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Sample records for targeted gene silencing

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

    Science.gov (United States)

    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. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

    Science.gov (United States)

    Fernando, Deepani D; Marr, Edward J; Zakrzewski, Martha; Reynolds, Simone L; Burgess, Stewart T G; Fischer, Katja

    2017-06-10

    Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools. We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi). We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls. A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

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

    Directory of Open Access Journals (Sweden)

    Sha Lu

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  5. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  6. Silence of the Genes

    Indian Academy of Sciences (India)

    Srimath

    The human genome codes for ~35,000 genes and all these genes are not expressed in every cell. The time and site of gene expression is very precisely regulated. In any cell, only. Silence of the Genes. 2006 Nobel Prize in Physiology or Medicine. Utpal Nath and Saumitra Das. Keywords. RNA interference, siRNA,.

  7. Silence of the Genes

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 4. Silence of the Genes - 2006 Nobel Prize in Physiology or Medicine. Utpal Nath Saumitra Das. General Article Volume 12 Issue 4 April 2007 pp 6-18. Fulltext. Click here to view fulltext PDF. Permanent link:

  8. Antisense gene silencing

    DEFF Research Database (Denmark)

    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...

  9. Posttranscriptional gene silencing in nuclei

    Science.gov (United States)

    Hoffer, Paul; Ivashuta, Sergey; Pontes, Olga; Vitins, Alexa; Pikaard, Craig; Mroczka, Andrew; Wagner, Nicholas; Voelker, Toni

    2011-01-01

    In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants. PMID:21173264

  10. Identification of novel Pax8 targets in FRTL-5 thyroid cells by gene silencing and expression microarray analysis.

    Directory of Open Access Journals (Sweden)

    Tina Di Palma

    Full Text Available The differentiation program of thyroid follicular cells (TFCs, by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown.To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation.This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies.

  11. Identification of Novel Pax8 Targets in FRTL-5 Thyroid Cells by Gene Silencing and Expression Microarray Analysis

    Science.gov (United States)

    Di Palma, Tina; Conti, Anna; de Cristofaro, Tiziana; Scala, Serena; Nitsch, Lucio; Zannini, Mariastella

    2011-01-01

    Background The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. Methodology/Principal Findings To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. Conclusions/Significance This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies. PMID:21966443

  12. RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes.

    Science.gov (United States)

    Oswald, Franz; Winkler, Michael; Cao, Ying; Astrahantseff, Kathy; Bourteele, Soizic; Knöchel, Walter; Borggrefe, Tilman

    2005-12-01

    Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jkappa/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes.

  13. RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene

    Directory of Open Access Journals (Sweden)

    Jaime Marcial-Quino

    2017-11-01

    Full Text Available The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA, which can silence expression of a target gene through the RNA interference (RNAi pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.

  14. RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene

    Science.gov (United States)

    Marcial-Quino, Jaime; Rufino-González, Yadira; Sierra-Palacios, Edgar; Vanoye-Carlo, America; González-Valdez, Abigail; Torres-Arroyo, Angélica; Oria-Hernández, Jesús; Reyes-Vivas, Horacio

    2017-01-01

    The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways. PMID:29099754

  15. Silencing of Target Chitinase Genes via Oral Delivery of dsRNA Caused Lethal Phenotypic Effects in Mythimna separata (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Cao, Budao; Bao, Wenhua; Wuriyanghan, Hada

    2017-02-01

    Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous, migratory corn pest. Outbreak of M. separata has led to severe damage to corn production recently in China. RNAi (RNA interference) is a gene silencing technology applied both in model and non-model organisms, and it is especially useful for the latter in which the reverse genetic research tools are not available. RNAi approach was broadly investigated in many plant pathogens and was used for the generation of anti-pest transgenic plants. We are proposing to use this technology to silence M. separata endogenous genes, thereby, providing a biocontrol method for this insect. Feeding of dsRNAs for target Chitinase genes resulted in substantial decreases of their transcript levels in M. separata. Furthermore, silencing of target Chitinase genes led to phenotypic effects such as reduced body weight and increased mortality. Our study provided both reverse genetic research tool and potential control strategy for this insect species.

  16. Personalized gene silencing therapeutics for Huntington disease.

    Science.gov (United States)

    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. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Targeted Gene-Silencing Reveals the Functional Significance of Myocardin Signaling in the Failing Heart

    Science.gov (United States)

    Torrado, Mario; Iglesias, Raquel; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T.

    2011-01-01

    Background Myocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear. Methodology/Principal Findings The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. Conclusions/Significance These data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view. PMID:22028870

  18. Targeted gene-silencing reveals the functional significance of myocardin signaling in the failing heart.

    Directory of Open Access Journals (Sweden)

    Mario Torrado

    Full Text Available BACKGROUND: Myocardin (MYOCD, a potent transcriptional coactivator of smooth muscle (SM and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF. However, the molecular and functional consequences of myocd upregulation in HF are still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox-induced diastolic HF (DHF model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1 a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2 amelioration of impaired diastolic dysfunction, and (3 higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that a moderate inhibition (e.g., normalization of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

  19. Circular siRNAs for Reducing Off-Target Effects and Enhancing Long-Term Gene Silencing in Cells and Mice.

    Science.gov (United States)

    Zhang, Liangliang; Liang, Duanwei; Chen, Changmai; Wang, Yuan; Amu, Gubu; Yang, Jiali; Yu, Lijia; Dmochowski, Ivan J; Tang, Xinjing

    2018-03-02

    Circular non-coding RNAs are found to play important roles in biology but are still relatively unexplored as a structural motif for chemically regulating gene function. Here, we investigated whether small interfering RNA (siRNA) with a circular structure can circumvent off-target gene silencing, a problem often observed with standard linear duplex siRNA. In the present work, we, for the first time, synthesized a series of circular siRNAs by cyclizing two ends of a single-stranded RNA (sense or antisense strand) to construct circular siRNAs that were more resistant to enzymatic degradation. Gene silencing of GFP and luciferase was successfully achieved using these circular siRNAs with circular sense strand RNAs and their complementary linear antisense strand RNAs. The off-target effect of sense strand RNAs was evaluated and no cross off-target effects were observed. In addition, we successfully achieved longer gene-silencing efficiency in mice with circular siRNAs than with linear siRNAs. These results indicate the promise of circular siRNAs for overcoming off-target effects of siRNAs and enhancing the possible long-term effect of siRNA gene silencing in basic research and drug development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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. Copyright © 2016 by the Genetics Society of America.

  1. RNAi mediated gene silencing of ITPA using a targeted nanocarrier: Apoptosis induction in SKBR3 cancer cells.

    Science.gov (United States)

    Charbgoo, Fahimeh; Behmanesh, Mehrdad; Nikkhah, Maryam; Kane, Eric G

    2017-08-01

    A pure nucleotide pool is required for high-fidelity DNA replication and prevention of carcinogenesis in living cells. Human inosine triphosphatase (ITPase), encoded by the ITPA gene, plays a critical role in maintaining the purity of the cellular nucleotide pool by excluding nucleotides that enhance mutagenesis. ITPase is a nucleoside triphosphate pyrophosphatase that hydrolyzes the non-canonical nucleotides inosine triphosphate (ITP) and xanthine triphosphate (XTP). The monophosphate products of ITPase reactions are subsequently excluded from the nucleotide pool and the improper substitution of ITP and XTP into DNA and RNA is prevented. Previous studies show that deficiency in ITPA can suppress cellular growth and enhance DNA instability. In this study, we evaluated the influence of effective ITPA down-regulation on the induction of apoptosis in a human cancer cell line using folate-single wall nanotubes (SWNT) as a targeted nanocarrier. We assessed whether SWNT enhances IPTA-siRNA transfection efficiency in cancer cells using folate as a homing device. Since folate receptor is considerably overexpressed in cancer cells, conjugation of SWNTs to folate could enhance their cancer-specific penetrance. We found that nanocarrier mediated ITPA-siRNA transfection into SKBR3 cells caused significant reduction of ITPA mRNA expression level and complete down-regulation of the ITPase protein product. The silencing of ITPA led to promotion of apoptosis in SWNT-treated SKBR3 cancer cells. © 2017 John Wiley & Sons Australia, Ltd.

  2. Silence of the Genes

    Indian Academy of Sciences (India)

    Srimath

    tary to the endogenous sense mRNA produced by the normal gene. The antisense strand binds to the sense strand and blocks protein synthesis. This method of gene inhibition was termed antisense technology. The antisense technology soon became a. RNA interference. (RNAi) is a novel mechanism for controlling gene.

  3. Bioinformatics tools for achieving better gene silencing in plants.

    Science.gov (United States)

    Ahmed, Firoz; Dai, Xinbin; Zhao, Patrick Xuechun

    2015-01-01

    RNA interference (RNAi) is one of the most popular and effective molecular technologies for knocking down the expression of an individual gene of interest in living organisms. Yet the technology still faces the major issue of nonspecific gene silencing, which can compromise gene functional characterization and the interpretation of phenotypes associated with individual gene knockdown. Designing an effective and target-specific small interfering RNA (siRNA) for induction of RNAi is therefore the major challenge in RNAi-based gene silencing. A 'good' siRNA molecule must possess three key features: (a) the ability to specifically silence an individual gene of interest, (b) little or no effect on the expressions of unintended siRNA gene targets (off-target genes), and (c) no cell toxicity. Although several siRNA design and analysis algorithms have been developed, only a few of them are specifically focused on gene silencing in plants. Furthermore, current algorithms lack a comprehensive consideration of siRNA specificity, efficacy, and nontoxicity in siRNA design, mainly due to lack of integration of all known rules that govern different steps in the RNAi pathway. In this review, we first describe popular RNAi methods that have been used for gene silencing in plants and their serious limitations regarding gene-silencing potency and specificity. We then present novel, rationale-based strategies in combination with computational and experimental approaches to induce potent, specific, and nontoxic gene silencing in plants.

  4. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    Energy Technology Data Exchange (ETDEWEB)

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Katiyar, Santosh K., E-mail: skatiyar@uab.edu [Birmingham Veterans Affairs Medical Center, Birmingham, AL 35294 (United States); Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    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

  5. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    International Nuclear Information System (INIS)

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K.

    2012-01-01

    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 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

  6. Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

    NARCIS (Netherlands)

    Romero, I.; Tikunov, Y.M.; Bovy, A.G.

    2011-01-01

    Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through

  7. Homology-dependent Gene Silencing in Paramecium

    Science.gov (United States)

    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

  8. Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study.

    Science.gov (United States)

    Bahn, Sabine; Mimmack, Michael; Ryan, Margaret; Caldwell, Maeve A; Jauniaux, Eric; Starkey, Michael; Svendsen, Clive N; Emson, Piers

    2002-01-26

    Identification of genes and characterisation of their function is an essential step towards understanding complex pathophysiological abnormalities in Down's syndrome. We did a study to investigate abnormalities in gene expression in human neuronal stem cells and progenitor cells from Down's syndrome and control post-mortem human fetal tissue. Indexing-based differential display PCR was done on neuronal precursor cells derived from the cortex of a fetus with Down's syndrome, and findings were compared with those of two control samples. Findings were validated against neurosphere preparations from three independent Down's syndrome fetuses and five independent controls by real-time quantitative PCR. Results of differential display PCR analysis showed that SCG10--a neuron--specific growth-associated protein regulated by the neuron-restrictive silencer factor REST-was almost undetectable in the Down's syndrome sample. This finding was validated by real-time PCR. We also found that other genes regulated by the REST transcription factor were selectively repressed, whereas non-REST-regulated genes with similar functions were unaffected. Changes in expression of several key developmental genes in the Down's syndrome stem-cell and progenitor-cell pool correlated with striking changes in neuron morphology after differentiation. Our findings suggest a link between dysregulation of the REST transcription factor and some of the neurological deficits seen in Down's syndrome. Experimental REST downregulation has been shown to trigger apoptosis, which could account for the striking and selective loss of neurons in the differentiated Down's syndrome cell preparations.

  9. Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Néstor García

    2017-12-01

    Full Text Available The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.

  10. Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing

    DEFF Research Database (Denmark)

    Nagasawa, T; Zhang, Q; Raghunath, P N

    2006-01-01

    )-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma...... promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed...... differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target...

  11. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

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

    Science.gov (United States)

    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. Copyright © 2016

  13. Gene Therapy for Neuropathic Pain by Silencing of TNF-α Expression with Lentiviral Vectors Targeting the Dorsal Root Ganglion in Mice

    Science.gov (United States)

    Ogawa, Nobuhiro; Kawai, Hiromichi; Terashima, Tomoya; Kojima, Hideto; Oka, Kazuhiro; Chan, Lawrence; Maegawa, Hiroshi

    2014-01-01

    Neuropathic pain can be a debilitating condition. Many types of drugs that have been used to treat neuropathic pain have only limited efficacy. Recent studies indicate that pro-inflammatory mediators including tumor necrosis factor α (TNF-α) are involved in the pathogenesis of neuropathic pain. In the present study, we engineered a gene therapy strategy to relieve neuropathic pain by silencing TNF-α expression in the dorsal root ganglion (DRG) using lentiviral vectors expressing TNF short hairpin RNA1-4 (LV-TNF-shRNA1-4) in mice. First, based on its efficacy in silencing TNF-α in vitro, we selected shRNA3 to construct LV-TNF-shRNA3 for in vivo study. We used L5 spinal nerve transection (SNT) mice as a neuropathic pain model. These animals were found to display up-regulated mRNA expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY), injury markers, and interleukin (IL)-6, an inflammatory cytokine in the ipsilateral L5 DRG. Injection of LV-TNF-shRNA3 onto the proximal transected site suppressed significantly the mRNA levels of ATF3, NPY and IL-6, reduced mechanical allodynia and neuronal cell death of DRG neurons. These results suggest that lentiviral-mediated silencing of TNF-α in DRG relieves neuropathic pain and reduces neuronal cell death, and may constitute a novel therapeutic option for neuropathic pain. PMID:24642694

  14. Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco

    NARCIS (Netherlands)

    Hutvágner, G.; Mlynarova, L.; Nap, J.P.H.

    2000-01-01

    Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total

  15. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    Directory of Open Access Journals (Sweden)

    Nobutaka Nakashima

    2014-02-01

    Full Text Available 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.

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

    Science.gov (United States)

    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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. The Impact of Gene Silencing on Horizontal Gene Transfer and Bacterial Evolution.

    Science.gov (United States)

    Navarre, W W

    2016-01-01

    The H-NS family of DNA-binding proteins is the subject of intense study due to its important roles in the regulation of horizontally acquired genes critical for virulence, antibiotic resistance, and metabolism. Xenogeneic silencing proteins, typified by the H-NS protein of Escherichia coli, specifically target and downregulate expression from AT-rich genes by selectively recognizing specific structural features unique to the AT-rich minor groove. In doing so, these proteins facilitate bacterial evolution; enabling these cells to engage in horizontal gene transfer while buffering potential any detrimental fitness consequences that may result from it. Xenogeneic silencing and counter-silencing explain how bacterial cells can evolve effective gene regulatory strategies in the face of rampant gene gain and loss and it has extended our understanding of bacterial gene regulation beyond the classic operon model. Here we review the structures and mechanisms of xenogeneic silencers as well as their impact on bacterial evolution. Several H-NS-like proteins appear to play a role in facilitating gene transfer by other mechanisms including by regulating transposition, conjugation, and participating in the activation of virulence loci like the locus of enterocyte effacement pathogenicity island of pathogenic strains of E. coli. Evidence suggests that the critical determinants that dictate whether an H-NS-like protein will be a silencer or will perform a different function do not lie in the DNA-binding domain but, rather, in the domains that control oligomerization. This suggests that H-NS-like proteins are transcription factors that both recognize and alter the shape of DNA to exert specific effects that include but are not limited to gene silencing. © 2016 Elsevier Ltd All rights reserved.

  18. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato.

    Science.gov (United States)

    Velásquez, André C; Chakravarthy, Suma; Martin, Gregory B

    2009-06-10

    RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host. Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript. Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS. Inoculation of Nicotiana benthamiana and tomato seedlings

  19. Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    Science.gov (United States)

    Bonoiu, Adela C.; Bergey, Earl J.; Ding, Hong; Hu, Rui; Kumar, Rajiv; Yong, Ken-Tye; Prasad, Paras N.; Mahajan, Supriya; Picchione, Kelly E.; Bhattacharjee, Arin; Ignatowski, Tracey A.

    2011-01-01

    Gold nanorods (GNRs), cellular imaging nanoprobes, have been used for drug delivery therapy to immunologically privileged regions in the brain. We demonstrate that nanoplexes formed by electrostatic binding between negatively charged RNA and positively charged GNRs, silence the expression of the target housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within the CA1 hippocampal region of the rat brain, without showing cytotoxicity. Fluorescence imaging with siRNACy3GAPDH and dark field imaging using plasmonic enhanced scattering from GNRs were used to monitor the distribution of the nanoplexes within different neuronal cell types present in the targeted hippocampal region. Our results show robust nanoplex uptake and slow release of the fluorescent gene silencer with significant impact on suppression of GAPDH gene expression (70% gene silencing, >10 days post-injection). The observed gene knockdown using nanoplexes in targeted regions of the brain opens a new era of drug treatment for neurological disorders. PMID:21718174

  20. H-NS silences gfp, the green fluorescent protein gene: gfpTCD is a genetically Remastered gfp gene with reduced susceptibility to H-NS-mediated transcription silencing and with enhanced translation.

    Science.gov (United States)

    Corcoran, Colin P; Cameron, Andrew D S; Dorman, Charles J

    2010-09-01

    The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

  1. Locus-specific ribosomal RNA gene silencing in nucleolar dominance.

    Directory of Open Access Journals (Sweden)

    Michelle S Lewis

    2007-08-01

    Full Text Available The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs, the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

  2. Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Rivka Bracha

    2006-05-01

    Full Text Available In a previous work we described the transcriptional silencing of the amoebapore A (AP-A gene (Ehap-a of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp of Ehap-a that included a truncated segment (140 bp of a short interspersed nuclear element (SINE1. Silencing remained in effect even after removal of the plasmid (clone G3. Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1 also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1 and the other, the cysteine proteinase 5 (EhCP-5. This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced

  3. Evidence for gene silencing by endogenous DNA methylation

    Science.gov (United States)

    Holliday, Robin; Ho, Thu

    1998-01-01

    Transformed cells can spontaneously silence genes by de novo methylation, and it is generally assumed that this is due to DNA methyltransferase activity. We have tested the alternative hypothesis that gene silencing could be due to the uptake of 5-methyl-dCMP into DNA, via the di- and triphosphonucleotides. 5-Methyl-dCMP would be present in cells from the ongoing repair of DNA. We have isolated a strain of Chinese hamster ovary (CHO) cells, designated HAM−, which spontaneously silences two tested genes at a very high frequency. We have shown that this strain incorporates 5-[3H]methyldeoxycytidine into 5-methylcytosine and thymine in DNA. It also has low 5-methyl-dCMP deaminase activity. Another HAM+ strain has high deaminase activity and a very low frequency of gene silencing. The starting strain, CHO K1, has a phenotype intermediate between HAM− and HAM+. PMID:9671746

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

    Science.gov (United States)

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin

    DEFF Research Database (Denmark)

    Carroll, Jeffrey B; Warby, Simon C; Southwell, Amber L

    2011-01-01

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild......-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease...

  6. Investigating Gene Function in Cereal Rust Fungi by Plant-Mediated Virus-Induced Gene Silencing.

    Science.gov (United States)

    Panwar, Vinay; Bakkeren, Guus

    2017-01-01

    Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant-fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.

  7. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

    Science.gov (United States)

    Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

    2017-09-26

    Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae -derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1 . Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

  8. Use of guanidinopropyl-modified siRNAs to silence gene expression.

    Science.gov (United States)

    Buff, Maximilian C R; Bernhardt, Stefan; Marimani, Musa D; Ely, Abdullah; Engels, Joachim W; Arbuthnot, Patrick

    2015-01-01

    Silencing gene expression by harnessing the RNA interference (RNAi) pathway with short interfering RNAs (siRNAs) has useful analytical and potentially therapeutic application. To augment silencing efficacy of siRNAs, chemical modification has been employed to improve stability, target specificity, and delivery to target tissues. siRNAs incorporating guanidinopropyl (GP) moieties have demonstrated enhanced target gene silencing in cell culture and in vivo models of hepatitis B virus replication. Here we describe the synthesis of GP-modified siRNAs and use of 5' rapid amplification of cDNA ends (5' RACE) to verify an RNAi-mediated mechanism of action of these novel chemically modified siRNAs.

  9. Disulfide bond-conjugated dual PEGylated siRNAs for prolonged multiple gene silencing.

    Science.gov (United States)

    Liao, Zi-Xian; Hsiao, Chun-Wen; Ho, Yi-Cheng; Chen, Hsin-Lung; Sung, Hsing-Wen

    2013-09-01

    Many human diseases carry at least two independent gene mutations, further exacerbating clinical disorders. In this work, disulfide bond-conjugated dual PEGylated siRNAs were synthesized, capable of specifically targeting and silencing two genes simultaneously. To achieve efficient delivery, the conjugated siRNAs were formulated with the cationic chitosan together with an anionic polymer, poly(γ-glutamic acid) (γPGA), to form a ternary complex. Experimental results indicate that the incorporated γPGA could significantly enhance their intracellular delivery efficiency, allowing for reduction of the disulfide bond-conjugated PEGylated siRNAs delivered to the PEGylated siRNAs in the reductive cytoplasmic environment. The PEGylated siRNAs could more significantly increase their enzymatic tolerability, effectively silence multiple genes, and prolong the duration of their gene silencing capability than the unmodified siRNAs could. Silencing of different genes simultaneously significantly contributes to the efforts to treat multiple gene disorders, and prolonged duration of gene silencing can reduce the need for frequent administrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Development of a gene silencing DNA vector derived from a broad host range geminivirus

    Directory of Open Access Journals (Sweden)

    Hancock Leandria C

    2009-07-01

    Full Text Available Abstract Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV, named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS, transketolase, the sulfur allele of magnesium chelatase (ChlI, and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

  11. Bioreducible polymers for gene silencing and delivery.

    Science.gov (United States)

    Son, Sejin; Namgung, Ran; Kim, Jihoon; Singha, Kaushik; Kim, Won Jong

    2012-07-17

    Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various

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

    Science.gov (United States)

    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 ...

  13. Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

    Science.gov (United States)

    Kant, Ravi; Dasgupta, Indranil

    2017-07-01

    Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v /F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

  14. RNAi-based silencing of genes encoding the vacuolar- ATPase ...

    African Journals Online (AJOL)

    RNAi-based silencing of genes encoding the vacuolar- ATPase subunits a and c in pink bollworm (Pectinophora gossypiella). Ahmed M. A. Mohammed. Abstract. RNA interference is a post- transcriptional gene regulation mechanism that is predominantly found in eukaryotic organisms. RNAi demonstrated a successful ...

  15. Virus-Induced Gene Silencing in Maize with a Foxtail mosaic virus Vector.

    Science.gov (United States)

    Mei, Yu; Whitham, Steven A

    2018-01-01

    Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation. This chapter provides a protocol for constructing FoMV VIGS clones and inoculating them into maize seedlings.

  16. Long Double-Stranded Multiplex siRNAs for Dual Genes Silencing

    Science.gov (United States)

    Peng, Wei; Chen, Jianxin; Qin, Yinchao; Yang, Zhenjun

    2013-01-01

    Simultaneous suppression of multiple oncogenes is an attractive strategy to treat cancers. Herein we present a series of long double-stranded multiplex small interfering RNAs (multi-siRNAs) that is suitable for dual genes silencing through a sequence-specific RNA interference process without inducing significant immune responses. A gap feature structurally designed in either of the nucleotide strands of the multi-siRNAs was proved to be essential toward silencing target genes and avoiding immune responses. Furthermore, the silencing effect of multi-siRNAs against SURVIVIN and BCL-2 genes was shown to be effective and resulted in up-regulation of caspase-3 related apoptosis and, in turn, inhibition of bladder cancer cell proliferation. Our observation suggested that the rationally designed multi-siRNAs would have great potential for therapeutic siRNA design. PMID:23656495

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

    Directory of Open Access Journals (Sweden)

    Sirjana Devi Shrestha

    Full Text Available 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.

  18. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    Science.gov (United States)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  19. RNAi-based silencing of genes encoding the vacuolar- ATPase ...

    African Journals Online (AJOL)

    2016-11-09

    Nov 9, 2016 ... Spodoptera exigua larval development by silencing chitin synthase gene with RNA interference. Bull. Entomol. Res. 98:613-619. Dow JAT (1999). The Multifunctional Drosophila melanogaster V-. ATPase is encoded by a multigene family. J. Bioenerg. Biomembr. 31:75-83. Fire A, Xu SQ, Montgomery MK, ...

  20. MGMT gene silencing and benefit from temozolomide in glioblastoma

    NARCIS (Netherlands)

    Hegi, ME; Diserens, A; Gorlia, T; Hamou, M; de Tribolet, N; Weller, M; Kros, JM; Hainfellner, JA; Mason, W; Mariani, L; Bromberg, JEC; Hau, P; Mirimanoff, RO; Cairncross, JG; Janzer, RC; Stupp, R

    2005-01-01

    BACKGROUND: Epigenetic silencing of the MGMT (O(sup 6)-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive alkylating agents. METHODS: We tested the relationship

  1. Nuclear architecture and gene silencing in olfactory sensory neurons.

    Science.gov (United States)

    Armelin-Correa, Lucia M; Nagai, Maíra H; Leme Silva, Artur G; Malnic, Bettina

    2014-01-01

    Odorants are discriminated by hundreds of odorant receptor (OR) genes, which are dispersed throughout the mammalian genome. The OR genes are expressed in a highly specialized type of cell, the olfactory sensory neuron. Each one of these neurons expresses one of the 2 alleles from one single OR gene type. The mechanisms underlying OR gene expression are unclear. Here we describe recent work demonstrating that the olfactory sensory neuron shows a particular nuclear architecture, and that the genomic OR loci are colocalized in silencing heterochromatin compartments within the nucleus. These discoveries highlight the important role played by epigenetic modifications and nuclear genome organization in the regulation of OR gene expression.

  2. [Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

    Science.gov (United States)

    Wang, Chi-Juan; Xu, Hua; Zhang, Hai-Rui; Wang, Jian; Lin, Ya-Ni; Pang, Tian-Xiang; Li, Qing-Hua

    2014-08-01

    This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

  3. A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes

    Directory of Open Access Journals (Sweden)

    Tanurdzic Milos

    2004-04-01

    Full Text Available Abstract Background Ceratopteris richardii is a useful experimental system for studying gametophyte development and sexual reproduction in plants. However, few tools for cloning mutant genes or disrupting gene function exist for this species. The feasibility of systemic gene silencing as a reverse genetics tool was examined in this study. Results Several DNA constructs targeting a Ceratopteris protoporphyrin IX magnesium chelatase (CrChlI gene that is required for chlorophyll biosynthesis were each introduced into young gametophytes by biolistic delivery. Their transient expression in individual cells resulted in a colorless cell phenotype that affected most cells of the mature gametophyte, including the meristem and gametangia. The colorless phenotype was associated with a 7-fold decrease in the abundance of the endogenous transcript. While a construct designed to promote the transient expression of a CrChlI double stranded, potentially hairpin-forming RNA was found to be the most efficient in systemically silencing the endogenous gene, a plasmid containing the CrChlI cDNA insert alone was sufficient to induce silencing. Bombarded, colorless hermaphroditic gametophytes produced colorless embryos following self-fertilization, demonstrating that the silencing signal could be transmitted through gametogenesis and fertilization. Bombardment of young gametophytes with constructs targeting the Ceratopteris filamentous temperature sensitive (CrFtsZ and uroporphyrin dehydrogenase (CrUrod genes also produced the expected mutant phenotypes. Conclusion A method that induces the systemic silencing of target genes in the Ceratopteris gametophyte is described. It provides a simple, inexpensive and rapid means to test the functions of genes involved in gametophyte development, especially those involved in cellular processes common to all plants.

