buzzatii on the expression of the CG13617 gene, whose coding region is located only 12 bp away from the inversion proximal breakpoint. This gene is transcribed into a 2.3-kb mRNA present in all D. buzzatii developmental stages. More importantly, the expression level of CG13617 is reduced 5-fold in embryos of lines homozygous for the 2j inversion compared with lines without the inversion. An antisense RNA that originates in the Foldback-like transposon Kepler inserted at the breakpoint junction in all of the 2j lines and that forms duplexes with the CG13617 mRNA in 2j embryos is most likely responsible for the near silencing of the gene. Few examples of RNA interference caused by transposable elements (TEs) have been previously described, but this mechanism might be prevalent in many organisms and illustrates the potential of TEs as a major source of genetic variation. In addition, because chromosomal rearrangements are usually induced by TEs, position effects might be more common than previously recognized and contribute significantly to the evolutionary success of inversions.

  5. Nanotherapeutic approach for opiate addiction using DARPP-32 gene silencing in an animal model of opiate addiction.

    PubMed

    Ignatowski, T A; Aalinkeel, R; Reynolds, J L; Nair, B B; Sykes, D E; Gleason, C P K; Law, W C; Mammen, M J; Prasad, P N; Schwartz, S A; Mahajan, Supriya D

    2015-03-01

    Opiates act on the dopaminergic system of the brain and perturb 32 kDa dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein (DARPP-32) function. The DARPP-32 mediated inhibition of protein phosphatase-1 (PP-1) and modulation of transcriptional factor CREB is critical to the changes in neuronal plasticity that result in behavioral responses during drug abuse. To investigate the role of DARPP-32 mediated signaling on withdrawal behavior in a rat model of opiate addiction, we used intracerebral administration of gold nanorods (GNR) complexed to DARPP-32 siRNA to silence DARPP-32 gene expression and measure its effects on the opiate withdrawal syndrome. We hypothesized that DARPP-32 siRNA will suppress the neurochemical changes underlying the withdrawal syndrome and therefore prevent conditioned place aversion by suppressing or removing the constellation of negative effects associated with withdrawal, during the conditioning procedure. Our results showed that opiate addicted animals treated with GNR-DARPP-32 siRNA nanoplex showed lack of condition place aversive behavior consequent to the downregulation of secondary effectors such as PP-1 and CREB which modify transcriptional gene regulation and consequently neuronal plasticity. Thus, nanotechnology based delivery systems could allow sustained knockdown of DARPP-32 gene expression which could be developed into a therapeutic intervention for treating drug addiction by altering reward and motivational systems and interfere with conditioned responses.

  6. Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling.

    PubMed

    Bouhouche, Khaled; Gout, Jean-François; Kapusta, Aurélie; Bétermier, Mireille; Meyer, Eric

    2011-05-01

    Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements. PMID:21216825

  7. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    SciTech Connect

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K.

    2012-08-15

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16{sup INK4a} and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. -- Highlights: ►Epigenetic modulations have been shown to have a role in cancer risk. ►Proanthocyanidins decrease the levels of DNA methylation and histone deacetylation. ►Proanthocyanidins inhibit histone deacetylase activity in skin cancer cells. ►Proanthocyanidins reactivate tumor suppressor genes in skin

  8. Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression.

    PubMed

    Liu, Ming; Wang, Dan; Li, Ning

    2016-04-22

    The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatin immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS.

  9. Aberrant silencing of the endocrine peptide gene tachykinin-1 in gastric cancer

    SciTech Connect

    David, Stefan; Kan, Takatsugu; Cheng, Yulan; Agarwal, Rachana; Jin, Zhe; Mori, Yuriko

    2009-01-16

    Tachykinin-1 (TAC1) is the precursor protein for neuroendocrine peptides, including substance P, and is centrally involved in gastric secretion, motility, mucosal immunity, and cell proliferation. Here we report aberrant silencing of TAC1 in gastric cancer (GC) by promoter hypermethylation. TAC1 methylation and mRNA expression in 47 primary GCs and 41 noncancerous gastric mucosae (NLs) were analyzed by utilizing real-time quantitative PCR-based assays. TAC1 methylation was more prevalent in GCs than in NLs: 21 (45%) of 47 GCs versus 6 (15%) of 41 NLs (p < 0.01). Microsatellite instability was also associated with TAC1 methylation in GCs. There was no significant association between TAC1 methylation and age, gender, stage, histological differentiation, or the presence of Helicobacter pylori. TAC1 mRNA was markedly downregulated in GCs relative to NLs. 5-Aza-2'-deoxycytidine-induced demethylation of the TAC1 promoter resulted in TAC1 mRNA upregulation. Further studies are indicated to elucidate the functional involvement of TAC1 in gastric carcinogenesis.

  10. Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

    PubMed

    Buchmann, R Cody; Asad, Shaheen; Wolf, Jamie N; Mohannath, Gireesha; Bisaro, David M

    2009-05-01

    Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS. PMID:19279102

  11. Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

    PubMed

    Mudge, Stephen R; Basnayake, Shiromi W V; Moyle, Richard L; Osabe, Kenji; Graham, Michael W; Morgan, Terence E; Birch, Robert G

    2013-05-01

    Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale. PMID:23297683

  12. Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in suga