  4. Klotho gene silencing promotes pathology in the mdx mouse model of Duchenne muscular dystrophy

    Science.gov (United States)

    Wehling-Henricks, Michelle; Li, Zhenzhi; Lindsey, Catherine; Wang, Ying; Welc, Steven S.; Ramos, Julian N.; Khanlou, Négar; Kuro-o, Makoto; Tidball, James G.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a lethal muscle disease involving progressive loss of muscle regenerative capacity and increased fibrosis. We tested whether epigenetic silencing of the klotho gene occurs in the mdx mouse model of DMD and whether klotho silencing is an important feature of the disease. Our findings show that klotho undergoes muscle-specific silencing at the acute onset of mdx pathology. Klotho experiences increased methylation of CpG sites in its promoter region, which is associated with gene silencing, and increases in a repressive histone mark, H3K9me2. Expression of a klotho transgene in mdx mice restored their longevity, reduced muscle wasting, improved function and greatly increased the pool of muscle-resident stem cells required for regeneration. Reductions of fibrosis in late, progressive stages of the mdx pathology achieved by transgene expression were paralleled by reduced expression of Wnt target genes (axin-2), transforming growth factor-beta (TGF-β1) and collagens types 1 and 3, indicating that Klotho inhibition of the profibrotic Wnt/TGFβ axis underlies its anti-fibrotic effect in aging, dystrophic muscle. Thus, epigenetic silencing of klotho during muscular dystrophy contributes substantially to lost regenerative capacity and increased fibrosis of dystrophic muscle during late progressive stages of the disease. PMID:27154199

  5. Two Chloroplastic Viroids Induce the Accumulation of Small RNAs Associated with Posttranscriptional Gene Silencing

    Science.gov (United States)

    Martínez de Alba, A. E.; Flores, R.; Hernández, C.

    2002-01-01

    In plants, posttranscriptional gene silencing (PTGS) has been reported for cytoplasmic RNAs from endogenous nuclear genes, transgenes, viruses, and, recently, for a viroid with nuclear replication and accumulation. However, phenomena of this kind have not been described for mitochondrial or chloroplastic RNAs. Here we show that viroids that replicate and accumulate in the chloroplast are also targets of PTGS and this process may control viroid titer. PMID:12438638

  6. MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense

    OpenAIRE

    Liu, Sheng-Rui; Zhou, Jing-Jing; Hu, Chun-Gen; Wei, Chao-Ling; Zhang, Jin-Zhi

    2017-01-01

    MicroRNAs (miRNAs) are non-coding RNAs of approximately 20–24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant–pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense ...

  7. WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

    OpenAIRE

    Delmas, Amber L.; Riggs, Bridget M.; Pardo, Carolina E.; Dyer, Lisa M.; Darst, Russell P.; Izumchenko, Eugene G.; Monroe, Mänette; Hakam, Ardeshir; Kladde, Michael P.; Siegel, Erin M.; Brown, Kevin D.

    2011-01-01

    Aberrant activation of the Wnt/β-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in norma...

  8. Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer—The Case for Combined Delivery?

    Directory of Open Access Journals (Sweden)

    Rita Mendes

    2017-03-01

    Full Text Available Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

  9. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

    Directory of Open Access Journals (Sweden)

    Kevin Pruitt

    2006-03-01

    Full Text Available The class III histone deactylase (HDAC, SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively, had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

  10. Gene Silencing Triggers Polycomb Repressive Complex 2 Recruitment to CpG Islands Genome Wide

    DEFF Research Database (Denmark)

    Riising, Eva Madi; Vacher-Comet, Itys; Leblanc, Benjamin Olivier

    2014-01-01

    Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show that knoc......Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show......-wide ectopic PRC2 recruitment to endogenous PcG target genes found in other tissues. PRC2 binding analysis shows that it is restricted to nucleosome-free CpG islands (CGIs) of untranscribed genes. Our results show that it is the transcriptional state that governs PRC2 binding, and we propose that it binds...

  11. Epigenetic silencing may aid evolution by gene duplication.

    Science.gov (United States)

    Rodin, Sergei N; Riggs, Arthur D

    2003-06-01

    Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151-1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from "pseudogenization." Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication-degeneration-complementation) process.

  12. Developing Gene Silencing for the Study and Treatment of Dystonia

    Science.gov (United States)

    2017-12-01

    that this is possible but using cells growing in a dish in the laboratory, not in living animals. In this project, we aim to answer several specific... important therapeutic implication of these experiments is that either the striatum is not the primary site of dysfunction in DYT1, or that the molecular...1 AWARD NUMBER: W81XWH-14-1-0282 TITLE: Developing Gene Silencing for the Study and Treatment of Dystonia PRINCIPAL INVESTIGATOR: Pedro

  13. Mobile gene silencing in Arabidopsis is regulated by hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Dacheng Liang

    2014-12-01

    Full Text Available In plants and nematodes, RNAi can spread from cells from which it is initiated to other cells in the organism. The underlying mechanism controlling the mobility of RNAi signals is not known, especially in the case of plants. A genetic screen designed to recover plants impaired in the movement but not the production or effectiveness of the RNAi signal identified RCI3, which encodes a hydrogen peroxide (H2O2-producing type III peroxidase, as a key regulator of silencing mobility in Arabidopsis thaliana. Silencing initiated in the roots of rci3 plants failed to spread into leaf tissue or floral tissue. Application of exogenous H2O2 reinstated the spread in rci3 plants and accelerated it in wild-type plants. The addition of catalase or MnO2, which breaks down H2O2, slowed the spread of silencing in wild-type plants. We propose that endogenous H2O2, under the control of peroxidases, regulates the spread of gene silencing by altering plasmodesmata permeability through remodelling of local cell wall structure, and may play a role in regulating systemic viral defence.

  14. RNA-mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea

    NARCIS (Netherlands)

    Patel, R.M.; Kan, van J.A.L.; Bailey, A.M.; Foster, G.D.

    2008-01-01

    Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD)

  15. Use of a Virus Gene Silencing Vector for Maize Functional Genomics Research.

    Science.gov (United States)

    Zhou, Tao; Liu, Xuedong; Fan, Zaifeng

    2018-01-01

    Virus-induced gene silencing (VIGS) is a genetic technology that exploits the RNA-mediated defense against virus. The method has great potential for plant reverse genetics as it could knock down gene expression in a rapid way, which is triggered by a replicating viral genome engineered to carry a fragment of host gene to be silenced. A number of efficient VIGS vectors are available for dicots, such as for model plant Nicotiana benthamiana; however, only a few of VIGS vectors for monocotyledonous cereal crops. Here, we describe the method for the use of a newly developed VIGS vector based on a maize-infecting Cucumber mosaic virus (CMV) strain ZMBJ-CMV for maize. The RNA2 of ZMBJ-CMV was modified as a vector pCMV201-2b N81 having multiple cloning sites for the insert of 100-300 bp fragment of target gene. Using a method of vascular puncture inoculation of maize seeds with crude sap prepared from Agrobacterium-infiltrated N. benthamiana leaves, silencing of target genes could be obtained in 4 weeks.

  16. Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies.

    Science.gov (United States)

    Nunes, Cristiano C; Dean, Ralph A

    2012-06-01

    Recent discoveries regarding small RNAs and the mechanisms of gene silencing are providing new opportunities to explore fungal pathogen-host interactions and potential strategies for novel disease control. Plant pathogenic fungi are a constant and major threat to global food security; they represent the largest group of disease-causing agents on crop plants on the planet. An initial understanding of RNA silencing mechanisms and small RNAs was derived from model fungi. Now, new knowledge with practical implications for RNA silencing is beginning to emerge from the study of plant-fungus interactions. Recent studies have shown that the expression of silencing constructs in plants designed on fungal genes can specifically silence their targets in invading pathogenic fungi, such as Fusarium verticillioides, Blumeria graminis and Puccinia striiformis f.sp. tritici. Here, we highlight the important general aspects of RNA silencing mechanisms and emphasize recent findings from plant pathogenic fungi. Strategies to employ RNA silencing to investigate the basis of fungal pathogenesis are discussed. Finally, we address important aspects for the development of fungal-derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control fungal disease. © 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.

  17. Trophozoites of Entamoeba histolytica epigenetically silenced in several genes are virulence-attenuated

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    Mirelman D.

    2008-09-01

    Full Text Available The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehap-a gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5’ upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5 gene and the light subunit of the Gal- lectin (Ehlgl1 gene, each under the 5’ upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB-9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.

  18. MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense.

    Science.gov (United States)

    Liu, Sheng-Rui; Zhou, Jing-Jing; Hu, Chun-Gen; Wei, Chao-Ling; Zhang, Jin-Zhi

    2017-01-01

    MicroRNAs (miRNAs) are non-coding RNAs of approximately 20-24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant-pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense mechanism. Here, we summarize the current knowledge and recent advances in understanding the roles of miRNAs involved in the plant defense against viruses and viral counter-defense. We also document the application of miRNAs in plant antiviral defense. This review discusses the current understanding of the mechanisms of miRNA-mediated gene silencing and provides insights on the never-ending arms race between plants and viruses.

  19. Strategy of gene silencing in cassava for validation of resistance genes

    International Nuclear Information System (INIS)

    Cortes, Simon; Lopez, Camilo

    2010-01-01

    Cassava (Manihot esculenta) is a major source of food for more than 1000 million people in the world and constitutes an important staple crop. Cassava bacterial blight, caused by the gram negative bacterium Xanthomonas axonopodis pv. manihotis, is one of the most important constraints for this crop. A candidate resistance gene against cassava bacterial blight, named RXam1, has been identified previously. In this work, we employed the gene silencing approach using the African cassava mosaic virus (ACMV) to validate the function of the RXam1 gene. We used as positive control the su gen, which produce photo blanching in leaves when is silenced. Plants from the SG10735 variety were bombardment with the ACMV-A-SU+ACMV-B y ACMV-A-RXam1+ACMV-B constructions. The silencing efficiency employing the su gene was low, only one of seven plants showed photo blanching. In the putative silenced plants for the RXam1 gene, no presence of siRNAs corresponding to RXam1 was observed; although a low diminution of the RXam1 gene expression was obtained. The growth curves for the Xam strain CIO136 in cassava plants inoculated showing a little but no significance difference in the susceptibility in the silenced plants compared to not silenced

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

    Directory of Open Access Journals (Sweden)

    Gyöngyi Munkácsy

    2016-01-01

    Full Text Available 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.

  1. HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis.

    Science.gov (United States)

    Nowara, Daniela; Gay, Alexandra; Lacomme, Christophe; Shaw, Jane; Ridout, Christopher; Douchkov, Dimitar; Hensel, Götz; Kumlehn, Jochen; Schweizer, Patrick

    2010-09-01

    Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

  2. Highly efficient virus-induced gene silencing in apple and soybean by apple latent spherical virus vector and biolistic inoculation.

    Science.gov (United States)

    Yamagishi, Noriko; Yoshikawa, Nobuyuki

    2013-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the analysis of the gene function in plants within a short time. However, in woody fruit tree like apple, some of Solanum crops, and soybean, it is generally difficult to inoculate virus vector by conventional inoculation methods. Here, we show efficient VIGS in apple and soybean by Apple latent spherical virus (ALSV) vector and biolistic inoculation. The plants inoculated with ALSV vectors by particle bombardment showed uniform silenced phenotypes of target genes within 2-3 weeks post inoculation.

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

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    Yael Garbian

    2012-12-01

    Full Text Available 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.

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Frøkjær-Jensen, Christian; Jain, Nimit; Hansen, Loren

    2016-01-01

    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 s...

  6. SETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila.

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    Dawei Gou

    2010-05-01

    Full Text Available DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9 by members of the Su(var3-9 family of histone methyltransferases (HMTs triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1 can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2 and Su(var205, the Drosophila ortholog of mammalian "Heterochromatin Protein 1", to target genes for dSETDB1. By enlisting Dnmt2 and Su(var205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.

  7. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

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    Praveen Guleria

    Full Text Available 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.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.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.

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

    Science.gov (United States)

    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

  9. Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum

    DEFF Research Database (Denmark)

    Grønlund, Mette; Olsen, Anne; Johansen, Elisabeth

    2010-01-01

    Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However, the avail...

  10. AMFR gene silencing inhibits the differentiation of porcine preadipocytes.

    Science.gov (United States)

    Chen, C Z; Zhu, Y N; Chai, M L; Dai, L S; Gao, Y; Jiang, H; Zhang, L J; Ding, Y; Liu, S Y; Li, Q Y; Lu, W F; Zhang, J B

    2016-04-07

    Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.

  11. RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.

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    Paul McVeigh

    2014-09-01

    Full Text Available 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 (dsRNA-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.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 (siRNAs. 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.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 development of multiple-throughput phenotypic screens for control

  12. RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp.

    Science.gov (United States)

    Flachowsky, Henryk; Tränkner, Conny; Szankowski, Iris; Waidmann, Sascha; Hanke, Magda-Viola; Treutter, Dieter; Fischer, Thilo C

    2012-01-01

    RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.

  13. WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

    Science.gov (United States)

    Delmas, Amber L; Riggs, Bridget M; Pardo, Carolina E; Dyer, Lisa M; Darst, Russell P; Izumchenko, Eugene G; Monroe, Mänette; Hakam, Ardeshir; Kladde, Michael P; Siegel, Erin M; Brown, Kevin D

    2011-11-01

    Aberrant activation of the Wnt/β-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in ∼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

  14. Flexible tools for gene expression and silencing in tomato.

    Science.gov (United States)

    Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

    2009-12-01

    As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

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

    Science.gov (United States)

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

    2016-03-02

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.

    Science.gov (United States)

    Qin, Li-Na; Cai, Fu-Rong; Dong, Xin-Rui; Huang, Zhen-Bang; Tao, Yong; Huang, Jian-Zhong; Dong, Zhi-Yang

    2012-04-01

    A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

    NARCIS (Netherlands)

    Chan, M.F.; van Amerongen, R.; Nijjar, T.; Cuppen, E.; Jones, P.A.; Laird, P.W.

    2001-01-01

    Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by

  18. Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum

    NARCIS (Netherlands)

    Zhang, Z.; Thomma, B.P.H.J.

    2014-01-01

    Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the

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

    Directory of Open Access Journals (Sweden)

    Masci Stefania

    2010-07-01

    Full Text Available Abstract 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.

  20. Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference

    Directory of Open Access Journals (Sweden)

    Masaki Takahashi

    2015-01-01

    Full Text Available The α-synuclein (SNCA gene is a responsible gene for Parkinson's disease (PD; and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi; however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named “expression-control RNAi” (ExCont-RNAi. ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.

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

    Science.gov (United States)

    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.

  2. A translational silencing function of MCPIP1/Regnase-1 specified by the target site context.

    Science.gov (United States)

    Behrens, Gesine; Winzen, Reinhard; Rehage, Nina; Dörrie, Anneke; Barsch, Monika; Hoffmann, Anne; Hackermüller, Jörg; Tiedje, Christopher; Heissmeyer, Vigo; Holtmann, Helmut

    2018-02-19

    The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.

  3. Designing of highly effective complementary and mismatch siRNAs for silencing a gene.

    Science.gov (United States)

    Ahmed, Firoz; Raghava, Gajendra P S

    2011-01-01

    In past, numerous methods have been developed for predicting efficacy of short interfering RNA (siRNA). However these methods have been developed for predicting efficacy of fully complementary siRNA against a gene. Best of author's knowledge no method has been developed for predicting efficacy of mismatch siRNA against a gene. In this study, a systematic attempt has been made to identify highly effective complementary as well as mismatch siRNAs for silencing a gene.Support vector machine (SVM) based models have been developed for predicting efficacy of siRNAs using composition, binary and hybrid pattern siRNAs. We achieved maximum correlation 0.67 between predicted and actual efficacy of siRNAs using hybrid model. All models were trained and tested on a dataset of 2182 siRNAs and performance was evaluated using five-fold cross validation techniques. The performance of our method desiRm is comparable to other well-known methods. In this study, first time attempt has been made to design mutant siRNAs (mismatch siRNAs). In this approach we mutated a given siRNA on all possible sites/positions with all possible nucleotides. Efficacy of each mutated siRNA is predicted using our method desiRm. It is well known from literature that mismatches between siRNA and target affects the silencing efficacy. Thus we have incorporated the rules derived from base mismatches experimental data to find out over all efficacy of mutated or mismatch siRNAs. Finally we developed a webserver, desiRm (http://www.imtech.res.in/raghava/desirm/) for designing highly effective siRNA for silencing a gene. This tool will be helpful to design siRNA to degrade disease isoform of heterozygous single nucleotide polymorphism gene without depleting the wild type protein.

  4. Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia

    Directory of Open Access Journals (Sweden)

    Sateesh Krishnamurthy

    2012-01-01

    Full Text Available The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE or human airway epithelia (HAE grown at the air–liquid interface (ALI, the delivery of a Dicer-substrate small-interfering RNA (DsiRNA duplex against hypoxanthine–guanine phosphoribosyltransferase (HPRT with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2–5-day post-seeding exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF, a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi responses.

  5. The potential of virus-induced gene silencing for speeding up functional characterization of plant genes

    NARCIS (Netherlands)

    Benedito, V.A.; Visser, P.B.; Angenent, G.C.; Krens, F.A.

    2004-01-01

    Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These

  6. Nanoparticles for siRNA-Based Gene Silencing in Tumor Therapy.

    Science.gov (United States)

    Babu, Anish; Muralidharan, Ranganayaki; Amreddy, Narsireddy; Mehta, Meghna; Munshi, Anupama; Ramesh, Rajagopal

    2016-12-01

    Gene silencing through RNA interference (RNAi) has emerged as a potential strategy in manipulating cancer causing genes by complementary base-pairing mechanism. Small interfering RNA (siRNA) is an important RNAi tool that has found significant application in cancer therapy. However due to lack of stability, poor cellular uptake and high probability of loss-of-function due to degradation, siRNA therapeutic strategies seek safe and efficient delivery vehicles for in vivo applications. The current review discusses various nanoparticle systems currently used for siRNA delivery for cancer therapy, with emphasis on liposome based gene delivery systems. The discussion also includes various methods availed to improve nanoparticle based-siRNA delivery with target specificity and superior efficiency. Further this review describes challenges and perspectives on the development of safe and efficient nanoparticle based-siRNA-delivery systems for cancer therapy.

  7. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea.

    Science.gov (United States)

    Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

    2015-05-06

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

  8. Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis.

    Science.gov (United States)

    Yu-Wai-Man, Cynthia; Tagalakis, Aristides D; Manunta, Maria D; Hart, Stephen L; Khaw, Peng T

    2016-02-24

    There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye.

  9. An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Zheng, Zhimin

    2010-01-06

    RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5′ overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5′ overhang and the partnering between RDM12 and RDR2. © 2010 Blackwell Publishing Ltd.

  10. Virus-induced gene silencing and Agrobacterium tumefaciens-mediated transient expression in Nicotiana tabacum.

    Science.gov (United States)

    Zhang, Zhao; Thomma, Bart P H J

    2014-01-01

    Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the corresponding Verticillium effector Ave1 through Agrobacterium tumefaciens-mediated transient transformation (agroinfiltration), we established a rapid system for assessing the requirement of candidate plant genes for Ve1-mediated immune signaling.

  11. Pathways of cellular internalisation of liposomes delivered siRNA and effects on siRNA engagement with target mRNA and silencing in cancer cells.

    Science.gov (United States)

    Alshehri, Abdullah; Grabowska, Anna; Stolnik, Snow

    2018-02-28

    Design of an efficient delivery system is a generally recognised bottleneck in translation of siRNA technology into clinic. Despite research efforts, cellular processes that determine efficiency of siRNA silencing achieved by different delivery formulations remain unclear. Here, we investigated the mechanism(s) of cellular internalisation of a model siRNA-loaded liposome system in a correlation to the engagement of delivered siRNA with its target and consequent silencing by adopting siRNA molecular beacon technology. Probing of cellular internalisation pathways by a panel of pharmacological inhibitors indicated that clathrin-mediated (dynamin-dependent) endocytosis, macropinocytosis (dynamine independent), and cell membrane cholesterol dependent process(es) (clathrin and caveolea-independent) all play a role in the siRNA-liposomes internalization. The inhibition of either of these entry routes was, in general, mirrored by a reduction in the level of siRNA engagement with its target mRNA, as well as in a reduction of the target gene silencing. A dramatic increase in siRNA engagement with its target RNA was observed on disruption of endosomal membrane (by chloroquine), accompanied with an increased silencing. The work thus illustrates that employing molecular beacon siRNA technology one can start to assess the target RNA engagement - a stage between initial cellular internalization and final gene silencing of siRNA delivery systems.

  12. Aflatoxin-free transgenic maize using host-induced gene silencing.

    Science.gov (United States)

    Thakare, Dhiraj; Zhang, Jianwei; Wing, Rod A; Cotty, Peter J; Schmidt, Monica A

    2017-03-01

    Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.

  13. Reduced weed seed shattering by silencing a cultivated rice gene: strategic mitigation for escaped transgenes.

    Science.gov (United States)

    Yan, Huanxin; Li, Lei; Liu, Ping; Jiang, Xiaoqi; Wang, Lei; Fang, Jia; Lin, Zhimin; Wang, Feng; Su, Jun; Lu, Bao-Rong

    2017-08-01

    Transgene flow form a genetically engineered (GE) crop to its wild relatives may result in unwanted environmental consequences. Mitigating transgenes via introducing a gene that is disadvantageous to wild relatives but beneficial to crops, and is tightly-linked with the target transgenes, may provide a promising solution to limit the spread of transgenes in wild/weedy populations. Here we demonstrate a novel system with significantly reduced seed shattering in crop-weed hybrid descendants by partially silenced expression of the seed-shattering gene SH4 in cultivated rice, using artificial microRNA and antisense RNA techniques. Accordingly, fewer seeds were found in the soil of the field plots where transgenic hybrid lineages were grown. However, no differences in productivity-related traits were detected between GE and non-GE cultivated rice. To silence seed-shattering genes provides a useful strategy to reduce the potential environmental impacts caused by transgene flow from commercial GE rice to weedy rice, in addition to the control of weedy rice.

  14. Gene Silencing of 4-1BB by RNA Interference Inhibits Acute Rejection in Rats with Liver Transplantation

    Directory of Open Access Journals (Sweden)

    Yang Shi

    2013-01-01

    Full Text Available 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.

  15. Virus-induced gene silencing (VIGS) as a reverse genetic tool to study development of symbiotic root nodules

    DEFF Research Database (Denmark)

    Kjær, Gabriela Didina Constantin; Grønlund, Mette; Stougaard, Jens

    2008-01-01

    Virus-induced gene silencing (VIGS) can provide a shortcut to plants with altered expression of specific genes. Here, we report that VIGS of the Nodule inception gene (Nin) can alter the nodulation phenotype and Nin gene expression in Pisum sativum. PsNin was chosen as target because of the disti......NinB, nodulation was reduced by at least 45%. Down-regulation of PsNin transcripts in plants inoculated with vectors carrying PsNin cDNA fragments was confirmed and these plants displayed a relative increase in the root/shoot ratio, as expected if nitrogen fixation had been impaired....

  16. Epidermal growth factor receptor: Target for delivery and silencing

    NARCIS (Netherlands)

    Santos Oliveira, S.|info:eu-repo/dai/nl/304841455

    2008-01-01

    Epidermal growth factor receptor in cancer therapy Recently, cancer research has been able to identify molecular targets that are specific for (or highly expressed by) cancer cells. These molecular targets serve as models for the development of rationally designed anticancer drugs that target

  17. Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats

    NARCIS (Netherlands)

    Fitzgerald, A.; Kan, van J.A.L.; Plummer, K.M.

    2004-01-01

    RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing

  18. Mannan-Binding Lectin-Associated Serine Protease 1/3 Cleavage of Pro-Factor D into Factor D In Vivo and Attenuation of Collagen Antibody-Induced Arthritis through Their Targeted Inhibition by RNA Interference-Mediated Gene Silencing

    Science.gov (United States)

    Banda, Nirmal K.; Acharya, Sumitra; Scheinman, Robert I.; Mehta, Gaurav; Coulombe, Marilyne; Takahashi, Minoru; Sekine, Hideharu; Thiel, Steffen; Fujita, Teizo; Holers, V Michael

    2016-01-01

    The complement system is proposed to play an important role in the pathogenesis of rheumatoid arthritis (RA). The complement system mannan-binding lectin associated serine proteases 1 and 3 (MASP-1/3) cleave proDf (inactive) into Df (active), but it is unknown where this cleavage occurs and whether inhibition of MASP-1/3 is a relevant therapeutic strategy for RA. We show herein that the cleavage of proDf into Df by MASP-1/3 can occur in the circulation and that inhibition of MASP-1/3 by gene silencing is sufficient to ameliorate collagen antibody-induced arthritis (CAIA) in mice. Specifically, to examine the cleavage of proDf into Df, MASP-1/3 producing Df−/− liver tissue (donor) was transplanted under the kidney capsule of MASP-1/3−/− (recipient) mice. Five weeks after the liver transplantation, cleaved Df was present in the circulation of MASP-1/3−/− mice. To determine the individual effects of MASP-1/3 and Df gene silencing on CAIA, mice were injected with scrambled, MASP-1/3 targeted, or Df targeted siRNAs. The mRNA levels for MASP-1 and 3 decreased in the liver to 62% and 58%, respectively, in mice injected with MASP-1/3 siRNAs, and Df mRNA decreased to 53% in the adipose tissue of mice injected with Df siRNAs; additionally, circulating MASP-1/3 and Df protein levels were decreased. In mice injected with both siRNAs the clinical disease activity, histopathologic injury scores, C3 deposition, and synovial macrophage/ neutrophil infiltration were significantly decreased. Thus MASP-1/3 is a new therapeutic target for the treatment of RA, likely through both direct effects on the LP and indirect through the AP. PMID:27707997

  19. Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

    Science.gov (United States)

    Dearnley, Megan; Reynolds, Nicholas P; Cass, Peter; Wei, Xiaohu; Shi, Shuning; Mohammed, A Aalam; Le, Tam; Gunatillake, Pathiraja; Tizard, Mark L; Thang, San H; Hinton, Tracey M

    2016-11-14

    The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

  20. Virus-Induced Gene Silencing as a Tool to Study Tomato Fruit Biochemistry.

    Science.gov (United States)

    Fantini, Elio; Giuliano, Giovanni

    2016-01-01

    Virus-Induced Gene Silencing (VIGS) is an excellent reverse genetic tool for the study of gene function in plants, based on virus infection. In this chapter, we describe a high-throughput approach based on VIGS for the study of tomato fruit biochemistry. It comprises the selection of the sequence for silencing using bioinformatics tools, the cloning of the fragment in the Tobacco Rattle Virus (TRV), and the agroinfiltration of tomato fruits mediated by Agrobacterium tumefaciens.

  1. Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shang-Lang [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chao, Chuck C.-K., E-mail: cckchao@mail.cgu.edu.tw [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Department of Medical Research and Development, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan (China)

    2015-06-16

    A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

  2. Heterochromatic genes undergo epigenetic changes and escape silencing in immunodeficiency, centromeric instability, facial anomalies (ICF syndrome.

    Directory of Open Access Journals (Sweden)

    Marie-Elisabeth Brun

    Full Text Available Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF syndrome is a rare autosomal recessive disorder that is characterized by a marked immunodeficiency, severe hypomethylation of the classical satellites 2 and 3 associated with disruption of constitutive heterochromatin, and facial anomalies. Sixty percent of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B gene, encoding a de novo DNA methyltransferase. In the present study, we have shown that, in ICF lymphoblasts and peripheral blood, juxtacentromeric heterochromatic genes undergo dramatic changes in DNA methylation, indicating that they are bona fide targets of the DNMT3B protein. DNA methylation in heterochromatic genes dropped from about 80% in normal cells to approximately 30% in ICF cells. Hypomethylation was observed in five ICF patients and was associated with activation of these silent genes. Although DNA hypomethylation occurred in all the analyzed heterochromatic genes and in all the ICF patients, gene expression was restricted to some genes, every patient having his own group of activated genes. Histone modifications were preserved in ICF patients. Heterochromatic genes were associated with histone modifications that are typical of inactive chromatin: they had low acetylation on H3 and H4 histones and were slightly enriched in H3K9Me(3, both in ICF and controls. This was also the case for those heterochromatic genes that escaped silencing. This finding suggests that gene activation was not generalized to all the cells, but rather was restricted to a clonal cell population that may contribute to the phenotypic variability observed in ICF syndrome. A slight increase in H3K27 monomethylation was observed both in heterochromatin and active euchromatin in ICF patients; however, no correlation between this modification and activation of heterochromatic genes was found.

  3. Heterochromatic Genes Undergo Epigenetic Changes and Escape Silencing in Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF) Syndrome

    Science.gov (United States)

    Brun, Marie-Elisabeth; Lana, Erica; Rivals, Isabelle; Lefranc, Gérard; Sarda, Pierre; Claustres, Mireille; Mégarbané, André; De Sario, Albertina

    2011-01-01

    Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF) syndrome is a rare autosomal recessive disorder that is characterized by a marked immunodeficiency, severe hypomethylation of the classical satellites 2 and 3 associated with disruption of constitutive heterochromatin, and facial anomalies. Sixty percent of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In the present study, we have shown that, in ICF lymphoblasts and peripheral blood, juxtacentromeric heterochromatic genes undergo dramatic changes in DNA methylation, indicating that they are bona fide targets of the DNMT3B protein. DNA methylation in heterochromatic genes dropped from about 80% in normal cells to approximately 30% in ICF cells. Hypomethylation was observed in five ICF patients and was associated with activation of these silent genes. Although DNA hypomethylation occurred in all the analyzed heterochromatic genes and in all the ICF patients, gene expression was restricted to some genes, every patient having his own group of activated genes. Histone modifications were preserved in ICF patients. Heterochromatic genes were associated with histone modifications that are typical of inactive chromatin: they had low acetylation on H3 and H4 histones and were slightly enriched in H3K9Me3, both in ICF and controls. This was also the case for those heterochromatic genes that escaped silencing. This finding suggests that gene activation was not generalized to all the cells, but rather was restricted to a clonal cell population that may contribute to the phenotypic variability observed in ICF syndrome. A slight increase in H3K27 monomethylation was observed both in heterochromatin and active euchromatin in ICF patients; however, no correlation between this modification and activation of heterochromatic genes was found. PMID:21559330

  4. An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs.

    Science.gov (United States)

    Shan, Zhixin; Lin, Qiuxiong; Deng, Chunyu; Li, Xiaohong; Huang, Wei; Tan, Honghong; Fu, Yongheng; Yang, Min; Yu, Xi-Yong

    2009-07-01

    Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.

  5. HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis[W][OA

    Science.gov (United States)

    Nowara, Daniela; Gay, Alexandra; Lacomme, Christophe; Shaw, Jane; Ridout, Christopher; Douchkov, Dimitar; Hensel, Götz; Kumlehn, Jochen; Schweizer, Patrick

    2010-01-01

    Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens. PMID:20884801

  6. Targeting Herpetic Keratitis by Gene Therapy

    Directory of Open Access Journals (Sweden)

    Hossein Mostafa Elbadawy

    2012-01-01

    Full Text Available Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1 can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.

  7. Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana.

    Science.gov (United States)

    Gama, Florinda; Saavedra, Teresa; Dandlen, Susana; de Varennes, Amarilis; Correia, Pedro J; Pestana, Maribela; Nolasco, Gustavo

    2017-05-01

    To evaluate the dynamic role of the ferric-chelate reductase enzyme (FCR) and to identify possible pathways of regulation of its activity in different plant organs an investigation was conducted by virus-induced gene silencing (VIGS) using tobacco rattle virus (TRV) to silence the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. Half of Nicotiana benthamiana plants received the VIGS vector and the rest remained as control. Four treatments were imposed: two levels of Fe in the nutrient solution (0 or 2.5 μM of Fe), each one with silenced or non-silenced (VIGS-0; VIGS-2.5) plants. Plants grown without iron (0; VIGS-0) developed typical symptoms of iron deficiency in the youngest leaves. To prove that FRO1 silencing had occurred, resupply of Fe (R) was done by adding 2.5 μM of Fe to the nutrient solution in a subset of chlorotic plants (0-R; VIGS-R). Twelve days after resupply, 0-R plants had recovered from Fe deficiency while plants containing the VIGS vector (VIGS-R) remained chlorotic and both FRO1 gene expression and FCR activity were considerably reduced, consequently preventing Fe uptake. With the VIGS technique we were able to silence the FRO1 gene in N. benthamiana and point out its importance in chlorophyll synthesis and Fe partition. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wang, X.M.; Gao, S.J.; Guo, X.F.; Sun, W.J. [Department of Oncology, The Second Affiliated Hospital, Harbin Medical University, Harbin (China); Yan, Z.Q. [Department of Breast Surgery, The Second Affiliated Hospital, Harbin Medical University, Harbin (China); Wang, W.X.; Xu, Y.Q.; Lu, D. [Department of Oncology, The Second Affiliated Hospital, Harbin Medical University, Harbin (China)

    2014-03-21

    Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

  9. Gene silencing by chemically modified siRNAs.

    Science.gov (United States)

    Engels, Joachim W

    2013-03-25

    RNA interference (RNAi) has not only already risen as a gold standard for validating gene function in basic science studies, but also holds great promise as a new therapeutic paradigm. Advantages of RNAi-based therapeutics include relatively fast initial screening and the ability to target proteins not yet addressable by traditional drug design strategies. In this review we describe the development of chemically modified small inhibiting siRNAs and their application as potential therapeutics during the past decade. Focus is on proper siRNA design, choice of chemical modification and how to circumvent immunogenicity as well as off-target effects. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis.

    Science.gov (United States)

    Cao, Mengji; Du, Peng; Wang, Xianbing; Yu, Yun-Qi; Qiu, Yan-Hong; Li, Wanxiang; Gal-On, Amit; Zhou, Changyong; Li, Yi; Ding, Shou-Wei

    2014-10-07

    Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of exoribonuclease4/thylene-insensitive5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.

  11. siRNA Versus miRNA as Therapeutics for Gene Silencing

    Directory of Open Access Journals (Sweden)

    Jenny K W Lam

    2015-01-01

    Full Text Available 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.

  12. Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato

    Science.gov (United States)

    Fu, Da-Qi; Meng, Lan-Huan; Zhu, Ben-Zhong; Zhu, Hong-Liang; Yan, Hua-Xue; Luo, Yun-Bo

    2016-12-01

    Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

  13. Mechanisms of epigenetic silencing of the c21 gene in Y1 adrenocortical tumor cells.

    Science.gov (United States)

    Szyf, M; Slack, A D

    2000-11-01

    We utilized Y1 adrenocortical carcinoma cell line as a model system to dissect the events regulating epigenomic gene silencing in tumor cells. We show here that the chromatin structure of c21 gene is inactive in Y1 cells and that it could be reconfigured to an active form by either expressing antisense mRNA to DNA methyltransferase 1 (dnmt1) or an attenuator of Ras protooncogenic signaling hGAP. Surprisingly however, the known inducer of active chromatin structure the histone deacetylase inhibitor trichostatin A TSA fails to induce expression of c21. These results suggest that the primary cause of c21 gene silencing is independent of histone deacetylation. We present a model to explain the possible roles of the different components of the epigenome and the DNA methylation and demethylation machineries in silencing c21 gene expression.

  14. Smuggling gold nanoparticles across cell types - A new role for exosomes in gene silencing.

    Science.gov (United States)

    Roma-Rodrigues, Catarina; Pereira, Francisca; Alves de Matos, António P; Fernandes, Marta; Baptista, Pedro V; Fernandes, Alexandra R

    2017-05-01

    Once released to the extracellular space, exosomes enable the transfer of proteins, lipids and RNA between different cells, being able to modulate the recipient cells' phenotypes. Members of the Rab small GTP-binding protein family, such as RAB27A, are responsible for the coordination of several steps in vesicle trafficking, including budding, mobility, docking and fusion. The use of gold nanoparticles (AuNPs) for gene silencing is considered a cutting-edge technology. Here, AuNPs were functionalized with thiolated oligonucleotides anti-RAB27A (AuNP@PEG@anti-RAB27A) for selective silencing of the gene with a consequent decrease of exosomes´ release by MCF-7 and MDA-MB-453 cells. Furthermore, communication between tumor and normal cells was observed both in terms of alterations in c-Myc gene expression and transportation of the AuNPs, mediating gene silencing in secondary cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available 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.

  16. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases

    DEFF Research Database (Denmark)

    Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath

    2011-01-01

    . The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally......, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci....

  17. Silencing the Honey Bee (Apis mellifera) Naked Cuticle Gene (nkd) Improves Host Immune Function and Reduces Nosema ceranae Infections.

    Science.gov (United States)

    Li, Wenfeng; Evans, Jay D; Huang, Qiang; Rodríguez-García, Cristina; Liu, Jie; Hamilton, Michele; Grozinger, Christina M; Webster, Thomas C; Su, Songkun; Chen, Yan Ping

    2016-11-15

    Nosema ceranae is a new and emerging microsporidian parasite of European honey bees, Apis mellifera, that has been implicated in colony losses worldwide. RNA interference (RNAi), a posttranscriptional gene silencing mechanism, has emerged as a potent and specific strategy for controlling infections of parasites and pathogens in honey bees. While previous studies have focused on the silencing of parasite/pathogen virulence factors, we explore here the possibility of silencing a host factor as a mechanism for reducing parasite load. Specifically, we used an RNAi strategy to reduce the expression of a honey bee gene, naked cuticle (nkd), which is a negative regulator of host immune function. Our studies found that nkd mRNA levels in adult bees were upregulated by N. ceranae infection (and thus, the parasite may use this mechanism to suppress host immune function) and that ingestion of double-stranded RNA (dsRNA) specific to nkd efficiently silenced its expression. Furthermore, we found that RNAi-mediated knockdown of nkd transcripts in Nosema-infected bees resulted in upregulation of the expression of several immune genes (Abaecin, Apidaecin, Defensin-1, and PGRP-S2), reduction of Nosema spore loads, and extension of honey bee life span. The results of our studies clearly indicate that silencing the host nkd gene can activate honey bee immune responses, suppress the reproduction of N. ceranae, and improve the overall health of honey bees. This study represents a novel host-derived therapeutic for honey bee disease treatment that merits further exploration. Given the critical role of honey bees in the pollination of agricultural crops, it is urgent to develop strategies to prevent the colony decline induced by the infection of parasites/pathogens. Targeting parasites and pathogens directly by RNAi has been proven to be useful for controlling infections in honey bees, but little is known about the disease impacts of RNAi silencing of host factors. Here, we demonstrate

  18. Dissection of tomato lycopene biosynthesis through virus-induced gene silencing.

    Science.gov (United States)

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

    2013-10-01

    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.

  19. V2 from a curtovirus is a suppressor of post-transcriptional gene silencing.

    Science.gov (United States)

    Luna, Ana P; Rodríguez-Negrete, Edgar A; Morilla, Gabriel; Wang, Liping; Lozano-Durán, Rosa; Castillo, Araceli G; Bejarano, Eduardo R

    2017-10-01

    The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway.

  20. [Antiproliferative effect of silencing LSD1 gene on Jurkat cell line and its mechanism].

    Science.gov (United States)

    Han, Shiwei; Huang, Yiqun; Zheng, Ruiji

    2016-01-01

    To investigate the effect of silencing LSD1 gene by RNA interference on the proliferation, apoptosis on human lymphocytic leukemia Jurkat cell line and its mechanism. The hairpin- like oligonucleotide sequences targeting LSD1 gene was transfected into Jurkat cells by lipofectamine(TM) 2000. The LSD1 mRNA and protein were detected by RQ- PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Bcl-2, Bax, procaspase- 3, and histone H3K4me, H3K4me2, H3K4me3, Act- H3, H3K9me were detected by Western blot. LSD1 mRNA was markedly suppressed by the shRNA targeting LSD1. LSD1 shRNA suppressed the proliferation and induced cells apoptosis of Jurkat cells. The cell apoptotic rate was (41.34±3.58)%, (3.45±1.54)%, (1.76±0.52)% in LSD1 shRNA, Neg-shRNA and Blank respectively, the difference among them was statistically significant (PJurkat cell line.

  1. Dentin sialophosphoprotein (DSPP gene-silencing inhibits key tumorigenic activities in human oral cancer cell line, OSC2.

    Directory of Open Access Journals (Sweden)

    Rajeshree Joshi

    2010-11-01

    Full Text Available 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.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}.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 down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced

  2. Transgene-induced gene silencing is not affected by a change in ploidy level.

    Directory of Open Access Journals (Sweden)

    Daniela Pignatta

    Full Text Available BACKGROUND: Whole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Diploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS inverted repeat (hairpin constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA. CONCLUSIONS/SIGNIFICANCE: Our results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

  3. Position-Dependent Methylation and Transcriptional Silencing of Transgenes in Inverted T-DNA Repeats: Implications for Posttranscriptional Silencing of Homologous Host Genes in Plants

    Science.gov (United States)

    Stam, Maike; Viterbo, Ada; Mol, Joseph N. M.; Kooter, Jan M.

    1998-01-01

    Posttranscriptional silencing of chalcone synthase (Chs) genes in petunia transformants occurs by introducing T-DNAs that contain a promoter-driven or promoterless Chs transgene. With the constructs we used, silencing occurs only by T-DNA loci which are composed of two or more T-DNA copies that are arranged as inverted repeats (IRs). Since we are interested in the mechanism by which these IR loci induce silencing, we have analyzed different IR loci and nonsilencing single-copy (S) T-DNA loci with respect to the expression and methylation of the transgenes residing in these loci. We show that in an IR locus, the transgenes located proximal to the IR center are much more highly methylated than are the distal genes. A strong silencing locus composed of three inverted T-DNAs bearing promoterless Chs transgenes was methylated across the entire locus. The host Chs genes in untransformed plants were moderately methylated, and no change in methylation was detected when the genes were silenced. Run-on transcription assays showed that promoter-driven transgenes located proximal to the center of a particular IR are transcriptionally more repressed than are the distal genes of the same IR locus. Transcription of the promoterless Chs transgenes could not be detected. In the primary transformant, some of the IR loci were detected together with an unlinked S locus. We observed that the methylation and expression characteristics of the transgenes of these S loci were comparable to those of the partner IR loci, suggesting that there has been cross talk between the two types of loci. Despite the similar features, S loci are unable to induce silencing, indicating that the palindromic arrangement of the Chs transgenes in the IR loci is critical for silencing. Since transcriptionally silenced transgenes in IRs can trigger posttranscriptional silencing of the host genes, our data are most consistent with a model of silencing in which the transgenes physically interact with the

  4. Surface functionalisation of PLGA nanoparticles for gene silencing

    DEFF Research Database (Denmark)

    Andersen, Morten Østergaard; Lichawska, Agata; Arpanaei, Ayyoob

    2010-01-01

    . In addition, particles containing cetylated-PEI achieved 64% silencing of TNFα in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.......This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using...

  5. Tumor targeted gene therapy

    International Nuclear Information System (INIS)

    Kang, Joo Hyun

    2006-01-01

    Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

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

    Science.gov (United States)

    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...

  7. KDR gene silencing inhibits proliferation of A549 cells and enhances their sensitivity to docetaxel.

    Science.gov (United States)

    Wei, R; Zang, J-P

    2015-11-23

    We investigated the effects of kinase-domain insert containing receptor (KDR) gene silencing on the proliferation of A549 cells and their sensitivity to docetaxel. After designing and synthesizing the KDR siRNA sequence, the sequence was transfected into A549 cells using Lipofectamine 2000. The expression of KDR mRNA and protein after KDR gene silencing was detected by reverse transcription-polymerase chain reaction and western blotting; A549 cell cycle was detected by flow cytometry. An MTT assay and colony formation was performed to determine the sensitivity of A549 cells to docetaxel after KDR gene silencing. After 48-h KDR gene silencing, KDR gene and protein expression significantly decreased (P A549 cell cycle was significantly arrested in G0/G1 phase, and the number of cells in S phase was reduced; the difference was statistically significant (P A549 cells to docetaxel showed a significant enhancement (P A549 cells, inhibit the proliferation of A549 cells, and enhance their sensitivity to docetaxel.

  8. Therapeutic effect of photodynamic therapy combined with targeted delivery of silencing vascular endothelial growth factor (Conference Presentation)

    Science.gov (United States)

    Hsu, Yih-Chih

    2016-03-01

    Photodynamic therapy is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates tumours oxygen-independent hypoxic conditions. Vascular endothelial growth factor (VEGF) is one of the primary factors that affect tumor angiogenesis. Another emerging treatment to cure cancer is the use of interference RNA to silence a specific mRNA sequence. Such treatment requires a delivery system such as liposomes. The nanoparticle size measured was about 30 nm. Cellular uptake study was performed to verify that the nanoparticles have a sigma receptor mediated pathway. Non-targeted LCP NPs did not show significant difference with or without haloperidol but has a lower intensity as than targeted LCP NPs. These results confirm that LCP NPs have a receptor mediated pathway. Cell viability was found to decrease at 25 nM of transfected VEGF siRNA. Combined therapy of PDT and VEGF siRNA showed significant response as compared with PDT and gene therapy alone. In vivo toxicity assay with mice treated with targeted LCP NPs containing control siRNA or VEGF siRNA and non-targeted LCP NPs containing VEGF siRNA did not show any significant difference with the PBS injected group which suggests that there is no toxicity with the dose. It suggests that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

  9. ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

    Science.gov (United States)

    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.

  10. Wip1 gene silencing enhances the chemosensitivity of human colon cancer cells.

    Science.gov (United States)

    Xia, Zhong-Sheng; Wu, Di; Zhong, Wa; Lu, Xi-Ji; Yu, Tao; Chen, Qi-Kui

    2017-08-01

    Colon cancer is one of the most common cancers in the world. Multidrug resistance is one of the main reasons for failure of therapy in patients with advanced colon cancer. In previous studies, multiple methods were investigated to reverse the multidrug resistance of colon cancer cells. However, to date, no clinical method has been identified to be satisfactory. Therefore, successful reversal of drug resistance in colon cancer cells still requires new therapeutic strategies or pharmaceuticals. Wild-type p53-induced phosphatase (Wip1), a member of the 2C type serine/threonine protein phosphatase family, is closely associated with the p53 gene, which is the most important tumor-suppressor gene. Wip1 was reported to be associated with the chemosensitivity of breast cancer cells. However, the correlation between the expression of Wip1 gene and the chemosensitivity of colon cancer cells has not been reported yet. In the present study, Wip1-811 small interfering RNA (siRNA) targeting Wip1 was investigated to reverse the multidrug resistance of colon cancer cells. The siRNA duplexes were transfected into RKO colon cancer cells. The messenger RNA (mRNA) expression of Wip1 was measured by reverse transcription-quantitative polymerase chain reaction. The protein level of Wip1 was detected by western blotting. The cell viability was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. Intracellular adriamycin cumulative concentration was determined using flow cytometry. Wip1-811 siRNA efficiently inhibited the expression of Wip1 at the mRNA and protein levels, and enhanced the sensitivity of RKO colon cancer cells towards chemotherapy, which was accompanied by increased cell apoptosis, following the inhibition of Wip1 gene expression. These results indicate that Wip1 gene silencing could enhance the chemosensitivity of colon cancer cells, which may provide a new potential approach for the reversal of multidrug resistance in colon cancer

  11. MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma.

    Directory of Open Access Journals (Sweden)

    Ombretta Melaiu

    Full Text Available 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.

  12. Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

    Directory of Open Access Journals (Sweden)

    Michelle F. Valentine

    2017-05-01

    Full Text Available Soybean [Glycine max (L. Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2, to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets.

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

    DEFF Research Database (Denmark)

    Mandegar, Mohammad A.; Huebsch, Nathaniel; Frolov, Ekaterina B.

    2016-01-01

    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...

  14. Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA

    NARCIS (Netherlands)

    Huang, Yi; Deng, F.; Hu, Z.H.; Vlak, J.M.; Wang, H.

    2007-01-01

    Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA

  15. RNAi-mediated gene silencing as a principle of action of venoms and poisons.

    Science.gov (United States)

    Pereira, Tiago Campos; Lopes-Cendes, Iscia

    2008-01-01

    RNA interference (RNAi) is a natural phenomenon in which double-stranded RNA molecules (dsRNAs) promote silencing of genes with similar sequence. It is noteworthy that in some instances the effects of gene silencing are similar to those caused by venoms and natural poisons (e.g., hemorrhage and low blood pressure). This observation raises the possibility that venomous/poisonous species in fact produce dsRNAs in their venoms/poisons and leading to the deleterious effects in the victim by RNAi-mediated gene silencing. Two approaches could be used to test this hypothesis, first, the neutralization of the dsRNAs and comparing to a non-treated venom sample; and second, to identify the dsRNA present in the venom and attempt to artificially reproduce its effects in the laboratory. In addition, we present three innovative treatment strategies for accidental interactions with venomous or poisonous species. RNAi has several roles in biological systems: gene regulation, antiviral defense, transposon silencing and heterochromatin formation. The hypothesis presented here provides a new role: a natural attack mechanism.

  16. Gene silencing of stearoyl-ACP desaturase enhances the stearic acid content in Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Jaeger, de L.; Springer, J.; Wolbert, E.J.H.; Martens, D.E.; Eggink, G.; Wijffels, R.H.

    2017-01-01

    In this study, stearoyl-ACP desaturase (SAD), the enzyme that converts stearic acid into oleic acid, is silenced by artificial microRNA in the green microalga Chlamydomonas reinhardtii. Two different constructs, which target different positions on the mRNA of stearoyl-ACP desaturase, were tested.

  17. GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Guillaume Jannot

    2016-12-01

    Full Text Available MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.

  18. Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

    Directory of Open Access Journals (Sweden)

    Verónica S Di Stilio

    Full Text Available Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS of a PHYTOENE DESATURASE ortholog (TdPDS can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05, as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.

  19. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  20. Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers.

    Science.gov (United States)

    Yan, Huijun; Shi, Shaochuan; Ma, Nan; Cao, Xiaoqian; Zhang, Hao; Qiu, Xianqin; Wang, Qigang; Jian, Hongying; Zhou, Ningning; Zhang, Zhao; Tang, Kaixue

    2018-01-01

    Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors. Virus-induced gene silencing (VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose, called 'graft-accelerated VIGS', where axillary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR1, RhAG, and RhNUDX1 in rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods. © 2017 Institute of Botany, Chinese Academy of Sciences.

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

    Science.gov (United States)

    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

  2. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. © 2016 American Society of Plant Biologists. All Rights Reserved.

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

    Science.gov (United States)

    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. PMID:24401541

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

    Science.gov (United States)

    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.

  7. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

    Science.gov (United States)

    Zhang, Hanbang; Ehrenkaufer, Gretchen M; Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  8. Identification of epigenetically silenced genes in tumor endothelial cells

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Melotte, Veerle; Vire, Emmanuelle; Langenkamp, Elise; Molema, Grietje; Fuks, Francois; Herman, James G.; Van Criekinge, Wim; Griffioen, Arjan W.; van Engeland, Manon

    2007-01-01

    Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated

  9. Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

    DEFF Research Database (Denmark)

    Hansen, Klavs R.; Burns, G.; Mata, J.

    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...... 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...

  10. Comparison of approaches for efficient gene silencing induced by microRNA-based short hairpin RNA and indicator gene expression.

    Science.gov (United States)

    Shan, Z X; Lin, Q X; Deng, C Y; Zhou, Z L; Tan, H H; Fu, Y H; Li, X H; Zhu, J N; Mai, L P; Kuang, S J; Lin, S G; Yu, X Y

    2010-04-01

    MicroRNA-based short hairpin RNAs (shRNAs) are natural inducers of RNA interference and have been increasingly used in shRNA expression strategies. In the present study, we compared the efficiencies of exogenous green fluorescence protein (GFP) and endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown and red fluorescent protein (RFP) indicator expression mediated by three differently designed plasmids. RFP was introduced either at the 5' end, at the 3' end of the human mir155-based target gene (TG) (e.g., GFP or GAPDH) shRNA expression cassette (EC), or at the 3' end of the chimeric intron-containing TG shRNA EC. Comparisons with the control vector showed an obvious reduction of GFP or GAPDH expression with the various shRNA expression plasmids (P < 0.05). When RFP was located at the 5' end or at the 3' end of the TG shRNA EC, RFP expression was low; whereas when RFP was connected with the chimeric intron-containing TG shRNA EC, RFP expression was high. Taken together, this study demonstrated an efficient plasmid design for both TG silencing induced by microRNA-based shRNA and indicator gene expression in vitro.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  12. A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    Full Text Available It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1-2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r or insulin-like growth factor 1 receptor (Igf-1r siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1-34 (rhPTH (1-34 or insulin-like growth factor-1 (IGF-1. Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD, microstructure, mineral apposition rates (MARs, and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05. Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01. The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05. This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of

  13. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

    Directory of Open Access Journals (Sweden)

    Jose C Garcia-Garcia

    2009-06-01

    Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

  14. Baculovirus-based gene silencing of Humanin for the treatment of pituitary tumors.

    Science.gov (United States)

    Gottardo, María Florencia; Pidre, Matías L; Zuccato, Camila; Asad, Antonela S; Imsen, Mercedes; Jaita, Gabriela; Candolfi, Marianela; Romanowski, Víctor; Seilicovich, Adriana

    2018-02-01

    Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells. In order to evaluate the effect of blockade of endogenous HNr expression in vivo, we constructed a recombinant baculovirus (BV) encoding shHNr (BV-shHNr). In vitro, BV-shRNA was capable of transducing more than 80% of GH3 cells and decreased HNr mRNA. Also, BV-shHNr increased apoptosis in transduced GH3 cells. Intratumor injection of BV-shHNr to nude mice bearing s.c. GH3 tumors increased the number of apoptotic cells, delayed tumor growth and enhanced survival rate, suggesting that endogenous HNr may be involved in pituitary tumor progression. These preclinical data suggests that the silencing of HN expression could have a therapeutic impact on the treatment of pituitary tumors.

  15. Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumor.

    Directory of Open Access Journals (Sweden)

    Anthony R Dallosso

    2009-11-01

    Full Text Available Wilms' tumour (WT is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively. This demonstrates that long-range epigenetic silencing (LRES occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits

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

    Science.gov (United States)

    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. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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

    2008-11-01

    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 (U87Delta) 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 U87Delta 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 U87Delta cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87Delta cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, beta-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87Delta cells with a decrease in G(1) and increase in S and G(2) fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87Delta 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.

  18. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Energy Technology Data Exchange (ETDEWEB)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F [Department of Pharmaceutical Science, Zhujiang Hospital, Southern Medical University, Guangzhou 510282 (China); Yang, B, E-mail: andrewfxu1998@gmail.co [Department of Chemistry, Indiana University-Bloomington, Bloomington, IN 47405 (United States)

    2009-10-07

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  19. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Science.gov (United States)

    Ji, A. M.; Su, D.; Che, O.; Li, W. S.; Sun, L.; Zhang, Z. Y.; Yang, B.; Xu, F.

    2009-10-01

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  20. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    International Nuclear Information System (INIS)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F; Yang, B

    2009-01-01

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  1. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

    Science.gov (United States)

    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. A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3' untranslated region and intronic cis-elements.

    Science.gov (United States)

    Muhle, Rebecca A; Adjalley, Sophie; Falkard, Brie; Nkrumah, Louis J; Muhle, Michael E; Fidock, David A

    2009-11-01

    Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitised erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilising the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var subtelomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronised parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may result from the integrated UpsA promoter being largely silenced by the neighbouring cg6 promoter. Our analyses also revealed that the DownsA 3' untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA-promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyse promoter activity of Group A var genes, which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of var

  3. Improved apple latent spherical virus-induced gene silencing in multiple soybean genotypes through direct inoculation of agro-infiltratedNicotiana benthamianaextract.

    Science.gov (United States)

    Gedling, C R; Ali, E M; Gunadi, A; Finer, J J; Xie, K; Liu, Y; Yoshikawa, N; Qu, F; Dorrance, A E

    2018-01-01

    Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [ Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase ( GmPDS1 ) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems. In this report, the ALSV VIGS vector was modified by mobilizing ALSV cDNAs into a binary vector compatible with Agrobacterium tumefaciens -mediated delivery, so that VIGS-triggering ALSV variants could be propagated in agro-infiltrated Nicotiana benthamiana leaves. Homogenate of these N. benthamiana leaves was then applied directly onto the unifoliate of young soybean seedlings to initiate systemic gene silencing. This rapid inoculation method bypassed the need for a particle bombardment apparatus. Among the 19 soybean genotypes evaluated with this new method, photo-bleaching indicative of GmPDS1 silencing was observed in nine, with two exhibiting photo-bleaching in 100% of the inoculated individuals. ALSV RNA was detected in pods, embryos, stems, leaves, and roots in symptomatic plants of four genotypes. This modified protocol allowed for inoculation of soybean plants via simple mechanical rubbing with the homogenate of N. benthamiana leaves agro-infiltrated with ALSV VIGS constructs. More importantly, inoculated plants showed no apparent virus disease symptoms which could otherwise interfere with VIGS phenotypes. This streamlined procedure expanded this functional genomics tool to nine soybean genotypes.

  4. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA...

  5. siRNA delivery using polyelectrolyte-gold nanoassemblies in neuronal cells for BACE1 gene silencing.

    Science.gov (United States)

    Chaudhary, Aparna; Garg, Sanjeev

    2017-11-01

    Small interfering RNA (siRNA) mediated RNA interference is a versatile therapeutic tool for many intractable genetic disorders. Various nanoassemblies specifically designed to deliver the siRNAs could be utilized for efficient siRNA delivery which is one of the major concern for the success of this therapeutic. Thus, in the present study, polyelectrolyte-gold nanoassemblies (PE-Gold NAs) were selected for siRNA delivery of an in vitro verified siRNA. Three different polyelectrolytes (polyethyleneimine, citraconic anhydride modified poly (allylamine) hydrochloride and poly l-arginine) were used to formulate the PE-Gold NAs using the layer-by-layer technique. Successful physico-chemical characterizations of these PE-Gold NAs were performed using UV-Visible, FTIR, 1 H-NMR spectroscopies, XRD, TEM, DLS and Zeta potential measurements. In vitro studies for the cytotoxicity, the uptake of these nanoassemblies and the gene silencing were carried out using these PE-Gold NAs in N2a and NB4 1A3 (murine neuronal) cell lines. The three selected PE-Gold NAs showed significant BACE1 (β-site APP cleaving enzyme 1) gene silencing (50-60%). This work demonstrates the potential of PE-Gold NAs to deliver siRNA targeting BACE1 in neuronal cells. Finally, it was concluded that different polyelectrolytes used in the PE-Gold NAs achieve different gene silencing due to the variation in their delivery efficiencies. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Heat-induced release of epigenetic silencing reveals the concealed role of an imprinted plant gene.

    Directory of Open Access Journals (Sweden)

    Diego H Sanchez

    2014-11-01

    Full Text Available Epigenetic mechanisms suppress the transcription of transposons and DNA repeats; however, this suppression can be transiently released under prolonged heat stress. Here we show that the Arabidopsis thaliana imprinted gene SDC, which is silent during vegetative growth due to DNA methylation, is activated by heat and contributes to recovery from stress. SDC activation seems to involve epigenetic mechanisms but not canonical heat-shock perception and signaling. The heat-mediated transcriptional induction of SDC occurs particularly in young developing leaves and is proportional to the level of stress. However, this occurs only above a certain window of absolute temperatures and, thus, resembles a thermal-sensing mechanism. In addition, the re-silencing kinetics during recovery can be entrained by repeated heat stress cycles, suggesting that epigenetic regulation in plants may conserve memory of stress experience. We further demonstrate that SDC contributes to the recovery of plant biomass after stress. We propose that transcriptional gene silencing, known to be involved in gene imprinting, is also co-opted in the specific tuning of SDC expression upon heat stress and subsequent recovery. It is therefore possible that dynamic properties of the epigenetic landscape associated with silenced or imprinted genes may contribute to regulation of their expression in response to environmental challenges.

  7. Targeting Aflatoxin Biosynthetic Genes.

    Science.gov (United States)

    Srour, Ali Y; Fakhoury, Ahmad M; Brown, Robert L

    2017-01-01

    Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities are mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requires early detection of the aflatoxin-causing organisms. Detection and quantification of aflatoxin-producing fungi has always been a challenge, especially within species of Aspergillus and Penicillium. Culture-based methods require a high level of expertise and a list of sophisticated equipment. Furthermore, even for a trained taxonomist, species that are identical in morphology, physiology, and nutritional aspects can be challenging to classify. Fungal taxonomy has changed over the past few decades; more species are being reclassified, and new species are being described due to advances in sequencing and genome assembly. These developments make the use of PCR-based approaches practical, rapid, and more reliable for the identification of fungi to the species level. This chapter presents a variety of protocols to detect and quantify aflatoxin-producing fungi using mycotoxin biosynthesis pathway genes.

  8. Artificial MiRNA Knockdown of Platelet Glycoprotein lbα: A Tool for Platelet Gene Silencing.

    Directory of Open Access Journals (Sweden)

    Tim Thijs

    Full Text Available In recent years, candidate genes and proteins implicated in platelet function have been identified by various genomic approaches. To elucidate their exact role, we aimed to develop a method to apply miRNA interference in platelet progenitor cells by using GPIbα as a proof-of-concept target protein. After in silico and in vitro screening of siRNAs targeting GPIbα (siGPIBAs, we developed artificial miRNAs (miGPIBAs, which were tested in CHO cells stably expressing GPIb-IX complex and megakaryoblastic DAMI cells. Introduction of siGPIBAs in CHO GPIb-IX cells resulted in 44 to 75% and up to 80% knockdown of GPIbα expression using single or combined siRNAs, respectively. Conversion of siGPIBAs to miGPIBAs resulted in reduced silencing efficiency, which could however be circumvented by tandem integration of two hairpins targeting different regions of GPIBA mRNA where 72% GPIbα knockdown was achieved. CHO GPIb-IX cells transfected with the miGPIBA construct displayed a significant decrease in their ability to aggregate characterized by lower aggregate numbers and size compared to control CHO GPIb-IX cells. More importantly, we successfully silenced GPIbα in differentiating megakaryoblastic DAMI cells that exhibited morphological changes associated with actin organization. In conclusion, we here report the successful use of miRNA technology to silence a platelet protein in megakaryoblastic cells and demonstrate its usefulness in functional assays. Hence, we believe that artificial miRNAs are suitable tools to unravel the role of a protein of interest in stem cells, megakaryocytes and platelets, thereby expanding their application to novel fields of basic and translational research.

  9. Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos.

    Science.gov (United States)

    Jelly, Noémie S; Schellenbaum, Paul; Walter, Bernard; Maillot, Pascale

    2012-12-01

    Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiR(CP)-1 or amiR(CP)-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiR(CP)-1 and amiR(CP)-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1 day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (G(CP)-1 and G(CP)-2) were obtained by fusing the target sequence of amiR(CP)-1 or amiR(CP)-2 to the 3' terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future.

  10. Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

    Directory of Open Access Journals (Sweden)

    Dashuai Mu

    Full Text Available Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3 was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%. To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.

  12. Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans.

    Science.gov (United States)

    Kalleda, Natarajaswamy; Naorem, Aruna; Manchikatla, Rajam V

    2013-01-01

    Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.

  13. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

    Science.gov (United States)

    Tang, Wei; Newton, Ronald J; Weidner, Douglas A

    2007-01-01

    An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

  14. Targeted methylation of the epithelial cell adhesion molecule (EpCAM promoter to silence its expression in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Suneetha Nunna

    Full Text Available The Epithelial Cell Adhesion Molecule (EpCAM is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4-8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60-80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.

  15. Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Zhanxiu; Zhao, Lili; Li, Hui; Wang, Suxia; Shen, Yong

    2014-04-15

    We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was significantly enhanced in the model group. After 8 weeks, the number of horseradish peroxidase-labeled nerve fibers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and significantly higher than in the model group. The newly formed nerve fibers and myelinated nerve fibers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

  16. Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Zhanxiu; Zhao, Lili; Li, Hui; Wang, Suxia; Shen, Yong

    2014-01-01

    We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was significantly enhanced in the model group. After 8 weeks, the number of horseradish peroxidase-labeled nerve fibers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and significantly higher than in the model group. The newly formed nerve fibers and myelinated nerve fibers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group. PMID:25206893

  17. Plasmodium falciparum var Gene Silencing Is Determined by cis DNA Elements That Form Stable and Heritable Interactions ▿

    Science.gov (United States)

    Swamy, Lakshmi; Amulic, Borko; Deitsch, Kirk W.

    2011-01-01

    Antigenic variation in the human malaria parasite Plasmodium falciparum depends on the transcriptional regulation of the var gene family. In each individual parasite, mRNA is expressed exclusively from 1 var gene out of ∼60, while the rest of the genes are transcriptionally silenced. Both modifications to chromatin structure and DNA regulatory elements associated with each var gene have been implicated in the organization and maintenance of the silent state. Whether silencing is established at the level of entire chromosomal regions via heterochromatin spreading or at the level of individual var promoters through the action of a silencing element within each var intron has been debated. Here, we consider both possibilities, using clonal parasite lines carrying chromosomally integrated transgenes. We confirm a previous finding that the loss of an adjacent var intron results in var promoter activation and further show that transcriptional activation of a var promoter within a cluster does not affect the transcriptional activity of neighboring var promoters. Our results provide more evidence for the hypothesis that var genes are primarily silenced at the level of an individual gene, rather than by heterochromatin spreading. We also tested the intrinsic directionality of an intron's silencing effect on upstream or downstream var promoters. We found that an intron is capable of silencing in either direction and that, once established, a var promoter-intron pair is stably maintained through many generations, suggesting a possible role in epigenetic memory. This study provides insights into the regulation of endogenous var gene clusters. PMID:21317310

  18. H-NS mediates the silencing of laterally acquired genes in bacteria.

    Directory of Open Access Journals (Sweden)

    Sacha Lucchini

    2006-08-01

    Full Text Available Histone-like nucleoid structuring protein (H-NS is a modular protein that is associated with the bacterial nucleoid. We used chromatin immunoprecipitation to determine the binding sites of H-NS and RNA polymerase on the Salmonella enterica serovar Typhimurium chromosome. We found that H-NS does not bind to actively transcribed genes and does not co-localize with RNA polymerase. This shows that H-NS principally silences gene expression by restricting the access of RNA polymerase to the DNA. H-NS had previously been shown to preferentially bind to curved DNA in vitro. In fact, at the genomic level we discovered that the level of H-NS binding correlates better with the AT-content of DNA. This is likely to have evolutionary consequences because we show that H-NS binds to many Salmonella genes acquired by lateral gene transfer, and functions as a gene silencer. The removal of H-NS from the cell causes un-controlled expression of several Salmonella pathogenicity islands, and we demonstrate that this has deleterious consequences for bacterial fitness. Our discovery of this novel role for H-NS may have implications for the acquisition of foreign genes by enteric bacteria.

  19. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

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    Tuttle John

    2012-08-01

    Full Text Available Abstract Background We previously developed a virus-induced gene silencing (VIGS vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV. The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens. We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

  20. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

    DEFF Research Database (Denmark)

    Pacak, Andrzej; Geisler, Katrin; Jørgensen, Bodil

    2010-01-01

    -expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created...... a need for tools to study gene function in these species. Results Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation...... the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments....

  1. Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells

    International Nuclear Information System (INIS)

    Qin Zhaoling; Zhao Ping; Zhang Xiaolian; Yu Jianguo; Cao Mingmei; Zhao Lanjuan; Luan Jie; Qi Zhongtian

    2004-01-01

    Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV

  2. Silencing of the Il2 gene transcription is regulated by epigenetic changes in anergic T cells.

    Science.gov (United States)

    Bandyopadhyay, Sanmay; Montagna, Cristina; Macian, Fernando

    2012-09-01

    Anergy is induced in T cells as a consequence of a partial or suboptimal stimulation. Anergic T cells become unresponsive and fail to proliferate and produce cytokines. We had previously shown that in anergic CD4(+) T cells, Ikaros participates in the transcriptional repression of the Il2 gene by recruiting histone deacetylases that cause core histone deacetylation at the Il2 promoter. Here we show that deacetylation at the Il2 promoter is the initial step in a process that leads to the stable silencing of the Il2 gene transcription in anergic T cells. We have found that anergy-induced deacetylation of the Il2 promoter permits binding of the histone methyl-transferase Suv39H1, which trimethylates lysine-9 of histone H3 (Me3H3-K9). Furthermore, the establishment of the Me3H3-K9 mark allows the recruitment of the heterochromatin protein HP1, allowing the silenced Il2 loci to reposition close to heterochromatin-rich regions. Our results indicate that silencing of Il2 transcription in anergic T cells is attained through a series of epigenetic changes that involve the establishment of repressive marks and the subsequent nuclear repositioning of the Il2 loci, which become juxtaposed to transcriptionally silent regions. This mechanism may account for the stable nature of the inhibition of IL-2 production in anergic cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    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

  4. In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

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    Arase Sachiko

    2012-03-01

    Full Text Available Abstract Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

  5. NIR-induced spatiotemporally controlled gene silencing by upconversion nanoparticle-based siRNA nanocarrier.

    Science.gov (United States)

    Chen, Guojun; Ma, Ben; Xie, Ruosen; Wang, Yuyuan; Dou, Kefeng; Gong, Shaoqin

    2017-12-27

    Spatiotemporal control over the release or activation of biomacromolecules such as siRNA remains a significant challenge. Light-controlled release has gained popularity in recent years; however, a major limitation is that most photoactivable compounds/systems respond only to UV irradiation, but not near-infrared (NIR) light that offers a deeper tissue penetration depth and better biocompatibility. This paper reports a simple NIR-to-UV upconversion nanoparticle (UCNP)-based siRNA nanocarrier for NIR-controlled gene silencing. siRNA is complexed onto a NaYF 4 :Yb/Tm/Er UCNP through an azobenzene (Azo)-cyclodextrin (CD) host-guest interaction. The UV emission generated by the NIR-activated UCNP effectively triggers the trans-to-cis photoisomerization of azobenzene, thus leading to the release of siRNA due to unmatched host-guest pairs. The UCNP-siRNA complexes are also functionalized with PEG (i.e., UCNP-(CD/Azo)-siRNA/PEG NPs), targeting ligands (i.e., EGFR-specific GE11 peptide), acid-activatable cell-penetrating peptides (i.e., TH peptide), and imaging probes (i.e., Cy5 fluorophore). The UCNP-(CD/Azo)-siRNA/PEG NPs with both GE11 and TH peptides display a high level of cellular uptake and an excellent endosomal/lysosomal escape capability. More importantly, NIR-controlled spatiotemporal knockdown of GFP expression is successfully achieved in both a 2D monolayer cell model and a 3D multicellular tumor spheroid model. Thus, this simple and versatile nanoplatform has great potential for the selective activation or release of various biomacromolecules. Copyright © 2017. Published by Elsevier B.V.

  6. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

    Science.gov (United States)

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

    2015-01-01

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  7. The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing.

    Science.gov (United States)

    Redrup, Lisa; Branco, Miguel R; Perdeaux, Elizabeth R; Krueger, Christel; Lewis, Annabelle; Santos, Fátima; Nagano, Takashi; Cobb, Bradley S; Fraser, Peter; Reik, Wolf

    2009-02-01

    Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.

  8. An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.

    Science.gov (United States)

    Hubner, Eric K; Lechler, Christian; Kohnke-Ertel, Birgit; Zmoos, Anne-Flore; Sage, Julien; Schmid, Roland M; Ehmer, Ursula

    2017-01-01

    Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice. After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system. Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

    Directory of Open Access Journals (Sweden)

    Nilsson Lena

    2010-11-01

    Full Text Available Abstract Background Gene silencing vectors based on Barley stripe mosaic virus (BSMV are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species have created a need for tools to study gene function in these species. Results Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa and diploid (A. strigosa oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component. Conclusions Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too

  10. Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

    Science.gov (United States)

    Ulrich, Julia; Dao, Van Anh; Majumdar, Upalparna; Schmitt-Engel, Christian; Schwirz, Jonas; Schultheis, Dorothea; Ströhlein, Nadi; Troelenberg, Nicole; Grossmann, Daniela; Richter, Tobias; Dönitz, Jürgen; Gerischer, Lizzy; Leboulle, Gérard; Vilcinskas, Andreas; Stanke, Mario; Bucher, Gregor

    2015-09-03

    Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

  11. Silencing of human T-cell leukemia virus type I gene transcription by epigenetic mechanisms

    Directory of Open Access Journals (Sweden)

    Mueller Nancy

    2005-10-01

    Full Text Available Abstract Background Human T-cell leukemia virus type I (HTLV-I causes adult T-cell leukemia (ATL after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. Results The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. Conclusion These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.

  12. Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. High-Throughput Screening Using iPSC-Derived Neuronal Progenitors to Identify Compounds Counteracting Epigenetic Gene Silencing in Fragile X Syndrome.

    Science.gov (United States)

    Kaufmann, Markus; Schuffenhauer, Ansgar; Fruh, Isabelle; Klein, Jessica; Thiemeyer, Anke; Rigo, Pierre; Gomez-Mancilla, Baltazar; Heidinger-Millot, Valerie; Bouwmeester, Tewis; Schopfer, Ulrich; Mueller, Matthias; Fodor, Barna D; Cobos-Correa, Amanda

    2015-10-01

    Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused in most of cases by epigenetic silencing of the Fmr1 gene. Today, no specific therapy exists for FXS, and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large-scale drug discovery screens since they conserve the Fmr1 gene silenced within the disease context. We have established a high-content imaging assay to run a large-scale phenotypic screen aimed to identify compounds that reactivate the silenced Fmr1 gene. A set of 50,000 compounds was tested, including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPSC generation, culture scale-up, and quality control and screening with a very sensitive high-content imaging assay assisted by single-cell image analysis and multiparametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of fragile X mental retardation protein (FMRP) and thus sets the basis for further large-scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention. © 2015 Society for Laboratory Automation and Screening.

  14. Inhibition of Taura syndrome virus replication in Litopenaeus vannamei through silencing the LvRab7 gene using double-stranded RNA.

    Science.gov (United States)

    Ongvarrasopone, Chalermporn; Saejia, Pipop; Chanasakulniyom, Mayuree; Panyim, Sakol

    2011-07-01

    Taura syndrome virus (TSV) is a major cause of high mortality in Pacific white shrimp (Litopenaeus vannamei, Lv). Previously, silencing of Penaeus monodon Rab7 (PmRab7) by injecting double-stranded RNA corresponding to PmRab7 (dsRNA-PmRab7) prevented white spot syndrome virus or yellow head virus infection. Rab7 is proposed to be involved in intracellular trafficking of the viruses. This study aimed to investigate whether knockdown of Rab7 in L. vannamei by dsRNA-PmRab7 could inhibit replication of TSV. RNA interference (RNAi) technology using dsRNA targeting the LvRab7 gene was used to silence the mRNA expression of LvRab7. The silencing of the LvRab7 gene inhibited TSV replication dramatically when compared to groups receiving dsRNA-GFP or NaCl. This is the first demonstration that dsRNA targeting the endogenous shrimp gene LvRab7 strongly reduces TSV replication. It provides further evidence that LvRab7 is involved in the endosomal trafficking pathway of viruses infecting penaeid shrimp.

  15. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells.

    Science.gov (United States)

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi

    2016-03-01

    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (PJurkat cells, thus inhibiting cell proliferation.

  16. Gene Silencing and Sex Determination by Programmed DNA Elimination in Parasitic Nematodes

    Science.gov (United States)

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

    2016-01-01

    Maintenance of genome integrity is essential. However, programmed DNA elimination removes specific DNA sequences from the genome during early 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 determines 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

  17. Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

    Czech Academy of Sciences Publication Activity Database

    Crhák Khaitová, Lucie; Fojtová, M.; Křížová, Kateřina; Lunerová Bedřichová, Jana; Fulneček, Jaroslav; Depicker, A.; Kovařík, Aleš

    2011-01-01

    Roč. 6, č. 5 (2011), s. 650-660 ISSN 1559-2294 R&D Projects: GA ČR(CZ) GD204/09/H002; GA MŠk(CZ) LC06004 Grant - others:GA ČR(CZ) GPP501/11/P667 Program:GP Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : transcriptional gene silencing * transgene epialleles * DNA methylation Subject RIV: BO - Biophysics Impact factor: 4.318, year: 2011

  18. Dissecting epigenetic silencing complexity in the mouse lung cancer suppressor gene Cadm1.

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    Stella Marie Reamon-Buettner

    Full Text Available Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3, and opposing histone modification marks (H3K4me3 and H3K27me3 all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and 'bivalent' histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

  19. Primary and secondary siRNAs in geminivirus-induced gene silencing.

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    Michael Aregger

    2012-09-01

    Full Text Available In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL proteins producing short interfering (siRNAs. 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.

  20. Primary and secondary siRNAs in geminivirus-induced gene silencing.

    Science.gov (United States)

    Aregger, Michael; Borah, Basanta K; Seguin, Jonathan; Rajeswaran, Rajendran; Gubaeva, Ekaterina G; Zvereva, Anna S; Windels, David; Vazquez, Franck; Blevins, Todd; Farinelli, Laurent; Pooggin, Mikhail M

    2012-09-01

    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.

  1. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

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    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

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

    Science.gov (United States)

    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...

  3. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

    DEFF Research Database (Denmark)

    Pacak, Andrzej; Geisler, Katrin; Jørgensen, Bodil

    2010-01-01

    -expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created...

  4. Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis

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    Zhimin Zheng

    2015-05-01

    Full Text Available Paramutation is an epigenetic phenomenon that has been observed in a number of multicellular organisms. The epigenetically silenced state of paramutated alleles is not only meiotically stable but also “infectious” to active homologous alleles. The molecular mechanism of paramutation remains unclear, but components involved in RNA-directed DNA methylation (RdDM are required. Here, we report a multi-copy pRD29A-LUC transgene in Arabidopsis thaliana that behaves like a paramutation locus. The silent state of LUC is induced by mutations in the DNA glycosylase gene ROS1. The silent alleles of LUC are not only meiotically stable but also able to transform active LUC alleles into silent ones, in the absence of ros1 mutations. Maintaining silencing at the LUC gene requires action of multiple pathways besides RdDM. Our study identified specific factors that are involved in the paramutation-like phenomenon and established a model system for the study of paramutation in Arabidopsis.

  5. Probing transcription factor binding activity and downstream gene silencing in living cells with a DNA nanoswitch.

    Science.gov (United States)

    Bertucci, Alessandro; Guo, Junling; Oppmann, Nicolas; Glab, Agata; Ricci, Francesco; Caruso, Frank; Cavalieri, Francesca

    2018-01-25

    Transcription factor DNA binding activity is of pivotal importance in living systems because of its primary involvement in the regulation of genetic machinery. The analysis of transient expression levels of transcription factors in response to a certain cell status is a powerful means for investigating cellular dynamics at the biomolecular level. Herein, a DNA-based molecular switch that enables probing of transcription factor DNA binding activity is directly used in living cells. We demonstrate that the DNA nanoswitch allows for dynamic fluorescence imaging of NF-κB and quantification of downstream gene silencing in real time. The present strategy is based on a functional DNA nanodevice that transduces, through a binding-induced conformational change, the recognition of a specific transcription factor into a fluorescent signal. In addition, stochastic optical resolution microscopy, a super-resolution microscopy technique, is used to track the internalization and intracellular trafficking of the DNA nanodevice with high spatial resolution. Overall, it has been shown that a rationally designed DNA nanodevice can be used to achieve rapid, simple, and cost-effective real-time determination of transcription factor binding activity and downstream gene silencing.

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

    Directory of Open Access Journals (Sweden)

    Lehto Kirsi

    2011-04-01

    Full Text Available Abstract 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

  7. Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

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    Maryam Zangi

    2016-07-01

    Full Text Available Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP when compared to the absence of P19 suppressor (0.2%TSP. Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

  8. Silencing Bag-1 gene via magnetic gold nanoparticle-delivered siRNA plasmid for colorectal cancer therapy in vivo and in vitro.

    Science.gov (United States)

    Huang, Wenbai; Liu, Zhan'ao; Zhou, Guanzhou; Ling, Jianmin; Tian, Ailing; Sun, Nianfeng

    2016-08-01

    Apoptosis disorder is generally regarded as an important mechanism of carcinogenesis. Inducement of tumor cell apoptosis can be an effectual way to treat cancer. Bcl-2-associated athanogene 1 (Bag-1) is a positive regulator of Bcl-2 which is an anti-apoptotic gene. Bag-1 is highly expressed in colorectal cancer, which plays a critical role in promoting metastasis, poor prognosis, especially in anti-apoptotic function, and is perhaps a valuable gene target for colorectal cancer therapy. Recently, we applied a novel non-viral gene carrier, magnetic gold nanoparticle, and mediated plasmid pGPH1/GFP/Neo-Bag-1-homo-825 silencing Bag-1 gene for treating colorectal cancer in vivo and in vitro. By mediating with magnetic gold nanoparticle, siRNA plasmid was successfully transfected into cell. In 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, magnetic gold nanoparticle had no significant cytotoxicity and by which delivered RNA plasmid inhibited cell viability significantly (P nanoparticle plasmid complex-transfected Balb c/nude tumor xenograft. In conclusion, Bag-1 is confirmed an anti-apoptosis gene that functioned in colorectal cancer, and the mechanism of Bag-1 gene causing colorectal cancer may be related to Wnt/β-catenin signaling pathway abnormality and suggested that magnetic gold nanoparticle-delivered siRNA plasmid silencing Bag-1 is an effective gene therapy method for colorectal cancer.

  9. Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number.

    Science.gov (United States)

    Khuong, Thi Thu Huong; Crété, Patrice; Robaglia, Christophe; Caffarri, Stefano

    2013-09-01

    An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions. Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.

  10. Frequent epigenetic silencing of the folate-metabolising gene cystathionine-beta-synthase in gastrointestinal cancer.

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    Hong Zhao

    Full Text Available Both gastric and colorectal cancers (CRC are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.To identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96 and 55% of gastric tumors (11 of 20. In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ(2-test. However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.A novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.

  11. RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

    Science.gov (United States)

    Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

    2014-07-01

    Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

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

    DEFF Research Database (Denmark)

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria

    2016-01-01

    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 ha...... of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6 days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing....

  13. Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts

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    Satoru Noguchi

    2014-01-01

    Full Text Available Ullrich congenital muscular dystrophy (UCMD is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

  14. A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3’ untranslated region and intronic cis-elements

    Science.gov (United States)

    Muhle, Rebecca A.; Adjalley, Sophie; Falkard, Brie; Nkrumah, Louis J.; Muhle, Michael E.; Fidock, David A.

    2009-01-01

    Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitized erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilizing the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var sub-telomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronized parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may well result from the integrated UpsA promoter being largely silenced by the neighboring cg6 promoter. Our analyses also revealed that the DownsA 3’ untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyze promoter activity of Group A var genes which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of

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

    Science.gov (United States)

    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.

  16. Simultaneous silencing of two arginine decarboxylase genes alters development in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Diana eSánchez-Rangel

    2016-03-01

    Full Text Available 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.

  17. Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

    Science.gov (United States)

    Gaglione, Maria; Mercurio, M. Emilia; Mosca, Nicola; Novellino, Ettore; Messere, Anna

    2014-01-01

    The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3′ end, carrying 5′-phosphate and 3′-hydroxyl groups (siRNAs). Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3′ overhang recognition by the PAZ domain and the 5′-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA) moieties at 5′, and at 3′ positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs. PMID:24791003

  18. Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

    Directory of Open Access Journals (Sweden)

    Maria Gaglione

    2014-01-01

    Full Text Available The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3′ end, carrying 5′-phosphate and 3′-hydroxyl groups (siRNAs. Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3′ overhang recognition by the PAZ domain and the 5′-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA moieties at 5′, and at 3′ positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Daino, Kazuhiro, E-mail: k_daino@nirs.go.jp [LCE/iRCM/DSV/CEA, route du Panorama, BP 6, 92265 Fontenay-aux-Roses Cedex (France); Roch-Lefevre, Sandrine [LDB/SRBE/DRPH/IRSN, 31, avenue de la Division Leclerc, BP 17, 92262 Fontenay-aux-Roses Cedex (France); Ugolin, Nicolas; Altmeyer-Morel, Sandrine; Guilly, Marie-Noelle; Chevillard, Sylvie [LCE/iRCM/DSV/CEA, route du Panorama, BP 6, 92265 Fontenay-aux-Roses Cedex (France)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Daino, K.; Ugolin, N.; Altmeyer-Morel, S.; Guilly, M.N.; Chevillard, S. [LCE/iRCM/DSV/CEA, route du Panorama, BP 6, 92265 Fontenay-aux-Roses Cedex (France); Roch-Lefevre, S. [LDB/SRBE/DRPH/IRSN, 31, avenue de la Division Leclerc, BP 17, 92262 Fontenay-aux-Roses Cedex (France)

    2009-07-01

    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 micro-array analyses, respectively. Among the previously identified genes that were down-regulated in radiation-induced rat OS tumors, Cited2 (Cbp/p300-interacting trans-activator, 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. (authors)

  1. ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

    Directory of Open Access Journals (Sweden)

    de Souza Emanuel M

    2009-03-01

    Full Text Available Abstract Background ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. Methods First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP. Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. Results The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM; tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC was 76.2% compared with 25.5% in invasive ductal carcinoma

  2. ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

    International Nuclear Information System (INIS)

    Seniski, Gerusa G; Zanata, Silvio M; Costa, Fabrício F; Klassen, Giseli; Camargo, Anamaria A; Ierardi, Daniela F; Ramos, Edneia AS; Grochoski, Mariana; Ribeiro, Enilze SF; Cavalli, Iglenir J; Pedrosa, Fabio O; Souza, Emanuel M de

    2009-01-01

    ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was

  3. Gene Therapy Targeting HIV Entry

    Directory of Open Access Journals (Sweden)

    Chuka Didigu

    2014-03-01

    Full Text Available Despite the unquestionable success of antiretroviral therapy (ART in the treatment of HIV infection, the cost, need for daily adherence, and HIV-associated morbidities that persist despite ART all underscore the need to develop a cure for HIV. The cure achieved following an allogeneic hematopoietic stem cell transplant (HSCT using HIV-resistant cells, and more recently, the report of short-term but sustained, ART-free control of HIV replication following allogeneic HSCT, using HIV susceptible cells, have served to both reignite interest in HIV cure research, and suggest potential mechanisms for a cure. In this review, we highlight some of the obstacles facing HIV cure research today, and explore the roles of gene therapy targeting HIV entry, and allogeneic stem cell transplantation in the development of strategies to cure HIV infection.

  4. Differential Cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds

    KAUST Repository

    Idris, Ali

    2010-12-01

    A Cotton leaf crumple virus (CLCrV)-based gene silencing vector containing a fragment of the Gossypium hirsutum Magnesium chelatase subunit I was used to establish endogenous gene silencing in cotton of varied genetic backgrounds. Biolistic inoculation resulted in systemic and persistent photo-bleaching of the leaves and bolls of the seven cultivars tested, however, the intensity of silencing was variable. CLCrV-VIGS-mediated expression of green fluorescent protein was used to monitor the in planta distribution of the vector, indicating successful phloem invasion in all cultivars tested. Acala SJ-1, one of the cotton cultivars, was identified as a particularly optimal candidate for CLCrV-VIGS-based cotton reverse-genetics. © 2010 Elsevier Ltd.

  5. Effects of kinase insert domain receptor (KDR) gene silencing on the sensitivity of A549 cells to erlotinib.

    Science.gov (United States)

    Zhu, W L; Liu, Y H

    2015-11-25

    We investigated the effects of kinase insert domain receptor (KDR) gene silencing on the proliferation of A549 cells and their sensitivity to erlotinib. A KDR small interfering RNA (siRNA) sequence was designed and synthesized; then, it was transfected into A549 cells using Lipofectamine(TM) 2000. KDR mRNA and protein expression after KDR gene silencing was detected by reverse transcription polymerase chain reaction and western blotting; the A549 cell cycle was detected by flow cytometry. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay were performed to determine the sensitivity of A549 cells to erlotinib after KDR gene silencing. After 48h of KDR gene silencing, there was a significant decrease in KDR gene and protein expression (P A549 cell cycle was arrested at the G0/G1 phase, and the number of cells in the S phase decreased; the difference was statistically significant (P A549 cells to erlotinib was significantly enhanced (P A549 cells, inhibit the proliferation of A549 cells, and enhance their sensitivity to erlotinib.

  6. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    Directory of Open Access Journals (Sweden)

    Justine M Pompey

    Full Text Available Dicer enzymes process double-stranded RNA (dsRNA into small RNAs that target gene silencing through the RNA interference (RNAi pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

  7. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    Science.gov (United States)

    Pompey, Justine M; Foda, Bardees; Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

  8. RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

    Science.gov (United States)

    Gilbert, Matthew K; Majumdar, Rajtilak; Rajasekaran, Kanniah; Chen, Zhi-Yuan; Wei, Qijian; Sickler, Christine M; Lebar, Matthew D; Cary, Jeffrey W; Frame, Bronwyn R; Wang, Kan

    2018-03-14

    Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

  9. Effects of cyclin D1 gene silencing on cell proliferation, cell cycle, and apoptosis of hepatocellular carcinoma cells.

    Science.gov (United States)

    Chen, Jin; Li, Xue; Cheng, Qi; Ning, Deng; Ma, Jie; Zhang, Zhi-Ping; Chen, Xiao-Ping; Jiang, Li

    2018-02-01

    This study aims to investigate the effects of Cyclin D1 silencing on cell cycle, cell proliferation, and apoptosis of hepatocellular carcinoma cells (HCC). Cells were divided into the blank group, negative control group (HCC cells transfected with control shRNA), Cyclin D1 shRNA group (HCC cells transfected with Cyclin D1 shRNA), and the normal group (human normal liver L-02 cells). Expressions of Cyclin D1, Caspase-3, Bcl-2, and C-myc were detected by RT-qPCR and Western blotting. Cell proliferation was detected by Cell Counting Kit-8. Cell cycle and apoptosis were detected by flow cytometry. Tumor xenograft in nude mice was performed to detect in vivo tumorigenesis. HCC tissues and HCC cells exhibited elevated expression levels of Cyclin D1. Cyclin D1 expression levels was found to be correlated with tumor size and tumor staging. Compared with the normal group, the blank group showed enhanced cell proliferation, a reduction in the amount of cells in G0/G1 phase, increased number cells in S and G2/M phase, reduced apoptosis, elevated expressions of Cyclin D1, Bcl-2, and C-myc, decreased Caspase-3 activity and significant tumorigenicity. In comparison with the blank group, the Cyclin D1 shRNA group revealed weakened cell proliferation, reduced cells in S and G2/M phase, increased cells in G0/G1 phase, increased Annexin V positive cell ratio, decreased expression of Cyclin D1, Bcl-2, and C-myc, elevated Caspase-3 activity and inhibited tumorigenicity. In conclusion, Cyclin D1 gene silencing suppresses cell proliferation and inhibits cell apoptosis, which may be a new target approach in the treatment and management for HCC. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  11. Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device

    Directory of Open Access Journals (Sweden)

    Robyn P Hickerson

    2013-01-01

    Full Text Available Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.

  12. Silencing the HaAK gene by transgenic plant-mediated RNAi impairs larval growth of Helicoverpa armigera.

    Science.gov (United States)

    Liu, Feng; Wang, Xiao-Dong; Zhao, Yi-Ying; Li, Yan-Jun; Liu, Yong-Chang; Sun, Jie

    2015-01-01

    Insect pests have caused noticeable economic losses in agriculture, and the heavy use of insecticide to control pests not only brings the threats of insecticide resistance but also causes the great pollution to foods and the environment. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been is currently developed for protection against insect pests. In this study, we used this technology to silence the arginine kinase (AK) gene of Helicoverpa armigera (HaAK), encoding a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrate. Transgenic Arabidopsis plants producing HaAK dsRNA were generated by Agrobacterium-mediated transformation. The maximal mortality rate of 55% was reached when H. armigera first-instar larvae were fed with transgenic plant leaves for 3 days, which was dramatically higher than the 18% mortality recorded in the control group. Moreover, the ingestion of transgenic plants significantly retarded larval growth, and the transcript levels of HaAK were also knocked down by up to 52%. The feeding bioassays further indicated that the inhibition efficiency was correlated with the integrity and concentration of the produced HaAK dsRNA in transgenic plants. These results strongly show that the resistance to H. armigera was improved in transgenic Arabidopsis plants, suggesting that the RNAi targeting of AK has the potential for the control of insect pests.

  13. Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata

    DEFF Research Database (Denmark)

    Grønlund, Mette; Kjær, Gabriela Didina Constantin; Piednoir, Elodie

    2008-01-01

    Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respec...

  14. The histone methyltransferases Set5 and Set1 have overlapping functions in gene silencing and telomere maintenance.

    Science.gov (United States)

    Jezek, Meagan; Gast, Alison; Choi, Grace; Kulkarni, Rushmie; Quijote, Jeremiah; Graham-Yooll, Andrew; Park, DoHwan; Green, Erin M

    2017-02-01

    Genes adjacent to telomeres are subject to transcriptional repression mediated by an integrated set of chromatin modifying and remodeling factors. The telomeres of Saccharomyces cerevisiae have served as a model for dissecting the function of diverse chromatin proteins in gene silencing, and their study has revealed overlapping roles for many chromatin proteins in either promoting or antagonizing gene repression. The H3K4 methyltransferase Set1, which is commonly linked to transcriptional activation, has been implicated in telomere silencing. Set5 is an H4 K5, K8, and K12 methyltransferase that functions with Set1 to promote repression at telomeres. Here, we analyzed the combined role for Set1 and Set5 in gene expression control at native yeast telomeres. Our data reveal that Set1 and Set5 promote a Sir protein-independent mechanism of repression that may primarily rely on regulation of H4K5ac and H4K8ac at telomeric regions. Furthermore, cells lacking both Set1 and Set5 have highly correlated transcriptomes to mutants in telomere maintenance pathways and display defects in telomere stability, linking their roles in silencing to protection of telomeres. Our data therefore provide insight into and clarify potential mechanisms by which Set1 contributes to telomere silencing and shed light on the function of Set5 at telomeres.

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

    Science.gov (United States)

    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

    2004-11-01

    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.

  16. Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

    Directory of Open Access Journals (Sweden)

    Fujun Jin

    Full Text Available Herpes simplex virus type 1 (HSV-1, a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18 and VP5 (UL19 individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

  17. Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

    Science.gov (United States)

    Jin, Fujun; Li, Shen; Zheng, Kai; Zhuo, Cuiqin; Ma, Kaiqi; Chen, Maoyun; Wang, Qiaoli; Zhang, Peizhuo; Fan, Jianglin; Ren, Zhe; Wang, Yifei

    2014-01-01

    Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

  18. Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria

    DEFF Research Database (Denmark)

    Zhang, Qingfeng; Siegel, T Nicolai; Martins, Rafael M

    2014-01-01

    Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral......-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non......-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of Pf...

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

    Directory of Open Access Journals (Sweden)

    Elisa Giorgetti

    2016-09-01

    Full Text Available 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.

  20. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  1. Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats

    Directory of Open Access Journals (Sweden)

    Guadalupe S. López-Álvarez

    2017-01-01

    Full Text Available The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4 using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.

  2. Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect of Silencing HIF-1α with Targeted Multifunctional ECO/siRNA Nanoparticles.

    Science.gov (United States)

    Malamas, Anthony S; Jin, Erlei; Gujrati, Maneesh; Lu, Zheng-Rong

    2016-07-05

    Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced MRI (DCE-MRI) with a biodegradable macromolecular MRI contrast agent was used to noninvasively assess the antiangiogenic effect of RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg of siRNA/kg every 3 days for 3 weeks compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silencing of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in tumor angiogenesis, glycolytic metabolism, and pH regulation. By possessing the ability to elicit a multifaceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment.

  3. Disruption of prefoldin-2 protein synthesis in root-knot nematodes via host-mediated gene silencing efficiently reduces nematode numbers and thus protects plants.

    Science.gov (United States)

    Ajjappala, Hemavathi; Chung, Ha Young; Sim, Joon-Soo; Choi, Inchan; Hahn, Bum-Soo

    2015-03-01

    The aim of this study is to demonstrate the feasibility of down-regulating endogeneous prefoldin-2 root-knot nematode transcripts by expressing dsRNA with sequence identity to the nematode gene in tobacco roots under the influence of strong Arabidopsis ubiquitin (UBQ1) promoter. Root-knot nematodes (RKNs) are sedentary endoparasites infecting a wide range of plant species. They parasitise the root system, thereby disrupting water and nutrient uptake and causing major reductions in crop yields. The most reliable means of controlling RKNs is via the use of soil fumigants such as methyl bromide. With the emergence of RNA interference (RNAi) technology, which permits host-mediated nematode gene silencing, a new strategy to control plant pathogens has become available. In the present study, we investigated host-induced RNAi gene silencing of prefoldin-2 in transgenic Nicotiana benthamiana. Reductions in prefoldin-2 mRNA transcript levels were observed when nematodes were soaked in a dsRNA solution in vitro. Furthermore, nematode reproduction was suppressed in RNAi transgenic lines, as evident by reductions in the numbers of root knots (by 34-60 % in independent RNAi lines) and egg masses (by 33-58 %). Endogenous expression of prefoldin-2, analysed via real-time polymerase chain reaction and Western blotting, revealed that the gene was strongly expressed in the pre-parasitic J2 stage. Our observations demonstrate the relevance and potential importance of targeting the prefoldin gene during the nematode life cycle. The work also suggests that further improvements in silencing efficiency in economically important crops can be accomplished using RNAi directed against plant-parasitic nematodes.

  4. Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles.

    Science.gov (United States)

    Nascimento, Ana Vanessa; Singh, Amit; Bousbaa, Hassan; Ferreira, Domingos; Sarmento, Bruno; Amiji, Mansoor M

    2017-01-01

    Efficiency of chemotherapy is often limited by low therapeutic index of the drug as well as emergence of inherent and acquired drug resistance in cancer cells. As a common strategy to overcome drug resistance, higher doses of chemo-agents are administered. However, adverse side effects are usually increased as a consequence. A potentially effective approach is to combine chemotherapy with other therapeutic strategies such as small interfering RNAs (siRNAs) that allow the use of lower yet efficient doses of the anticancer drugs. We previously developed epidermal growth factor receptor (EGFR)-targeted chitosan (CS) nanoparticles as a versatile delivery system for silencing the essential mitotic checkpoint gene Mad2, and induce cell death. Here, we tested this system as a single therapy and in combination with cisplatin in cisplatin sensitive and resistant lung cancer models, and characterized its in vivo efficacy and safety. Combination treatment resulted in significant improvement in tumor inhibition that was strikingly more effective in cisplatin-resistant tumors. Importantly, effective cisplatin dosage was dramatically reduced in the co-therapy regimen resulting in negligible toxic effects from the drug as confirmed by parameters such as body weight gain, biochemical markers of hepatic and renal function, and histopathology of liver/kidney/spleen tissues. Overall, we demonstrate that the combination of Mad2 siRNA-loaded CS nanoparticles strategy with chemotherapeutic agents such as cisplatin constitutes an efficient and safe approach for the treatment of drug resistant tumors. Lung cancer remains one of the leading killers in the United States and around the world. Platinum agents, including cisplatin, are the first line treatment in lung cancer, including non-small cell lung cancer (NSCLC), which is the predominant form of lung cancer. In this study, we have evaluated Mad2 cell-cycle checkpoint gene silencing using small interfering RNA (siRNA) delivered

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

    Directory of Open Access Journals (Sweden)

    Tatsuya eKon

    2014-11-01

    Full Text Available 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 CaMV 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 0 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.

  6. LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

    Science.gov (United States)

    James, Victoria; Zhang, Yining; Foxler, Daniel E; de Moor, Cornelia H; Kong, Yi Wen; Webb, Thomas M; Self, Tim J; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M; Morley, Simon J; Longmore, Gregory D; Bushell, Martin; Sharp, Tyson V

    2010-07-13

    In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.

  7. Targeted integration of genes in Xenopus tropicalis

    DEFF Research Database (Denmark)

    Shi, Zhaoying; Tian, Dandan; Xin, Huhu

    2017-01-01

    With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous...... recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis....

  8. Silencing of ecdysone receptor, insect intestinal mucin and sericotropin genes by bacterially produced double-stranded RNA affects larval growth and development in Plutella xylostella and Helicoverpa armigera.

    Science.gov (United States)

    Israni, B; Rajam, M V

    2017-04-01

    RNA interference mediated gene silencing, which is triggered by double-stranded RNA (dsRNA), has become a important tool for functional genomics studies in various systems, including insects. Bacterially produced dsRNA employs the use of a bacterial strain lacking in RNaseIII activity and harbouring a vector with dual T7 promoter sites, which allow the production of intact dsRNA molecules. Here, we report an assessment of the functional relevance of the ecdysone receptor, insect intestinal mucin and sericotropin genes through silencing by dsRNA in two lepidopteran insect pests, Helicoverpa armigera and Plutella xylostella, both of which cause serious crop losses. Oral feeding of dsRNA led to significant reduction in transcripts of the target insect genes, which caused significant larval mortality with various moulting anomalies and an overall developmental delay. We also found a significant decrease in reproductive potential in female moths, with a drop in egg laying and compromised egg hatching from treated larvae as compared to controls. dsRNA was stable in the insect gut and was efficiently processed into small interfering RNAs (siRNAs), thus accounting for the phenotypes observed in the present work. The study revealed the importance of these genes in core insect processes, which are essential for insect development and survival. © 2016 The Royal Entomological Society.

  9. HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes.

    Science.gov (United States)

    Zalewski, Wojciech; Orczyk, Wacław; Gasparis, Sebastian; Nadolska-Orczyk, Anna

    2012-11-07

    CKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium. The mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T(1) lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T(0) plants and T(1) lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T(1) lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively

  10. Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

    Science.gov (United States)

    Saeed, Muhammad; Krczal, Gabi; Wassenegger, Michael

    2015-04-01

    Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and βC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or βC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and βC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or βC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

  11. Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Guosong Jiang

    Full Text Available Heparanase (HPA, an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA induces transcriptional gene silencing (TGS in human cells. In this study, transfection of siRNA against -9/+10 bp (siH3, but not -174/-155 bp (siH1 or -134/-115 bp (siH2 region relative to transcription start site (TSS locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2, histone H3 lysine 27 trimethylation (H3K27me3 or active chromatin marker acetylated histone H3 (AcH3. The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB, but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (-9/+10 bp into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells.

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

    Science.gov (United States)

    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. © (2010) Max Planck Society. Journal compilation © New Phytologist Trust (2010).

  13. Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

    Science.gov (United States)

    Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

    2015-12-01

    Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides

  14. Heterochromatin-Mediated Gene Silencing Facilitates the Diversification of Olfactory Neurons

    Directory of Open Access Journals (Sweden)

    David B. Lyons

    2014-11-01

    Full Text Available 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 olfactory receptor (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, our data suggest that, in addition to its previously known functions, heterochromatin creates an epigenetic platform that affords stochastic, mutually exclusive gene choices and promotes cellular diversity.

  15. The drug target genes show higher evolutionary conservation than non-target genes.

    Science.gov (United States)

    Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

    2016-01-26

    Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

  16. Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

    Directory of Open Access Journals (Sweden)

    Qiu Ri-Xiang

    2010-11-01

    Full Text Available Abstract Background Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD with polyethylenimine (PEI in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA interference therapy targeting human survivin by this novel technique. Methods Two different expression vectors (pCMV-LUC and pSIREN were incubated with PEI to prepare cationic complexes (PEI/DNA and confirmed by the gel retardation assay. Human cervical carcinoma (Hela tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected. Results Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression. Conclusions This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration

  17. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

    Directory of Open Access Journals (Sweden)

    Shen-shen ZHI

    2011-02-01

    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  18. Loss of Major DNase I Hypersensitive Sites in Duplicatedglobin Gene Cluster Incompletely Silences HBB Gene Expression

    Czech Academy of Sciences Publication Activity Database

    Reading, N. S.; Shooter, C.; Song, J.; Miller, R.; Agarwal, A.; Láníková, Lucie; Clark, B.; Thein, S.L.; Divoký, V.; Prchal, J.T.

    2016-01-01

    Roč. 37, č. 11 (2016), s. 1153-1156 ISSN 1059-7794 R&D Projects: GA MŠk(CZ) LH15223 Institutional support: RVO:68378050 Keywords : globin gene s * regulation * sickle cell disease * HBB duplication Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 4.601, year: 2016

  19. Human γ-globin genes silenced independently of other genes in the β-globin locus.

    NARCIS (Netherlands)

    N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1991-01-01

    textabstractErythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5'

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

    Science.gov (United States)

    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.

  1. Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

    Science.gov (United States)

    Bandaranayake, Pradeepa C G; Yoder, John I

    2013-05-01

    Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene

  2. Development of Gold Nanoparticle towards Radioenhancement Therapy, Renal Clearance, siRNA Delivery and Light-Controlled Gene Silencing

    Science.gov (United States)

    Wang, Jianxin

    Gold nanoparticles (GNPs) have been widely studied and used in research for diagnostic, prophylactic or therapeutic purposes. However, they still face many technical challenges before they can be used to effectively address unmet biomedical needs. The theme of this dissertation is focused on addressing challenges of GNPs in clinical translation, and to improve their potential for application in radioenhancement therapy and siRNA delivery. We demonstrate the facile self-assembly of micellar gold nanocapsules using zwitterionic surfactants, with hydrodynamic diameters below 10 nm, which holds promise for good renal clearance to promote the excretion of GNPs in human body. We also prepared PEI- and PEG-coated GNPs and demonstrated their uptake into HeLa cells with exposure to soft X-rays (120 kVp), based on the consideration that the proximity of GNPs to nuclear DNA may be beneficial for enhancing low-energy ionizing radiotherapy. GNP-mediated siRNA delivery may be challenged by nonspecific siRNA desorption during circulation, which can cause off-target effects and immunogenicity. The use of gold nanorods (GNRs) for siRNA delivery also faces challenges like reduced dispersion stability during siRNA functionalization. We developed an effective way to load siRNA onto GNRs at high density, using oleylsulfobetaine (OSB) as an intermediate surfactant and dithiocarbamates (DTCs) as desorption-resistant anchors for siRNA. The GNR?siRNA complexes provided excellent control for laser-triggered gene silencing.

  3. A sexual shift induced by silencing of a single insulin-like gene in crayfish: ovarian upregulation and testicular degeneration.

    Directory of Open Access Journals (Sweden)

    Ohad Rosen

    2010-12-01

    Full Text Available 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.

  4. DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells.

    Science.gov (United States)

    Murphy, Shawn P; Holtz, Renae; Lewandowski, Nicole; Tomasi, Thomas B; Fuji, Hiroshi

    2002-09-15

    MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.

  5. Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato.

    Science.gov (United States)

    Xiong, Ai-Sheng; Yao, Quan-Hong; Peng, Ri-He; Li, Xian; Han, Pei-Lai; Fan, Hui-Qin

    2005-02-01

    RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.

  6. Early exposure to paraquat sensitizes dopaminergic neurons to subsequent silencing of PINK1 gene expression in mice.

    Science.gov (United States)

    Zhou, Hongxia; Huang, Cao; Tong, Jianbin; Xia, Xu-Gang

    2011-01-01

    Environmental exposure, genetic modification, and aging are considered risky for Parkinson's disease (PD). How these risk factors cooperate to induce progressive neurodegeneration in PD remains largely unknown. Paraquat is an herbicide commonly used for weed and grass control. Exposure to paraquat is associated with the increased incidence of PD. In contrast to familial PD, most sporadic PD cases do not have genetic mutation, but may suffer from partial dysfunction of neuron-protective genes as aging. Using conditional transgenic RNAi, we showed that temporal silencing of PINK1 expression in adult mice increased striatal dopamine, the phenotype that could not be induced by constitutive gene silencing. Moreover, early exposure to paraquat sensitized dopaminergic neurons to subsequent silencing of PINK1 gene expression, leading to a significant loss of dopaminergic neurons. Our findings suggest a novel pathogenesis of PD: exposure to environmental toxicants early in the life reduces the threshold of developing PD and partial dysfunction of neuron-protective genes later in the life initiates a process of progressive neurodegeneration to cross the reduced threshold of disease onset.

  7. ETS transcription factors control transcription of EZH2 and epigenetic silencing of the tumor suppressor gene Nkx3.1 in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Paolo Kunderfranco

    2010-05-01

    Full Text Available ETS transcription factors regulate important signaling pathways involved in cell differentiation and development in many tissues and have emerged as important players in prostate cancer. However, the biological impact of ETS factors in prostate tumorigenesis is still debated.We performed an analysis of the ETS gene family using microarray data and real-time PCR in normal and tumor tissues along with functional studies in normal and cancer cell lines to understand the impact in prostate tumorigenesis and identify key targets of these transcription factors. We found frequent dysregulation of ETS genes with oncogenic (i.e., ERG and ESE1 and tumor suppressor (i.e., ESE3 properties in prostate tumors compared to normal prostate. Tumor subgroups (i.e., ERG(high, ESE1(high, ESE3(low and NoETS tumors were identified on the basis of their ETS expression status and showed distinct transcriptional and biological features. ERG(high and ESE3(low tumors had the most robust gene signatures with both distinct and overlapping features. Integrating genomic data with functional studies in multiple cell lines, we demonstrated that ERG and ESE3 controlled in opposite direction transcription of the Polycomb Group protein EZH2, a key gene in development, differentiation, stem cell biology and tumorigenesis. We further demonstrated that the prostate-specific tumor suppressor gene Nkx3.1 was controlled by ERG and ESE3 both directly and through induction of EZH2.These findings provide new insights into the role of the ETS transcriptional network in prostate tumorigenesis and uncover previously unrecognized links between aberrant expression of ETS factors, deregulation of epigenetic effectors and silencing of tumor suppressor genes. The link between aberrant ETS activity and epigenetic gene silencing may be relevant for the clinical management of prostate cancer and design of new therapeutic strategies.

  8. Hydrophobicity of methylated DNA as a possible mechanism for gene silencing

    International Nuclear Information System (INIS)

    Kaur, Parminder; Lindsay, Stuart; Plochberger, Birgit; Costa, Peter; Cope, Stephanie M; Vaiana, Sara M

    2012-01-01

    AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes. (paper)

  9. Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

    Science.gov (United States)

    Sun, Xiaowen; Wu, Hefang; Zhao, Genhai; Li, Zhemin; Wu, Xihua; Liu, Hui; Zheng, Zhiming

    2018-04-02

    The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

  10. Host-induced silencing of essential genes in Puccinia triticina through transgenic expression of RNAi sequences reduces severity of leaf rust infection in wheat.

    Science.gov (United States)

    Panwar, Vinay; Jordan, Mark; McCallum, Brent; Bakkeren, Guus

    2018-05-01

    Leaf rust, caused by the pathogenic fungus Puccinia triticina (Pt), is one of the most serious biotic threats to sustainable wheat production worldwide. This obligate biotrophic pathogen is prevalent worldwide and is known for rapid adaptive evolution to overcome resistant wheat varieties. Novel disease control approaches are therefore required to minimize the yield losses caused by Pt. Having shown previously the potential of host-delivered RNA interference (HD-RNAi) in functional screening of Pt genes involved in pathogenesis, we here evaluated the use of this technology in transgenic wheat plants as a method to achieve protection against wheat leaf rust (WLR) infection. Stable expression of hairpin RNAi constructs with sequence homology to Pt MAP-kinase (PtMAPK1) or a cyclophilin (PtCYC1) encoding gene in susceptible wheat plants showed efficient silencing of the corresponding genes in the interacting fungus resulting in disease resistance throughout the T 2 generation. Inhibition of Pt proliferation in transgenic lines by in planta-induced RNAi was associated with significant reduction in target fungal transcript abundance and reduced fungal biomass accumulation in highly resistant plants. Disease protection was correlated with the presence of siRNA molecules specific to targeted fungal genes in the transgenic lines harbouring the complementary HD-RNAi construct. This work demonstrates that generating transgenic wheat plants expressing RNAi-inducing transgenes to silence essential genes in rust fungi can provide effective disease resistance, thus opening an alternative way for developing rust-resistant crops. © 2017 Her Majesty the Queen in Right of Canada. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Suicide genes or p53 gene and p53 target genes as targets for cancer gene therapy by ionizing radiation

    International Nuclear Information System (INIS)

    Liu Bing; Chinese Academy of Sciences, Beijing; Zhang Hong

    2005-01-01

    Radiotherapy has some disadvantages due to the severe side-effect on the normal tissues at a curative dose of ionizing radiation (IR). Similarly, as a new developing approach, gene therapy also has some disadvantages, such as lack of specificity for tumors, limited expression of therapeutic gene, potential biological risk. To certain extent, above problems would be solved by the suicide genes or p53 gene and its target genes therapies targeted by ionizing radiation. This strategy not only makes up the disadvantage from radiotherapy or gene therapy alone, but also promotes success rate on the base of lower dose. By present, there have been several vectors measuring up to be reaching clinical trials. This review focused on the development of the cancer gene therapy through suicide genes or p53 and its target genes mediated by IR. (authors)

  12. Silencing of a unique integrated domain nucleotide-binding leucine-rich repeat gene in wheat abolishes Diuraphis noxia resistance.

    Science.gov (United States)

    Nicolis, Vittorio Filippo; Venter, Eduard

    2018-03-13

    Plants respond in a similar manner to aphid feeding as to pathogen attack. Diuraphis noxia is a specialist aphid, feeding only on selected grasses that include wheat, barley, and oats. The wheat-Diuraphis noxia interaction is characterized by very similar responses as seen in wheat-pathogen interactions with none of the underlying resistance pathways and genes characterized yet. From wheat harboring the Dn1 resistance gene, we have identified a nucleotide-binding leucine-rich repeat (NLR) gene containing two integrated domains (IDs). These are three C-terminus ankyrin repeat-domains and an N-terminus WRKY domain. The NLR core of the gene can be traced through speciation events within the grass family, with a recent WRKY domain integration that is Triticum specific. Virus induced gene silencing of the gene in a resistant wheat line resulted in the abolishment of the resistance response and induced a highly susceptible phenotype. Silenced plants supported a higher number of aphids similar to the susceptible NIL and the intrinsic rate of increase of the aphids matched that of aphids feeding on the susceptible NIL. The presence of the gene is necessary for Dn1 resistance and we have named the gene Associated with Dn resistance 1 (Adr1) to reflect this function.

  13. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes.

    Science.gov (United States)

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-09-30

    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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Cystathionine-γ-lyase gene silencing with siRNA in monocytes ...

    Indian Academy of Sciences (India)

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture ...

  15. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.

    Science.gov (United States)

    Cao, Ru; Tsukada, Yu-Ichi; Zhang, Yi

    2005-12-22

    Polycomb group (PcG) proteins exist in at least two biochemically distinct protein complexes, the EED-EZH2 complex and the PRC1 complex, that respectively possess H3-K27 methyltransferase and H2A-K119 ubiquitin E3 ligase activities. How the enzymatic activities are regulated and what their role is in Hox gene silencing are not clear. Here, we demonstrate that Bmi-1 and Ring1A, two components of the PRC1 complex, play important roles in H2A ubiquitylation and Hox gene silencing. We show that both proteins positively regulate H2A ubiquitylation. Chromatin immunoprecipitation (ChIP) assays demonstrate that Bmi-1 and other components of the two PcG complexes bind to the promoter of HoxC13. Knockout Bmi-1 results in significant loss of H2A ubiquitylation and upregulation of Hoxc13 expression, whereas EZH2-mediated H3-K27 methylation is not affected. Our results suggest that EZH2-mediated H3-K27 methylation functions upstream of PRC1 and establishes a critical role for Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing.

  16. SHARP-2 gene silencing by lentiviral-based short hairpin RNA interference prolonged rat kidney transplant recipients' survival time.

    Science.gov (United States)

    Shou, Z; Xiao, H; Xu, Y; Wang, Y; Yang, Y; Jiang, H; Chen, J; Yamada, K; Miyamoto, K

    2009-01-01

    Split- and hairy-related protein-2 (SHARP-2) controls the expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), which both play a key role in transplant rejection. This study was designed to investigate whether SHARP-2 short hairpin RNA interference (shRNAi) could prolong the survival of rat kidney transplant recipients. A lentiviral-based shRNAi construct, LV-SHARP-2iC, showed a SHARP-2 gene silencing efficiency of 84% in normal rat kidney cells. In activated T-cells, SHARP-2 gene silencing with the LV-SHARP-2iC construct resulted in 61% and 69% down-regulation of IL-2 and IFN-gamma, respectively, compared with a scramble control construct. When donor kidney was perfused with 5 x 10(7) transforming units of the LV-SHARP-2iC construct, the median survival time of the transplant recipients was prolonged by 4 - 5 days compared with control groups. In conclusion, recombinant lentiviral LV-SHARP-2iC construct effectively silenced SHARP-2 gene expression, which reduced IL-2 and IFN-gamma mRNA expression and prolonged rat kidney transplant recipients' survival.

  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*

    Science.gov (United States)

    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

  18. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    Science.gov (United States)

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.

    Directory of Open Access Journals (Sweden)

    Cheng Yuan

    Full Text Available Barley stripe mosaic virus (BSMV is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS, magnesium chelatase subunit H (ChlH, and plastid transketolase (TK gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5 also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

  20. TGIF1 Gene Silencing in Tendon-Derived Stem Cells Improves the Tendon-to-Bone Insertion Site Regeneration

    Directory of Open Access Journals (Sweden)

    Liyang Chen

    2015-11-01

    Full Text Available Background/Aims: The slow healing process of tendon-to-bone junctions can be accelerated via implanted tendon-derived stem cells (TDSCs with silenced transforming growth interacting factor 1 (TGIF1 gene. Tendon-to-bone insertion site is the special form of connective tissues derivatives of common connective progenitors, where TGF-β plays bidirectional effects (chondrogenic or fibrogenic through different signaling pathways at different stages. A recent study revealed that TGF-β directly induces the chondrogenic gene Sox9. However, TGIF1 represses the expression of the cartilage master Sox9 gene and changes its expression rate against the fibrogenesis gene Scleraxis (Scx. Methods: TGIF1 siRNA was transduced or TGIF1 was over-expressed in tendon-derived stem cells. Following suprapinatus tendon repair, rats were either treated with transduced TDSCs or nontransduced TDSCs. Histologic examination and Western blot were performed in both groups. Results: In this study, the silencing of TGIF1 significantly upregulated the chondrogenic genes and markers. Similarly, TGIF1 inhibited TDSC differentiation into cartilage via interactions with TGF-β-activated Smad2 and suppressed the phosphorylation of Smad2. The area of fibrocartilage at the tendon-bone interface was significantly increased in the TGIF1 (- group compared with the control and TGIF1-overexpressing groups in the early stages of the animal model. The interface between the tendon and bone showed a increase of new bone and fibrocartilage in the TGIF1 (- group at 4 weeks. Fibrovascular scar tissue was observed in the TGIF1-overexpressing group and the fibrin glue only group. Low levels of fibrocartilage and fibrovascular scar tissue were found in the TDSCs group. Conclusion: Collectively, this study shows that the tendon-derived stem cell modified with TGIF1 gene silencing has promising effects on tendon-to-bone healing which can be further explored as a therapeutic tool in regenerative medicine.

  1. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

    Directory of Open Access Journals (Sweden)

    Kil Hyun Kim

    2016-04-01

    Full Text Available 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 (OD₆₀₀ 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.

  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

    International Nuclear Information System (INIS)

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-01-01

    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. The Anti-Adipogenic Potential of COUP-TFII Is Mediated by Downregulation of the Notch Target Gene Hey1.

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    Ilse Scroyen

    Full Text Available Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII belongs to the steroid/thyroid hormone receptor superfamily and may contribute to the pathogenesis of obesity. It has not conclusively been established, however, whether its role is pro- or anti-adipogenic.Gene silencing of Coup-tfII in 3T3-F442A preadipocytes resulted in enhanced differentiation into mature adipocytes. This was associated with upregulation of the Notch signaling target gene Hey1. A functional role of Hey1 was confirmed by gene silencing in 3T3-F442A preadipocytes, resulting in impaired differentiation. In vivo, de novo fat pad formation in NUDE mice was significantly stimulated following injection of preadipocytes with Coup-tfII gene silencing, but impaired with Hey1 gene silencing. Moreover, expression of Coup-tfII was lower and that of Hey1 higher in isolated adipocytes of obese as compared to lean adipose tissue.These in vitro and in vivo data support an anti-adipogenic role of COUP-TFII via downregulating the Notch signaling target gene Hey1.

  4. Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation.

    Science.gov (United States)

    Rosser, James M; An, Wenfeng

    2010-05-15

    Recent transgenic studies on L1 retrotransposons have afforded exciting insights into L1 biology, and a unique opportunity to model their function and regulation in vivo. Thus far, the majority of the transgenic L1 mouse lines are constructed via pronuclear microinjection, a procedure that typically results in the integration of tandem arrayed transgenes. Transgene arrays are susceptible to repeat-induced gene silencing (RIGS) in both plants and animals. In order to examine the potential impact of RIGS on L1 retrotransposition, we derived a cohort of animals carrying reduced copies of ORFeus transgene at the same genomic locus by Cre-mediated recombination. The copy number reduction of ORFeus transgenes did not decrease the overall retrotransposition activity. Using a sensitive and reproducible quantitative PCR assay, an average frequency of 0.45 insertions per cell was observed for animals carrying the donor transgene at a single copy, representing a 9-fold increase of retrotransposition frequency on a per-copy basis. DNA methylation analyses revealed that the observed retrotransposition activity was correlated with differential CpG methylation at the heterologous promoter: the promoter region was largely methylated in animals with the high-copy array but significantly hypomethylated in animals with the single-copy counterpart. In contrast, the ORF2 region, which represents the body of the ORFeus transgene, and the 3' end of the transgene showed high level of methylation in both high-copy and single-copy samples. The observed methylation patterns were metastable across generations. In summary, our data suggest that tandem arrayed L1 transgenes are subject to RIGS, and transgenes present at a single copy in the genome are thus recommended for modeling L1 in animals. Copyright 2010 Elsevier B.V. All rights reserved.

  5. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

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    Bianco Linda

    2009-11-01

    Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

  6. Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.

    Science.gov (United States)

    Manhães, Ana Marcia E de A; de Oliveira, Marcos V V; Shan, Libo

    2015-01-01

    Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.

  7. Silencing of Abcd1 and Abcd2 genes sensitizes astrocytes for inflammation: implication for X-adrenoleukodystrophy*

    Science.gov (United States)

    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

  8. Silencing of Abcd1 and Abcd2 genes sensitizes astrocytes for inflammation: implication for X-adrenoleukodystrophy.

    Science.gov (United States)

    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-kappaB, 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.

  9. An Alpha Motif at Tas3C Terminus Mediates RITS Cis Spreading and Promotes Heterochromatic Gene Silencing

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

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    Changho Eun

    Full Text Available RNA-directed DNA methylation (RdDM is a small interfering RNA (siRNA-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

  11. Targeted Gene Therapy for Breast Cancer

    Science.gov (United States)

    1999-08-01

    or immunotoxin therapy, natural vector-host tropisms must be altered. Recent improvements in monoclonal antibody (mAb) engineering have expanded the...endocytosis. To achieve targeted gene therapy or immunotoxin therapy, natural vector-host tropisms must be altered. Recent improvements in monoclonal...trafficking of monoclonal antibody- antigen to an endolysosomal pathway is important. After altering targeting specificities, prokaryotic and plant

  12. Inhibition of FSS-induced actin cytoskeleton reorganization by silencing LIMK2 gene increases the mechanosensitivity of primary osteoblasts.

    Science.gov (United States)

    Yang, Zhi; Tan, Shuyi; Shen, Yun; Chen, Rui; Wu, Changjing; Xu, Yajuan; Song, Zijun; Fu, Qiang

    2015-05-01

    Mechanical stimulation plays an important role in bone cell metabolic activity. However, bone cells lose their mechanosensitivity upon continuous mechanical stimulation (desensitization) and they can recover the sensitivity with insertion of appropriate rest period into the mechanical loading profiles. The concrete molecular mechanism behind the regulation of cell mechanosensitivity still remains unclear. As one kind of mechanosensitive cell to react to the mechanical stimulation, osteoblasts respond to fluid shear stress (FSS) with actin cytoskeleton reorganization, and the remodeling of actin cytoskeleton is closely associated with the alteration of cell mechanosensitivity. In order to find out whether inhibiting the actin cytoskeleton reorganization by silencing LIM-kinase 2 (LIMK2) gene would increase the mechanosensitivity of primary osteoblasts, we attenuated the formation of actin stress fiber under FSS in a more specific way: inhibiting the LIMK2 expression by RNA interference. We found that inhibition of LIMK2 expression by RNA interference attenuated the formation of FSS-induced actin stress fiber, and simultaneously maintained the integrity of actin cytoskeleton in primary osteoblasts. We confirmed that the decreased actin cytoskeleton reorganization in response to LIMK2 inhibition during FSS increased the mechanosensitivity of the osteoblasts, based on the increased c-Fos and COX-2 expression as well as the enhanced proliferative activity in response to FSS. These data suggest that osteoblasts can increase their mechanosensitivity under continuous mechanical stimulation by reducing the actin stress fiber formation through inhibiting the LIMK2 expression. This study provides us with a new and more specific method to regulate the osteoblast mechanosensitivity, and also a new therapeutic target to cure bone related diseases, which is of importance in maintaining bone mass and promoting osteogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Small RNA-Mediated Epigenetic Myostatin Silencing

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    Thomas C Roberts

    2012-01-01

    Full Text Available Myostatin (Mstn is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

  14. Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

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    Yusuke Ohnishi

    Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense

  15. Differential expression of store-operated calcium- and proliferation-related genes in hepatocellular carcinoma cells following TRPC1 ion channel silencing.

    Science.gov (United States)

    Selli, Cigdem; Pearce, Dominic A; Sims, Andrew H; Tosun, Metiner

    2016-09-01

    TRPC1 and store-operated Ca(2+) (SOC) entry have previously been associated with hepatocellular carcinoma cell proliferation. The aim of the study was to determine genes and processes associated with TRPC1 down-regulation and the resulting increase of SOC entry and decrease in hepatocellular carcinoma cell proliferation. For this purpose, transcriptome analysis was performed to determine differentially expressed genes in TRPC1-silenced Huh7 cells. SOC entry- and proliferation-related genes correlated with TRPC1 down-regulation were also examined. Changes in SOC entry and cell proliferation were monitored in the TRPC1-silenced and parental cells and found to be significantly increased and decreased, respectively, in TRPC1-silenced cells. A total of 71 genes were significantly differentially expressed (40 up- and 31 down-regulated), including four mitogen-activated protein kinase (MAPK) signalling-associated genes. STIM1 levels were significantly up-regulated and negatively correlated with TRPC1 levels. In addition, expression of two cell cycle regulation genes, CDK11A/11B and URGCP, was observed to decrease, whereas ERBB3 and FGFR4, pro-survival genes, increased significantly in TRPC1-silenced cells. In conclusion, these results suggest reciprocal alterations in TRPC1 and STIM1 levels and a role for STIM1 in the regulation of SOC entry in TRPC1-silenced Huh7 cells. In addition to TRPC1, STIM1 may participate in Huh7 cell proliferation by regulating SOC entry. Alterations in MAPK signalling genes may be involved in diminished cell proliferation in TRPC1-silenced Huh7 cells. Similarly, changes in cell cycle regulating genes in TRPC1-silenced cells indicate possible cell cycle arrest along with compensatory up-regulation of ERBB3 growth factor receptor-amongst others-to maintain hepatocellular carcinoma cell proliferation.

  16. RNA interference-mediated silencing of eukaryotic translation initiation factor 3, subunit B (EIF3B) gene expression inhibits proliferation of colon cancer cells.

    Science.gov (United States)

    Wang, Zheng; Chen, Jinxian; Sun, Jianhua; Cui, Zhe; Wu, Hui

    2012-06-26

    A key factor underlying the control of the cellular growth, size and proliferation involves the regulation of the total protein synthesis. Most often, the initial stages of mRNA translation are rate limiting, which involves a group of eukaryotic translation initiation factors (EIFs). Research advances focused on the inhibition of their expression and activity hold the key to the initiation and progression of tumor and tumor prognosis. We performed RNA interference (RNAi) with the lentivirus vector system to silence the EIF3B gene using the colon cancer cell strain SW1116. The negative control included the normal target cells infected with the negative control virus whereas the knockdown cells included the normal target cells transfected with the RNAi target virus. We tested the inhibition resulting from the decreased expression of EIF3B gene on the proliferation rate of SW1116 cells, including the cell cycle, apoptosis and clonability. Compared with the negative control, the impact of EIF3B gene expression in SW1116 cells on the levels of mRNA and protein in the knockdown group, was significantly inhibited (P cell proliferation rate and clonability were also significantly inhibited (P cells in the G1 phase (P cells.

  17. AAC as a Potential Target Gene to Control Verticillium dahliae

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    Xiaofeng Su

    2017-01-01

    Full Text Available Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC. Compared with wild-type V. dahliae (Vd wt, ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C. Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

  18. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

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    David B West

    2016-02-01

    Full Text Available The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP; and 29 deletion alleles (DEL, usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

  19. Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant.

    Science.gov (United States)

    Aly, Radi; Cholakh, Hila; Joel, Daniel M; Leibman, Diana; Steinitz, Benjamin; Zelcer, Aaron; Naglis, Anna; Yarden, Oded; Gal-On, Amit

    2009-08-01

    Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

  20. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers

    Science.gov (United States)

    Veigl, Martina L.; Kasturi, Lakshmi; Olechnowicz, Joseph; Ma, AiHong; Lutterbaugh, James D.; Periyasamy, Sumudra; Li, Guo-Min; Drummond, James; Modrich, Paul L.; Sedwick, W. David; Markowitz, Sanford D.

    1998-01-01

    Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism. PMID:9671741

  1. Effect of tyrosine hydroxylase gene silencing in CD4+ T lymphocytes on differentiation and function of helper T cells.

    Science.gov (United States)

    Liu, Yan; Huang, Yan; Wang, Xiao-Qin; Peng, Yu-Ping; Qiu, Yi-Hua

    2012-01-01

    We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs. CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis. CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA. TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.

  2. Viral small interfering RNAs target host genes to mediate disease symptoms in plants.

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    Neil A Smith

    2011-05-01

    Full Text Available The Cucumber mosaic virus (CMV Y-satellite RNA (Y-Sat has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco. How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.

  3. Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

    International Nuclear Information System (INIS)

    Fan, Qi; Wang, Xuedi; Zhang, Hanguang; Li, Chuanwei; Fan, Junhua; Xu, Jing

    2012-01-01

    Highlights: ► Cat S is highly expressed in HCC cells with high metastatic potential. ► Knockdown of Cat S inhibits growth and invasion of HCC cells. ► Knockdown of Cat S inhibits HCC-associated angiogenesis. ► Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

  4. Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: Enhanced gene silencing and reduced adversed effects in vitro

    DEFF Research Database (Denmark)

    Zeng, Xianghui; de Groot, A. M.; Sijts, Alice

    2015-01-01

    giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did...... not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA–peptidomimetic nanocomplex core...

  5. Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

    2016-03-18

    Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.

  6. Development of male sterility by silencing Bcp1 gene of Arabidopsis ...

    African Journals Online (AJOL)

    The development of male sterility is one of the most important steps in hybrid seed production. Several methods for the abortion of pollens based on conventional as well as genetic engineering are reported for the various crop species. Here we have investigated the use of RNA interference (RNAi) technology to silence a ...

  7. Gene silencing activity of siRNA polyplexes based on biodegradable polymers.

    Science.gov (United States)

    Varkouhi, Amir K; Lammers, Twan; Schiffelers, Raymond M; van Steenbergen, Mies J; Hennink, Wim E; Storm, Gert

    2011-04-01

    Cationic polymers are used as non-viral vectors for nucleic acid delivery. In this study, two biodegradable cationic polymers were evaluated for the purpose of siRNA delivery: pHPMA-MPPM (poly((2-hydroxypropyl) methacrylamide 1-methyl-2-piperidine methanol)) and TMC (O-methyl-free N,N,N-trimethylated chitosan). The silencing activity and the cellular cytotoxicity of polyplexes based on these biodegradable polymers were compared with those based on non-biodegradable pDMAEMA (poly(2-dimethylamino)ethyl methacrylate) and PEI (polyethylenimine) and with the regularly used lipidic transfection agent Lipofectamine. To promote endosomal escape, either the endosomolytic peptide diINF-7 was added to the formulations or photochemical internalization (PCI) was applied. Incubation of H1299 human lung cancer cells expressing firefly luciferase with polyplexes based on pHPMA-MPPM and TMC showed 30-40% silencing efficiency. This silencing activity was equal to or better than that obtained with the standard transfectants. Under all experimental conditions tested, the cytotoxicity of the biodegradable polymers was low. The application of PCI, as well as the addition of the diINF-7 peptide to the formulations increased their silencing activity up to 70-80%. This demonstrates that pHPMA-MPPM- and TMC-based polyplexes benefit substantially from endosomal escape enhancement. Importantly, the polyplexes retained their silencing activity in the presence of serum, and they showed low cytotoxicity. These biodegradable vectors are therefore attractive systems for further in vivo evaluations. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Composite potato plants with transgenic roots on non-transgenic shoots: a model system for studying gene silencing in roots.

    Science.gov (United States)

    Horn, Patricia; Santala, Johanna; Nielsen, Steen Lykke; Hühns, Maja; Broer, Inge; Valkonen, Jari P T

    2014-12-01

    Composite potato plants offer an extremely fast, effective and reliable system for studies on gene functions in roots using antisense or inverted-repeat but not sense constructs for gene inactivation. Composite plants, with transgenic roots on a non-transgenic shoot, can be obtained by shoot explant transformation with Agrobacterium rhizogenes. The aim of this study was to generate composite potato plants (Solanum tuberosum) to be used as a model system in future studies on root-pathogen interactions and gene silencing in the roots. The proportion of transgenic roots among the roots induced was high (80-100%) in the four potato cultivars tested (Albatros, Desirée, Sabina and Saturna). No wild-type adventitious roots were formed at mock inoculation site. All strains of A. rhizogenes tested induced phenotypically normal roots which, however, showed a reduced response to cytokinin as compared with non-transgenic roots. Nevertheless, both types of roots were infected to a similar high rate with the zoospores of Spongospora subterranea, a soilborne potato pathogen. The transgenic roots of composite potato plants expressed significantly higher amounts of β-glucuronidase (GUS) than the roots of a GUS-transgenic potato line event. Silencing of the uidA transgene (GUS) was tested by inducing roots on the GUS-transgenic cv. Albatros event with strains of A. rhizogenes over-expressing either the uidA sense or antisense transcripts, or inverted-repeat or hairpin uidA RNA. The three last mentioned constructs caused 2.5-4.0 fold reduction in the uidA mRNA expression. In contrast, over-expression of uidA resulted in over 3-fold increase in the uidA mRNA and GUS expression, indicating that sense-mediated silencing (co-suppression) was not functional in roots. The results suggest that composite plants offer a useful experimental system for potato research, which has gained little previous attention.

  9. Silencing β3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and Metastasis of Triple Negative Breast Cancer

    Science.gov (United States)

    Mack, Margaret A.; Schiemann, William P.; Lu, Zheng-Rong

    2015-01-01

    Metastatic breast cancer is the second leading cause of cancer-related deaths amongst women. Triple-negative breast cancer (TNBC) is a highly aggressive subcategory of breast cancer and currently lacks well-defined molecular targets for effective targeted therapies. Disease relapse, metastasis, and drug resistance render standard chemotherapy ineffective in the treatment of TNBC. Since previous studies coupled β3 integrin to epithelial-mesenchymal transition (EMT) and metastasis, we exploited β3 integrin as a therapeutic target to treat TNBC by delivering β3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siβ3). Treatment of TNBC cells with ECO/siβ3 was sufficient to effectively silence β3 integrin expression, attenuate TGF-β-mediated EMT and invasion, restore TGF-β-mediated cytostasis, and inhibit 3-dimensional organoid growth. Modification of ECO/siβ3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siβ3 nanoparticles in vivo alleviated primary tumor burden, and more importantly, significantly inhibited metastasis. Mice bearing orthotopic, TGF-β-pre-stimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siβ3 nanoparticles were free of metastases and relapse after primary tumor resection and 4 weeks after release from the treatment, in comparison to untreated mice. Collectively, these results highlight ECO/siβ3 nanoparticles as a promising therapeutic regimen to combat TNBC. PMID:25858145

  10. Recent patents in RNA silencing in plants: constructs, methods and applications in plant biotechnology.

    Science.gov (United States)

    López-Gomollón, Sara; Dalmay, Tamas

    2010-11-01

    RNA silencing is a recently discovered mechanism to regulate gene expression at transcriptional and posttranscriptional levels. It is based on the recognition and methylation of target genes or cleavage of target mRNAs by small RNA molecules, with length varying from 21 to 24 nucleotides. RNA silencing plays an important role modulating most of the important cell processes, such as growth, development or stress response. During the past few years, diverse strategies have been applied to exploit RNA silencing as a tool to create plants with enhanced economical properties or able to cope with pathogens or abiotic stress. This review describes the most important patents related to RNA silencing in plants, which disclose vectors designed to induce RNA silencing by hairpin RNAs, amplicons or virus-based plasmids, methods for detection and quantification of silencing as well as general uses in plant biotechnology.

  11. Improved Gene Targeting through Cell Cycle Synchronization.

    Directory of Open Access Journals (Sweden)

    Vasiliki Tsakraklides

    Full Text Available Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

  12. Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression

    Directory of Open Access Journals (Sweden)

    Carlos R. Morales

    2014-01-01

    Full Text Available Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

  13. PTEN gene silencing contributes to airway remodeling and induces airway smooth muscle cell proliferation in mice with allergic asthma.

    Science.gov (United States)

    Wen, Xin; Yan, Jing; Han, Xin-Rui; Zheng, Gui-Hong; Tang, Ran; Liu, Li-Fang; Wu, Dong-Mei; Lu, Jun; Zheng, Yuan-Lin

    2018-01-01

    Allergic asthma is a complex genetic disorder that involves interactions between genetic and environmental factors. Usage of PTEN may be a good therapeutic strategy for the management of allergic inflammation. Thus, the present study aims to explore the effects of phosphatase and tensin homolog ( PTEN ) gene silencing on airway remodeling and proliferation of airway smooth muscle cells (ASMCs) in a mouse model of allergic asthma. A total of 56 healthy female BABL/c mice (weighing between 16 to 22 grams) were selected and were assigned on random into ovalbumin (OVA; mice were stimulated with OVA to induce allergic asthma), OVA + si-PTEN, normal saline (NS; mice were treated with normal saline) and NS + si-PTEN groups. Masson staining was employed in order to observe lung tissue sections. Immunohistochemical staining was used to detect the expression of α-SMA + . Gene silencing was conducted in the NS + si-PTEN and OVA + si-PTEN groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were used to detect the mRNA and protein expressions of PTEN in ASMCs of each group. CCK-8 assay and flow cytometry were performed to determine the cell proliferation rate and cell cycle. Airway remodeling and changes of smooth muscle layer were found in allergic asthmatic mice with thick airway walls. The expression of alpha smooth muscle actin (α-SMA + ) was significantly higher in ASMCs of the OVA, OVA + si-PTEN and NS + si-PTEN groups compared with ASMCs of the NS group. The mRNA and protein expressions of PTEN reduced in the OVA, OVA + si-PTEN and NS + si-PTEN groups. The rate of ASMCs proliferation in OVA, OVA + si-PTEN and NS + si-PTEN groups were significantly higher than the NS group. The proportion of ASMCs in S and G2 stages increased, while the number of cells in the G1 stage decreased after PTEN gene silencing. These results demonstrated that PTEN gene silencing might promote proliferation of ASMCs and airway remodeling in a

  14. Gene-silencing potency of symmetric and asymmetric lipid-conjugated siRNAs and its correlation with dicer recognition.

    Science.gov (United States)

    Kubo, Takanori; Yanagihara, Kazuyoshi; Sato, Yuichiro; Nishimura, Yoshio; Kondo, Shinichi; Seyama, Toshio

    2013-12-18

    Three types of siRNAs and three types of left-overhang siRNAs (LoRNAs) were synthesized along with their conjugations with palmitic acid (C16) to investigate the correlation between Dicer recognition and gene-silencing potency. The siRNA types were composed of 21-nucleotide (nt), 23-nt, and 25-nt lengths of sense and antisense strands with a 2-nt overhang at each 3'-end. The three LoRNA types were composed of a 21-nt, a 23-nt, and a 25-nt length of sense strand with a 2-nt DNA at the 3'-blunt-end and a 23-nt, a 25-nt, and a 27-nt length of antisense strand with a 2-nt overhang at the 3'-end. Additionally, each of these siRNAs and LoRNAs was modified with a C16 at the 5'- or 3'-end of the sense strand; these were named C16-siRNAs and C16-LoRNAs, respectively. The siRNAs and C16-siRNAs were barely cleaved by Dicer, and their gene-silencing efficacies were not excellent, contrary to our expectations. In contrast, most of the LoRNAs and C16-LoRNAs became substrates of Dicer, and they showed both strong gene-silencing efficacies and high nuclease resistance. Among the LoRNAs, the 25D-C16/27-nt LoRNA, which is composed of a 25-nt sense strand with a 2-nt DNA conjugated with C16 at the 3'-end and a 27-nt antisense strand with a 2-nt overhang at the 3'-end, showed an excellent gene-silencing effect with high cell membrane permeability and strong resistance against nuclease degradation. Additionally, the Lo25D-C16/27RNA excelled in all three aspects, nuclease resistance, cell membrane permeability, and RNAi efficacy, compared with the cholesterol conjugation. We are certain that Lo25D-C16/27RNA can be useful as a new generation of RNAi molecules with which to overcome some of the limitations of RNAi technology.

  15. siRNA-mediated Erc gene silencing suppresses tumor growth in Tsc2 mutant renal carcinoma model.

    Science.gov (United States)

    Imamura, Osamu; Okada, Hiroaki; Takashima, Yuuki; Zhang, Danqing; Kobayashi, Toshiyuki; Hino, Okio

    2008-09-18

    Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery of siRNAs for stable treatment except short hairpin RNAs (shRNAs). On the other hand, there are many reports of systemic delivery of siRNAs for transient treatment using liposome carriers and others. With regard to shRNAs, a report showed fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Therefore, we decided to use original siRNA microspheres instead of shRNA for stable treatment of disease. In this study, we designed rat-specific siRNA sequences for Erc/mesothelin, which is a tumor-specific gene expressed in the Eker (Tsc2 mutant) rat model of hereditary renal cancer and confirmed the efficacy of gene silencing in vitro. Then, by using siRNA microspheres, we found that the suppression of Erc/mesothelin caused growth inhibition of Tsc2 mutant renal carcinoma cells in tumor implantation experiments in mice.

  16. Identification and gene-silencing of a putative odorant receptor transcription factor in Varroa destructor: possible role in olfaction.

    Science.gov (United States)

    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. © 2016 The Royal Entomological Society.

  17. MutT Homolog 1 (MTH1): The Silencing of a Target.

    Science.gov (United States)

    Papeo, Gianluca

    2016-03-24

    MTH1 is a member of the nudix phosphohydrolase superfamily of enzymes, and it is involved in nucleotide pool homeostasis. The protein exerts its scavenging action by hydrolyzing oxidized nucleotides, thus avoiding their misincorporation into replicating DNA. Recent reports have validated its inhibition as a potential broad-spectrum target in oncology and have described small molecules able to engage this target with antiproliferative effect. In the accompanying paper (DOI: 10.1021/acs.jmedchem.5b01760 ), a team at AstraZeneca led by Jason G. Kettle elegantly demonstrated the complete lack of antiproliferative activity of cancer cells to highly potent MTH1 inhibitors.

  18. Silencing of the major family of NBS-LRR-encoding genes in lettuce results in the loss of multiple resistance specificities.

    Science.gov (United States)

    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.

  19. Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

    International Nuclear Information System (INIS)

    Moskalev, Evgeny A; Pötz, Oliver; Joos, Thomas O; Hoheisel, Jörg D; Luckert, Katrin; Vorobjev, Ivan A; Mastitsky, Sergey E; Gladkikh, Aleena A; Stephan, Achim; Schrenk, Marita; Kaplanov, Kamil D; Kalashnikova, Olga B

    2012-01-01

    The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples. Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect. The results suggest an

  20. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    Directory of Open Access Journals (Sweden)

    Morral Núria

    2011-01-01

    Full Text Available Abstract Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA or microRNA (miRNA. New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells as well as double stranded RNA (>90% with siRNA or microRNA. In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.

  1. Transgenic sugarcane resistant to Sorghum mosaic virus based on coat protein gene silencing by RNA interference.

    Science.gov (United States)

    Guo, Jinlong; Gao, Shiwu; Lin, Qinliang; Wang, Hengbo; Que, Youxiong; Xu, Liping

    2015-01-01

    As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

  2. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    Directory of Open Access Journals (Sweden)

    Jinlong Guo

    2015-01-01

    Full Text Available As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV and/or Sorghum mosaic virus (SrMV, with additional differences in viral strains. RNA interference (RNAi is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

  3. Expression of cancer stem markers could be influenced by silencing of p16 gene in HeLa cervical carcinoma cells.

    Science.gov (United States)

    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.

  4. Identification and targeting of genes in atherosclerosis

    NARCIS (Netherlands)

    van Capelleveen, J.C.

    2017-01-01

    The aim of the studies described in this thesis was to assess the relevance of variations in known and novel genes for CVD associated endpoints in order to ultimately identify therapeutic targets for the treatment of atherosclerosis. The type of research is truly translational; it nourishes on the

  5. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    Science.gov (United States)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  6. [Silencing of tumor metastasis suppressor gene 1 promotes invasion of prostate cancer cell in vitro and its molecular mechanisms].

    Science.gov (United States)

    Xu, Xiao-yan; You, Jiang-feng; Pei, Fei; Zhang, Bo

    2011-12-18

    To explore the effect of small interference RNA (siRNA) targeting homosapiens longevity assurance homologue 2(LASS2, or TMSG1) on the invasion of PC-3M-2B4 (a variant subline of human prostate carcinoma cell line PC-3M with low metastatic potential) and its molecular mechanisms. PC-3M-2B4 cells were transfected with siRNA by using lipofectamine 2000. The expression of LASS2 mRNA and protein was detected after transfection by real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot to screen the effective siRNA fragment. The V-ATPase activity of PC-3M-2B4 cells was detected by V-ATPase activity assay kit. The concentration of extracellular hydrogen ion was measured by pH-sensitive fluorescence probe bis-carboxyethyl-carboxyfluorescein (BCECF). The matrix metalloproteinase-2 (MMP-2) protein in the supernatant and cells was analyzed by Western blot. The activity of MMP-2 was examined by Gelatin zymography. Furthermore, the migration and invasion of cells were evaluated by in vitro wound migration assay and invasion assay. RFQ-PCR and Western blot revealed dramatic reduction (84.5% and 60% ) in the levels of LASS2 mRNA and protein after transfection of siRNA-2 in PC-3M-2B4 cells. The V-ATPases activity and extracellular hydrogen ion concentration were significantly increased in PC-3M-2B4 cells transfected with the siRNA-2 compared with other control groups (P<0.05); There were no differences in the expression and secretion of MMP-2 protein between LASS2-siRNA treated cells and other control groups. However, the activity of MMP-2 was up-regulated in LASS2-siRNA treated cells compared with other control groups( P<0.05); and the capacity for migration and invasion in LASS2-siRNA treated cells was significantly higher than in other control groups (P<0.05). Silencing of LASS2 can promote invasion of prostate cancer cells in vitro through the increase of the V-ATPases activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2

  7. Reversal of galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 cells.

    Science.gov (United States)

    Zhang, Lei; Liu, Xuegang; Tang, Zhen; Li, Xiaojun; Wang, Gengming

    2016-10-01

    This study aims to investigate reversal of Galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 (or A549/DDP) in vivo and in vitro. The stably transfected lentivirus vector was used to silence Galectin-1 in human lung adenocarcinoma cell line A549 and A549/DDP cells and the cell lines were cultured and passaged. RT-PCR and western blot assay were used to test A549, A549/DDP cells, silenced Galectin-1A549 (A549/I) cells, Galectin-1 mRNA and protein expression levels, respectively, in A549/DDP (A549/DDP/I) cells. CCK8 assay was used to measure median inhibitory concentration (IC50) in each group and resistant index of A549/DDP cells and A549/DDP/I cells. Tumor model in nude mice was established by armpit injection of A549, A549/DDP, A549/I, A549/DDP/I cells. Cisplatin was injected intraperitoneally in tumor models and growth of tumor was observed in vivo model. Four weeks later, nude mice were killed and tumor weight and diameter was measured. mRNA and protein expression of Galectin-1 in A549/DDP cells was higher than that in A549 cells. mRNA and protein expression of Galectin-1 in A549/DDP/I cells was lower than that in A549/DDP cells. Moreover, IC50 values ​​and resistance index in A549/DDP cells was higher than that in A549 cells group and IC50 values ​​and resistance index A549/DDP/I cell group were lower than that in A549/DDP cells. Additionally, tumor weight and volume in A549/DDP/I cell group were lower than that in A549/DDP. In conclusion, Galectin-1 gene silencing would improve the sensitivity of A549/DDP cells to cisplatin in vivo and in vitro. Copyright © 2016. Published by Elsevier Masson SAS.

  8. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    Directory of Open Access Journals (Sweden)

    Karin Groten

    Full Text Available 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 (p2-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

  9. Silence multiple

    DEFF Research Database (Denmark)

    Søndergaard, Katia Dupret

    The article highlights the importance of silences in the processes of innovation in organizations, and the claim is that silence and the absence of talk distribute authority, responsibility and decisions. The act of silencing is conceptualised as a central “configurating actor”. Using an Actor......-Network Theoretical approach to organization studies silence is conceptualised as both a means and an effect of innovative efforts. It is a way of ordering practices. Thus silencing is thought of as a central potential change agent both in composing a kind of specific organizational collectivity and in composing new...... working practices more generally. In line with the approach to destabilise the mundane, invisible and taken-for-granted aspects of innovative efforts in organisations (crucial for ANT and foucauldian post-structuralism more broadly), this article suggests to non-silence the silence and make...

  10. Genetic nanomedicine: gene delivery by targeted lipoplexes.

    Science.gov (United States)

    Düzgüneş, Nejat; de Ilarduya, Conchita Tros

    2012-01-01

    Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  12. Targeted BIRC5 silencing using YM155 causes cell death in neuroblastoma cells with low ABCB1 expression

    NARCIS (Netherlands)

    Lamers, Fieke; Schild, Linda; Koster, Jan; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.

    2012-01-01

    The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing

  13. Targeting Conserved Genes in Alternaria Species.

    Science.gov (United States)

    Pavón, Miguel Ángel; López-Calleja, Inés María; González, Isabel; Martín, Rosario; García, Teresa

    2017-01-01

    Real-time polymerase chain reaction (PCR) is a molecular biology technique based on the detection of the fluorescence produced by a reporter molecule, which increases as the reaction proceeds proportionally to the accumulation of the PCR product within each amplification cycle. The fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e., SYBR ® Green) or sequence-specific probes (i.e., Molecular Beacons or TaqMan ® Probes). Real-time PCR provides a tool for accurate and sensitive quantification of target fungal DNA. Here, we describe a TaqMan real-time PCR method for specific detection and quantification of Alternaria spp. The method uses Alternaria-specific primers and probe, targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene.

  14. Silencing the SpMPK1, SpMPK2, and SpMPK3 Genes in Tomato Reduces Abscisic Acid—Mediated Drought Tolerance

    Directory of Open Access Journals (Sweden)

    Yan Liang

    2013-11-01

    Full Text Available Drought is a major threat to agriculture production worldwide. Mitogen-activated protein kinases (MAPKs play a pivotal role in sensing and converting stress signals into appropriate responses so that plants can adapt and survive. To examine the function of MAPKs in the drought tolerance of tomato plants, we silenced the SpMPK1, SpMPK2, and SpMPK3 genes in wild-type plants using the virus-induced gene silencing (VIGS method. The results indicate that silencing the individual genes or co-silencing SpMPK1, SpMPK2, and SpMPK3 reduced the drought tolerance of tomato plants by varying degrees. Co-silencing SpMPK1 and SpMPK2 impaired abscisic acid (ABA-induced and hydrogen peroxide (H2O2-induced stomatal closure and enhanced ABA-induced H2O2 production. Similar results were observed when silencing SpMPK3 alone, but not when SpMPK1 and SpMPK2 were individually silenced. These data suggest that the functions of SpMPK1 and SpMPK2 are redundant, and they overlap with that of SpMPK3 in drought stress signaling pathways. In addition, we found that SpMPK3 may regulate H2O2 levels by mediating the expression of CAT1. Hence, SpMPK1, SpMPK2, and SpMPK3 may play crucial roles in enhancing tomato plants’ drought tolerance by influencing stomatal activity and H2O2 production via the ABA-H2O2 pathway.

  15. Utilizing virus-induced gene silencing for the functional characterization of maize genes during infection with the fungal pathogen Ustilago maydis.

    Science.gov (United States)

    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.

  16. miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity.

    Science.gov (United States)

    Reid, C A; Boye, S L; Hauswirth, W W; Lipinski, D M

    2017-08-01

    The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22-23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3'-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.

  17. Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia

    Science.gov (United States)

    2013-01-01

    Background Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. Results We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. Conclusions The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these

  18. The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Tan, E-Jean; Kahata, Kaoru; Idås, Oskar; Thuault, Sylvie; Heldin, Carl-Henrik; Moustakas, Aristidis

    2015-01-01

    The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial-mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2'-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

    Directory of Open Access Journals (Sweden)

    Simona Kamenšek

    2015-06-01

    Full Text Available 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.

  20. Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex.

    Directory of Open Access Journals (Sweden)

    Marianna Feretzaki

    2016-03-01

    Full Text Available 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.

  1. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    Energy Technology Data Exchange (ETDEWEB)

    Kubo, Takanori, E-mail: kubo-t@yasuda-u.ac.jp [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Yanagihara, Kazuyoshi [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Takei, Yoshifumi [Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumi-cho, Showa-ku, Nagoya 466-8550 (Japan); Mihara, Keichiro [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, Yuichiro; Seyama, Toshio [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan)

    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.

  2. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  3. Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in "Elstar" apple and the effect of grafting

    DEFF Research Database (Denmark)

    Krath, Britta; Eriksen, Folmer Damsted; Pedersen, Bjarne H.

    2009-01-01

    allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus x domestica Borkh., 'Elstar') with decreased levels......Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross-allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple. Apples with significantly reduced levels of the allergen, Mal d 1, may...... silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type 'Elstar', two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to 10,000-fold) in Mal d 1 gene expression. These levels...

  4. Polycomb repressor complex 1 promotes gene silencing through H2AK119 mono-ubiquitination in acinar-to-ductal metaplasia and pancreatic cancer cells.

    Science.gov (United States)

    Benitz, Simone; Regel, Ivonne; Reinhard, Tobias; Popp, Anna; Schäffer, Isabell; Raulefs, Susanne; Kong, Bo; Esposito, Irene; Michalski, Christoph W; Kleeff, Jörg

    2016-03-08

    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.

  5. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Dietrich, Nikolaj; Pasini, Diego

    2006-01-01

    The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find...... that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2...... that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes....

  6. Engineering liposomal nanoparticles for targeted gene therapy.

    Science.gov (United States)

    Zylberberg, C; Gaskill, K; Pasley, S; Matosevic, S

    2017-08-01

    Recent mechanistic studies have attempted to deepen our understanding of the process by which liposome-mediated delivery of genetic material occurs. Understanding the interactions between lipid nanoparticles and cells is still largely elusive. Liposome-mediated delivery of genetic material faces systemic obstacles alongside entry into the cell, endosomal escape, lysosomal degradation and nuclear uptake. Rational design approaches for targeted delivery have been developed to reduce off-target effects and enhance transfection. These strategies, which have included the modification of lipid nanoparticles with target-specific ligands to enhance intracellular uptake, have shown significant promise at the proof-of-concept stage. Control of physical and chemical specifications of liposome composition, which includes lipid-to-DNA charge, size, presence of ester bonds, chain length and nature of ligand complexation, is integral to the performance of targeted liposomes as genetic delivery agents. Clinical advances are expected to rely on such systems in the therapeutic application of liposome nanoparticle-based gene therapy. Here, we discuss the latest breakthroughs in the development of targeted liposome-based agents for the delivery of genetic material, paying particular attention to new ligand and cationic lipid design as well as recent in vivo advances.

  7. Silencing of the CaCP Gene Delays Salt- and Osmotic-Induced Leaf Senescence in Capsicum annuum L.

    Directory of Open Access Journals (Sweden)

    Huai-Juan Xiao

    2014-05-01

    Full Text Available 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.

  8. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    Science.gov (United States)

    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

  9. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle.

    Science.gov (United States)

    Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

    2013-06-07

    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.

  10. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    Directory of Open Access Journals (Sweden)

    Alejandra García-Gasca

    2013-06-01

    Full Text Available 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.

  11. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  12. A new piece of the Shigella Pathogenicity puzzle: spermidine accumulation by silencing of the speG gene [corrected].

    Directory of Open Access Journals (Sweden)

    Marialuisa Barbagallo

    Full Text Available The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation

  13. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  14. Efficient and nontoxic biological response carrier delivering TNF-α shRNA for gene silencing in a murine model of rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Jialin Song

    2016-08-01

    Full Text Available Small interfering RNA (siRNA is an effective and specific method for silencing genes. However, an efficient and nontoxic carrier is needed to deliver the siRNA into the target cells. Tumor necrosis factor α (TNF-α plays a central role in the occurrence and progression of rheumatoid arthritis. In this study, we pre-synthetized a degradable cationic polymer (PDAPEI from 2,6-pyridinedicarboxaldehyde and low molecular weight polyethyleneimine (PEI, Mw=1.8 kDa as a gene vector for the delivery of TNF-α shRNA. The PDAPEI/pDNA complex showed a suitable particle size and stable zeta potential for transfection. In vitro study of the PDAPEI/pDNA complex revealed a lower cytotoxicity and higher transfection efficiency when transfecting TNF-α shRNA to macrophages by significantly down-regulating the expression of TNF-α. Moreover, the complex was extremely efficient in decreasing the severity of arthritis in mice with collagen-induced arthritis (CIA. PDAPEI delivered TNF-α shRNA has great potential in the treatment of rheumatoid arthritis.

  15. Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

    Science.gov (United States)

    Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

    2017-03-21

    This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

  16. Silencing of the HER2/neu Gene by siRNA Inhibits Proliferation and Induces Apoptosis in HER2/neu-Overexpressing Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Timo Faltus

    2004-11-01

    Full Text Available In eukaryotes, double-stranded (ds RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi. We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SKBR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neuoverexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as anti proliferative agents that display activity against neoplastic cells at very low doses.

  17. Gene transfer technology and genetic radioisotope targeting therapy

    International Nuclear Information System (INIS)

    Wang Jiaqiong; Wang Zizheng

    2004-01-01

    With deeper cognition about mechanisms of disease at the cellular and molecular level, gene therapy has become one of the most important research fields in medical molecular biology at present. Gene transfer technology plays an important role during the course of gene therapy, and further improvement should be made about vectors carrying target gene sequences. Also, gene survey is needed during gene therapy, and gene imaging is the most effective method. The combination of gene therapy and targeted radiotherapy, that is, 'Genetic Radioisotope Targeting Therapy', will be a novel approach to tumor gene therapy

  18. Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines

    OpenAIRE

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2007-01-01

    AIM: To identify the relationship between DNA hyper-methylation and histone modification at a hyperme-thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification.

  19. Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci.

    NARCIS (Netherlands)

    Stam, M.; de Bruin, R.A.M.; van Blokland, H.J.M.; van der Hoorn, R.; Mol, J.N.M.; Kooter, J.M.

    2000-01-01

    The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between

  20. Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines.

    Science.gov (United States)

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2007-12-14

    To identify the relationship between DNA hyper-methylation and histone modification at a hyperme-thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. We used chromatin immunoprecipitation (ChIP) assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and mutL homolog 1 (MLH1) genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation-specific PCR (MSP) to evaluate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC), trichostatin A (TSA) or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. For the p16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza-dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected after TSA treatment, and increased moderately at the silenced loci after 5-Aza-dC treatment. Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate with DNA methylation

  1. Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress

    Directory of Open Access Journals (Sweden)

    Li Xiao Hui

    2015-09-01

    Full Text Available S-adenosylhomocysteine hydrolase (SAHH, catalyzing the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

  2. Gene silencing of CCD7 and CCD8 in Phelipanche aegyptiaca by tobacco rattle virus system retarded the parasite development on the host.

    Science.gov (United States)

    Aly, Radi; Dubey, Neeraj Kumar; Yahyaa, Mosaab; Abu-Nassar, Jackline; Ibdah, Mwafaq

    2014-01-01

    Strigolactones are phytohormones that stimulate seed germination of parasitic plants including Phelipanche aegyptiaca. Strigolactones are derived from carotenoids via a pathway involving the carotenoid cleavage dioxygenases CCD7 and CCD8. We report here identification of PaCCD7 and PaCCD8 orthologous genes from P. aegyptiaca. Expression analysis of PaCCD7 and PaCCD8 genes showed significant variation in their transcript levels in seeds and tubercles of P. aegyptiaca at different developmental stages. These two parasitic PaCCD7 and PaCCD8 genes were silenced in P. aegyptiaca using a trans-silencing approach in Nicotiana benthamiana. The transient knock-down of PaCCD7 and PaCCD8 inhibited tubercle development and the infestation process in host plants. Our results suggest an important role of the strigolactone associated genes (PaCCD7 and PaCCD8) in the parasite life cycle.

  3. Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii.

    Directory of Open Access Journals (Sweden)

    Wenfeng Li

    Full Text Available In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH, gene expression of CHH in the crayfish (Procambarus clarkii was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA, followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide, in the eyestalk ganglia of CHH dsRNA-injected (DSI animals were significantly decreased at 24, 48, and 72 hour post injection (hpi, with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ and hemolymph. Green fluorescence protein (GFP dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene expression reveal that metabolic functions of CHH

  4. Differential effects of silencing crustacean hyperglycemic hormone gene expression on the metabolic profiles of the muscle and hepatopancreas in the crayfish Procambarus clarkii.

    Science.gov (United States)

    Li, Wenfeng; Chiu, Kuo-Hsun; Tien, Yi-Chun; Tsai, Shih-Fu; Shih, Li-Jane; Lee, Chien-Hsun; Toullec, Jean-Yves; Lee, Chi-Ying

    2017-01-01

    In order to functionally characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), gene expression of CHH in the crayfish (Procambarus clarkii) was knocked down by in vivo injection of CHH double-stranded RNA (dsRNA), followed by metabolomic analysis of 2 CHH target tissues (the muscle and hepatopancreas) using nuclear magnetic resonance spectroscopy. Compared to the levels in untreated and saline-injected (SAI) animals, levels of CHH transcript, but not those of molt-inhibiting hormone (a CHH-family peptide), in the eyestalk ganglia of CHH dsRNA-injected (DSI) animals were significantly decreased at 24, 48, and 72 hour post injection (hpi), with concomitant changes in levels of CHH peptide in the sinus gland (a neurohemal organ) and hemolymph. Green fluorescence protein (GFP) dsRNA failed to affect levels of CHH transcript in the eyestalk ganglia of GFP DSI animals. Number of metabolites whose levels were significantly changed by CHH dsRNA was 149 and 181 in the muscle and 24 and 12 in the hepatopancreas, at 24 and 48 hpi, respectively. Principal component analysis of these metabolites show that metabolic effects of silencing CHH gene expression were more pronounced in the muscle (with the cluster of CHH DSI group clearly being separated from that of SAI group at 24 hpi) than in the hepatopancreas. Moreover, pathway analysis of the metabolites closely related to carbohydrate and energy metabolism indicate that, for CHH DSI animals at 24 hpi, metabolic profile of the muscle was characterized by reduced synthesis of NAD+ and adenine ribonucleotides, diminished levels of ATP, lower rate of utilization of carbohydrates through glycolysis, and a partially rescued TCA cycle, whereas that of the hepatopancreas by unaffected levels of ATP, lower rate of utilization of carbohydrates, and increased levels of ketone bodies. The combined results of metabolic changes in response to silenced CHH gene expression reveal that metabolic functions of CHH on the

  5. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the ...

  6. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this.

  7. Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Heikkinen Liisa

    2008-06-01

    Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

  8. Influence of silencing the MC4R gene by lentivirusmediated RNA ...

    African Journals Online (AJOL)

    ... expression systems were an efficient approach to the knockdown of the MC4R gene expression in bovine fibroblast cells and they provided a new molecular basis for understanding the relationship of MC4R and other genes, which were responsible for the regulation of energy homeostasis by the melanocortin system.

  9. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    International Nuclear Information System (INIS)

    Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

    2012-01-01

    Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  10. Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

    Science.gov (United States)

    Ohno, Sho; Hosokawa, Munetaka; Kojima, Misa; Kitamura, Yoshikuni; Hoshino, Atsushi; Tatsuzawa, Fumi; Doi, Motoaki; Yazawa, Susumu

    2011-11-01

    Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

  11. Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; 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. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.

  12. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Law, Alice Y.S. [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong); Wong, Chris K.C., E-mail: ckcwong@hkbu.edu.hk [Department of Biology, Hong Kong Baptist University, Kowloon Tong (Hong Kong)

    2010-02-01

    Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1{alpha} dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

  13. RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

    Science.gov (United States)

    Saema, Syed; Rahman, Laiq ur; Niranjan, Abhishek; Ahmad, Iffat Zareen; Misra, Pratibha

    2015-01-01

    Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera. PMID:26357855

  14. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity.

    Science.gov (United States)

    Kubo, Takanori; Yanagihara, Kazuyoshi; Takei, Yoshifumi; Mihara, Keichiro; Sato, Yuichiro; Seyama, Toshio

    2012-10-05

    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. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Biolistics-based gene silencing in plants using a modified particle inflow gun.

    Science.gov (United States)

    Davies, Kevin M; Deroles, Simon C; Boase, Murray R; Hunter, Don A; Schwinn, Kathy E

    2013-01-01

    RNA interference (RNAi) is one of the most commonly used techniques for examining the function of genes of interest. In this chapter we present two examples of RNAi that use the particle inflow gun for delivery of the DNA constructs. In one example transient RNAi is used to show the function of an anthocyanin regulatory gene in flower petals. In the second example stably transformed cell cultures are produced with an RNAi construct that results in a change in the anthocyanin hydroxylation pattern.

  16. [Fruit-specific RNAi-mediated Lcy gene enhances content of lycopene in tomatoes silencing].

    Science.gov (United States)

    Wan, Qun; Zhang, Xing-Guo; Song, Ming

    2007-05-01

    Tomatoes ( Lycopersicon esculentum Mill.) are the principal dietary source of Lycopene which is one of carotenoid and is highly beneficial in preventing some diseases such as the cancer and the heart disease. Suppressing the expression of Lcy gene, the main gene regulating the transformation of the lycopene, is a convenient and effective way to enhance the content of lycopene. The primers were designed according to the gene sequence(U46919)and (X86452) in GenBank. The fruit-specific promoter--phytoene desaturase gene(Pds) promoter and the DNA segment of the Lcy gene were isolated from the genome DNA of tomatoes. The 3'end of Lcy DNA segment was connected together by an intron to inform the RNA interferential segment then which was inserted in the expression vector with the Pds promoter to inform the fruit-specific expression vector. The vector was transformed into the tomatoes through the Agrobacterium tumefaciens. Five transformants were obtained. And the PCR proved that the extra-gene was integrated into the tomato genome. The lycopene in the transgenic tomatoes fruit was increased significantly through analysing the contents of lycopene. These results show that regutating biosynthetic enzyme in carotenoid pathway by RNAi can improve the lycopene content of plant-derived products.

  17. Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

    Science.gov (United States)

    Rubins, Kathleen H.; Hensley, Lisa E.; Relman, David A.; Brown, Patrick O.

    2011-01-01

    Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection. PMID:21267444

  18. Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Kathleen H Rubins

    2011-01-01

    Full Text Available Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV, an emerging human pathogen, and Vaccinia virus (VAC, a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA, or poly (I-C was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

  19. The down regulation of target genes by photo activated DNA nanoscissors.

    Science.gov (United States)

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7.

    Science.gov (United States)

    Bi, Yanzhao; Zhang, Yifan; Cui, Chunying; Ren, Lulu; Jiang, Xueyun

    Nanodiamond (ND) is a renowned material in nonviral small interfering RNA (siRNA) carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH 2 ) 2 NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR) assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V-fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA nanoparticle with 60-110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH 2 ) 2 NH-VDGR was suggested to be used in siRNA delivery system and in cancer treatments.

  2. Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    Directory of Open Access Journals (Sweden)

    Bi YZ

    2016-11-01

    Full Text Available Yanzhao Bi, Yifan Zhang, Chunying Cui, Lulu Ren, Xueyun Jiang School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China Abstract: Nanodiamond (ND is a renowned material in nonviral small interfering RNA (siRNA carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH22NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR assay, enzyme-linked immunosorbent assay (ELISA, flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH22NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V–fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH22NH-VDGR/survivin-siRNA nanoparticle with 60–110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH22NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH22NH-VDGR was suggested

  3. Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing

    Directory of Open Access Journals (Sweden)

    Chen Shou-Yi

    2011-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max is limited, and global identification of the related miRNA targets has not been reported in previous research. Results In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3 was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

  4. Silencing of SPRY1 Triggers Complete Regression of Rhabdomyosarcoma Tumors Carrying a Mutated RAS Gene

    NARCIS (Netherlands)

    Schaaf, Gerben; Hamdi, Mohamed; Zwijnenburg, Danny; Lakeman, Arjan; Geerts, Dirk; Versteeg, Rogier; Kool, Marcel

    2010-01-01

    RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative

  5. Small RNAs and Argonaute proteins: key players in post-transcriptional gene silencing

    NARCIS (Netherlands)

    Steiner, F.A.

    2007-01-01

    Small RNAs are important transcriptional and post-transcriptional regulators of gene expression. Many classes of small RNAs have been discovered, each carrying out specialized functions. siRNAs and miRNAs are best studies. siRNAs function in the process of RNAi and are thought to defend the genome

  6. RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

    KAUST Repository

    Cernilogar, Filippo M.

    2013-06-13

    Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

  7. RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells

    International Nuclear Information System (INIS)

    Beck, Markus; Strand, Michael R.

    2003-01-01

    The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes

  8. Retracted: Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via Induction of Cell Cycle Arrest and Apoptosis

    Science.gov (United States)

    2017-11-17

    Retraction: Retracted: Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via Induction of Cell Cycle Arrest and Apoptosis Asian Pacific Journal of Cancer Prevention has retracted the article titled “Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via Induction of Cell Cycle Arrest and Apoptosis”(1) for reason of similarity with a series of articles identified by Byrne and Labbé (2). Xue-Mei Wang, Jiu-Wei Cui1&, Wei Li , Lu Cai, Wei Song , Guan-Jun Wang 1. Xue-Mei Wang, Jiu-Wei Cui1&, Wei Li , Lu Cai, Wei Song , Guan-Jun Wang. Silencing of the COPS3 Gene by siRNA Reduces Proliferation of Lung Cancer Cells Most Likely via Induction of Cell Cycle Arrest and Apoptosis. Asian Pac J Cancer Prev. 2012;13(5):1823-7. 2. J. A. Byrne and C. Labbé, “Striking similarities between publications from China describing single gene knockdown experiments in human cancer cell lines,” Scientometrics, vol. 110, no. 3, pp. 1471–1493, 2017. Authors did not respond to request for comment.

  9. Antisense silencing of the creA gene in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Bautista, L. F.; Aleksenko, Alexei Y.; Hentzer, Morten

    2000-01-01

    Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was appr...... was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected....

  10. Post-transcriptional gene silencing is involved in resistance of transgenic papayas to Papaya Ringspot Virus

    Czech Academy of Sciences Publication Activity Database

    Ruanjan, P.; Kertbundit, Sunee; Juříček, Miloslav

    2007-01-01

    Roč. 51, č. 3 (2007), s. 517-520 ISSN 0006-3134 Grant - others:BIOTEC, NASDA(TH) BT-B-06-PG-14-4503 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : Carica papaya * reverse transcription PCR * COAT PROTEIN GENE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.259, year: 2007

  11. The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with unique activities

    International Nuclear Information System (INIS)

    Mangwende, Tichaona; Wang Mingli; Borth, Wayne; Hu, John; Moore, Paul H.; Mirkov, T. Erik; Albert, Henrik H.

    2009-01-01

    The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses (Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS

  12. Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1 -> 3)-glucanase activity at the onset of tobacco defence reactions

    DEFF Research Database (Denmark)

    Riviere, M.P.; Marais, A.; Ponchet, M.

    2008-01-01

    silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic...... protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence......The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were...

  13. Chromosomal position effects on AAV-mediated gene targeting.

    Science.gov (United States)

    Cornea, Anda M; Russell, David W

    2010-06-01

    The effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = -0.55, P targeting frequencies at the target loci (r = 0.52, P targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells.

  14. Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

    Science.gov (United States)

    Rodrigues, Thais B; Duan, Jian J; Palli, Subba R; Rieske, Lynne K

    2018-03-22

    Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

  15. Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection

    Science.gov (United States)

    Walton, Senta M.; Liao, Tingting; Stubbs, Keith A.; Marshall, Barry J.; Fulurija, Alma; Benghezal, Mohammed

    2017-01-01

    Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress. PMID:28644872

  16. Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

    Directory of Open Access Journals (Sweden)

    Aleksandra W Debowski

    2017-06-01

    Full Text Available Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

  17. Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat

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    Guoyu Liu

    2016-09-01

    Full Text Available The function of a wheat starch regulator 1 (TaRSR1 in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus—virus induced gene-silencing (BSMV-VIGS method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1 at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes.

  18. CGG-repeat dynamics andFMR1gene silencing in fragile X syndrome stem cells and stem cell-derived neurons.

    Science.gov (United States)

    Zhou, Yifan; Kumari, Daman; Sciascia, Nicholas; Usdin, Karen

    2016-01-01

    Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different

  19. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

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    Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

    2010-12-01

    Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

  20. Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3-ketoacyl-CoA Thiolase Isozymes.

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    Nakazawa, Masami; Andoh, Hiroko; Koyama, Keiichiro; Watanabe, Yomi; Nakai, Takeo; Ueda, Mitsuhiro; Sakamoto, Tatsuji; Inui, Hiroshi; Nakano, Yoshihisa; Miyatake, Kazutaka

    2015-05-01

    Euglena gracilis produces wax ester under hypoxic and anaerobic culture conditions with a net synthesis of ATP. In wax ester fermentation, fatty acids are synthesized by reversing beta-oxidation in mitochondria. A major species of wax ester produced by E. gracilis is myristyl myristate (14:0-14:0Alc). Because of its shorter carbon chain length with saturated compounds, biodiesel produced from E. gracilis wax ester may have good cold flow properties with high oxidative stability. We reasoned that a slight metabolic modification would enable E. gracilis to produce a biofuel of ideal composition. In order to produce wax ester with shorter acyl chain length, we focused on isozymes of the enzyme 3-ketoacyl-CoA thiolase (KAT), a condensing enzyme of the mitochondrial fatty acid synthesis pathway in E. gracilis. We performed a gene silencing study of KAT isozymes in E. gracilis. Six KAT isozymes were identified in the E. gracilis EST database, and silencing any three of them (EgKAT1-3) altered the wax ester amount and composition. In particular, silencing EgKAT1 induced a significant compositional shift to shorter carbon chain lengths in wax ester. A model fuel mixture inferred from the composition of wax ester in EgKAT1-silenced cells showed a significant decrease in melting point compared to that of the control cells.

  1. RNAi-mediated silencing of vitellogenin gene function turns honeybee ( Apis mellifera) workers into extremely precocious foragers

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    Marco Antonio, David Santos; Guidugli-Lazzarini, Karina Rosa; Do Nascimento, Adriana Mendes; Simões, Zilá Luz Paulino; Hartfelder, Klaus

    2008-10-01

    The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee ( Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (>10 min) to earlier in life (by 3 4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (<10 min), which bees usually perform for orientation and cleaning, were not affected. Additionally, we found that the JH titer in dsVg bees collected after 7 days was not significantly different from the controls. The finding that depletion of the vitellogenin titer can drive young bees to become extremely precocious foragers could imply that vitellogenin is the primary switch signal. At this young age, downregulation of vitellogenin gene activity apparently had little effect on the JH titer. As this unexpected finding stands in contrast with previous results on the vitellogenin/JH interaction at a later age, when bees normally become foragers, we propose a three-step sequence in the constellation of physiological parameters underlying behavioral development.

  2. Assessment of RNAi-induced silencing in banana (Musa spp.).

    Science.gov (United States)

    Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

    2014-09-18

    In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target

  3. Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

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    Ling Zhang

    2014-01-01

    Full Text Available The Delta-12 oleate desaturase gene (FAD2-1, which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71% and a reduction in palmitic acid (to <3% in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

  4. The essential function of B. subtilis RNase III is to silence foreign toxin genes.

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    Sylvain Durand

    Full Text Available RNase III-related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi-RNAs and small interfering (si-RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III-mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.

  5. Epigenetic Silencing of the Proapoptotic Gene BIM in Anaplastic Large Cell Lymphoma through an MeCP2/SIN3a Deacetylating Complex12

    Science.gov (United States)

    Piazza, Rocco; Magistroni, Vera; Mogavero, Angela; Andreoni, Federica; Ambrogio, Chiara; Chiarle, Roberto; Mologni, Luca; Bachmann, Petra S; Lock, Richard B; Collini, Paola; Pelosi, Giuseppe; Gambacorti-Passerini, Carlo

    2013-01-01

    BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL) cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2) corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation. PMID:23633923

  6. Epigenetic Silencing of the Proapoptotic Gene BIM in Anaplastic Large Cell Lymphoma through an MeCP2/SIN3a Deacetylating Complex

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    Rocco Piazza

    2013-05-01

    Full Text Available BIM is a proapoptotic member of the Bcl-2 family. Here, we investigated the epigenetic status of the BIM locus in NPM/ALK+ anaplastic large cell lymphoma (ALCL cell lines and in lymph node biopsies from NPM/ALK+ ALCL patients. We show that BIM is epigenetically silenced in cell lines and lymph node specimens and that treatment with the deacetylase inhibitor trichostatin A restores the histone acetylation, strongly upregulates BIM expression, and induces cell death. BIM silencing occurs through recruitment of MeCP2 and the SIN3a/histone deacetylase 1/2 (HDAC1/2 corepressor complex. This event requires BIM CpG methylation/demethylation with 5-azacytidine that leads to detachment of the MeCP2 corepressor complex and reacetylation of the histone tails. Treatment with the ALK inhibitor PF2341066 or with an inducible shRNA targeting NPM/ALK does not restore BIM locus reacetylation; however, enforced expression of NPM/ALK in an NPM/ALK-negative cell line significantly increases the methylation at the BIM locus. This study demonstrates that BIM is epigenetically silenced in NPM/ALK-positive cells through recruitment of the SIN3a/HDAC1/2 corepressor complex and that NPM/ALK is dispensable to maintain BIM epigenetic silencing but is able to act as an inducer of BIM methylation.

  7. Nano and Microtechnologies for the Delivery of Oligonucleotides with Gene Silencing Properties

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    Giuseppe De Rosa

    2009-07-01

    Full Text Available Oligonucleotides (ONs are synthetic fragments of nucleic acid designed to modulate the expression of target proteins. DNA-based ONs (antisense, antigene, aptamer or decoy and more recently a new class of RNA-based ONs, the small interfering RNAs (siRNAs, have gained great attention for the treatment of different disease states, such as viral infections, inflammation, diabetes, and cancer. However, the development of therapeutic strategies based on ONs is hampered by their low bioavailability, poor intracellular uptake and rapid degradation in biological fluids. The use of a non-viral carrier can be a powerful tool to overcome these drawbacks. Lipid or polymer-based nanotechnologies can improve biological stability and cellular uptake of ONs, with possibility of tissue and/or cellular targeting. The use of polymeric devices can also produce a prolonged release of the ON, thus reducing the need of frequent administrations. This review summarizes advantages and issues related to the main non-viral vectors used for ON delivery.

  8. Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival.

    Science.gov (United States)

    Ahmed, Wareed; Menon, Shruti; Godbole, Adwait Anand; Karthik, Pullela V D N B; Nagaraja, Valakunja

    2014-04-01

    Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M. tuberculosis topoisomerase I (TopoI(Mt) ) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M. tuberculosis growth and open up new avenues for targeting the enzyme. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  9. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence.

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    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T; Cui, Jiajia; Cheng, Christopher J; Saltzman, W Mark

    2015-12-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.

  10. Functionalized graphene oxide mediated adriamycin delivery and miR-21 gene silencing to overcome tumor multidrug resistance in vitro.

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    Feng Zhi

    Full Text Available Multidrug resistance (MDR is a major impediment to successful cancer chemotherapy. Co-delivery of novel MDR-reversing agents and anticancer drugs to cancer cells holds great promise for cancer treatment. MicroRNA-21 (miR-21 overexpression is associated with the development and progression of MDR in breast cancer, and it is emerging as a novel and promising MDR-reversing target. In this study, a multifunctional nanocomplex, composed of polyethylenimine (PEI/poly(sodium 4-styrenesulfonates (PSS/graphene oxide (GO and termed PPG, was prepared using the layer-by-layer assembly method to evaluate the reversal effects of PPG as a carrier for adriamycin (ADR along with miR-21 targeted siRNA (anti-miR-21 in cancer drug resistance. ADR was firstly loaded onto the PPG surface (PPGADR by physical mixing and anti-miR-21 was sequentially loaded onto PPGADR through electric absorption to form (anti-miR-21PPGADR. Cell experiments showed that PPG significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line and exhibited much higher cytotoxicity than free ADR, suggesting that PPG could effectively reverse ADR resistance of MCF-7/ADR. Furthermore, the enhanced therapeutic efficacy of PPG could be correlated with effective silencing of miR-21 and with increased accumulation of ADR in drug-resistant tumor cells. The endocytosis study confirmed that PPG could effectively carry drug molecules into cells via the caveolae and clathrin-mediated endocytosis pathways. These results suggest that this PPG could be a potential and efficient non-viral vector for reversing MDR, and the strategy of combining anticancer drugs with miRNA therapy to overcome MDR could be an attractive approach in cancer treatment.

  11. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

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    Chernousova, S; Epple, M

    2017-05-01

    The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

  12. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors

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    Wu Yiqing

    2010-03-01

    Full Text Available Abstract Background Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. Methods 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. Results Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. Conclusions These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of

  13. Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors

    International Nuclear Information System (INIS)

    Li, Zidong; Chen, Bo; Wu, Yiqing; Jin, Feng; Xia, Yongjing; Liu, Xiangjun

    2010-01-01

    Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer

  14. The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms

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    F. Jerry Reen

    2015-07-01

    Full Text Available Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs. However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

  15. Active chromatin marks are retained on X chromosomes lacking gene or repeat silencing despite XIST/Xist expression in somatic cell hybrids.

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    Nancy P Thorogood

    2010-05-01

    Full Text Available X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

  16. UHRF1 gene silencing inhibits cell proliferation and promotes cell apoptosis in human cervical squamous cell carcinoma CaSki cells.

    Science.gov (United States)

    Ge, Ting-Ting; Yang, Meng; Chen, Zhuo; Lou, Ge; Gu, Tao

    2016-07-19

    Up-regulation of UHRF1 has been observed in a variety of cancers and appears to serve as an independent prognostic factor. To explore the effect of UHRF1 gene silencing on apoptosis and proliferation of cervical squamous cell carcinoma (CSCC) CaSki cells. This study consisted of 47 CSCC tissues and 40 normal cervical tissues. The CaSki cells were assigned into Blank group (CaSki cells not transfected), NC group (CaSki cells transfected with control siRNA), and UHRF1 Silence group (CaSki cells transfected with UHRF1 siRNA). qRT-PCR and Western blot were used for UHRF1 mRNA and protein expressions, CKK-8 assay for cell proliferation, flow cytometry for cell cycle and apoptosis, Western blot for expressions of apoptosis-related proteins. Nude mice tumor transplant experiment was performed. UHRF1 exhibited higher mRNA and protein expressions in the CSCC tissues than normal cervical tissues (both P cell proliferation ability in the UHRF1 Silence group was reduced when compared with the Blank group and the NC group, the cells at S-G2M stage in the UHRF1 Silence group were dropped when compared with the Blank group and the NC group (P cells at G0/G1 stage were elevated (P cells in the UHRF1 Silence group was increased in comparison with the Blank group and the NC group (P proliferation and enhance apoptosis of the CaSki cells.

  17. The mechanism of gene targeting in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Yinan Kan

    2014-04-01

    Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  18. Gene therapy and radionuclides targeting therapy in mammary carcinoma

    International Nuclear Information System (INIS)

    Song Jinhua

    2003-01-01

    Breast carcinoma's gene therapy is a hotspot in study of the tumor's therapy in the recent years. Currently the major therapy methods that in the experimentative and primary clinical application phases include immunological gene therapy, multidrug resistance gene therapy, antisense oligonucleotide therapy and suicide gene therapy. The gene targeting brachytherapy, which is combined with gene therapy and radiotherapy has enhanced the killer effects of the suicide gene and nuclide in tumor cells. That has break a new path in tumor's gene therapy. The further study in this field will step up it's space to the clinical application

  19. Transcriptionally targeted gene therapy to detect and treat cancer

    OpenAIRE

    Wu, Lily; Johnson, Mai; Sato, Makoto

    2003-01-01

    The greatest challenge in cancer treatment is to achieve the highest levels of specificity and efficacy. Cancer gene therapy could be designed specifically to express therapeutic genes to induce cancer cell destruction. Cancer-specific promoters are useful tools to accomplish targeted expression; however, high levels of gene expression are needed to achieve therapeutic efficacy. Incorporating an imaging reporter gene in tandem with the therapeutic gene will allow tangible proof of principle t...

  20. A developmental window of opportunity for imprinted gene silencing mediated by DNA methylation and the Kcnq1ot1 noncoding RNA.

    Science.gov (United States)

    Green, Kelly; Lewis, Annabelle; Dawson, Claire; Dean, Wendy; Reinhart, Bonnie; Chaillet, J Richard; Reik, Wolf

    2007-01-01

    The Kcnq1 imprinted domain encodes a paternally expressed noncoding RNA Kcnq1ot1 and several paternally repressed protein-coding genes. Transcriptional regulation is controlled by the Kcnq1ot1 gene whose maternal germline methylation imprint overlaps with the Kcnq1ot1 promoter. The domain can be divided into two groups of genes. One group is imprinted in all lineages and is reliant on DNA methylation for its imprinting. The other group contains genes that are imprinted specifically in the placenta and retain their imprinting in the absence of Dnmt1, the primary DNA maintenance methylase. In the placenta paternal Kcnq1ot1 expression is associated with the acquisition of repressive histone modifications throughout the domain. Using the Dnmt1o knockout, we have analyzed the effect of removing DNA maintenance methylation at the eight-cell stage on the Kcnq1 imprinted domain. In the placenta the expression of the normally silent maternal Kcnq1ot1 allele leads to reduced expression of the surrounding maternally expressed genes. This repression is seen in both the placental-specific imprinted genes and the ubiquitously imprinted genes. Conversely, reduction of functional Dnmt1 results solely in reduced expression of the ubiquitously imprinted genes in the placenta. This suggests that Kcnq1ot1 expression can epigenetically silence placentally imprinted genes in the cluster only during a specific developmental window. This highlights the possibility that Kcnq1ot1-mediated repression is temporally regulated leading to epigenetic silencing of placental-specific genes. We show that allele-specific histone modifications are still present in the Dnmt1 ( -/- ) trophoblast at placental-specific imprinted loci and are likely responsible for maintaining the imprinting of these genes in the absence of DNA methylation.

  1. CXCL12 gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer.

    Science.gov (United States)

    Ma, J; Su, H; Yu, B; Guo, T; Gong, Z; Qi, J; Zhao, X; Du, J

    2018-01-05

    To investigate the effect of CXCL12 gene silencing on proliferation,invasion, angiogenesis and the relationship of MAPK/PI3K/AP-1 signaling pathway in colon cancer cells. RT-PCR and Western-blot were used to detect the expression of CXCL12 mRNA and protein in four colon cancer cell lines. Human colon cancer cells were transfected with CXCL12 siRNA carrying by Lipofectamine 2000. The expression of CXCL12 protein was confirmed by immunoblotting. WST-1, invasion and angiogenesis assay were used to examine the effect on proliferation, invasion and angiogenesis in colon cancer cells after CXCL12 siRNA silence, respectively. The phosphorylation of MAPK/PI3K/AP-1 protein levels was detected by Western blotting in CXCL12 siRNA suppression DLD-1 cell. CXCL12 mRNA and proteins were only expressed in DLD-1 colon cancer cell lines. CXCL12 siRNA were transfected into DLD-1 cells, the expression CXCL12 proteins was significantly inhibited (P colon cancer cell. The silencing CXCL12 gene significantly inhibits the proliferation, invasion and angiogenesis ability of some types colon carcinoma cells through down-regulation of MAPK/PI3K/AP-1 signaling pathway.

  2. Molecular targeting of gene therapy and radiotherapy

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Kufe, D.W.; Advani, S.J.; Roizman, B.

    2001-01-01

    The full promise of gene therapy has been limited by the lack of specificity of vectors for tumor tissue as well as the lack of antitumor efficacy of transgenes encoded by gene delivery systems. In this paper we review our studies investigating two modifications of gene therapy combined with radiotherapy. The first investigations described include studies of radiation inducible gene therapy. In this paradigm, radio-inducible DNA sequences from the CarG elements of the Egr-1 promoter are cloned upstream of a cDNA encoding TNFa. The therapeutic gene (TNFa) is induced by radiation within the tumor microenvironment. In the second paradigm, genetically engineered herpes simplex virus (HSV-1) is induced by ionizing radiation to proliferate within the tumor volume. These modifications of radiotherapy and gene therapy may enhance the efficacy of both treatments

  3. Effects of perinuclear chromosome tethers in the telomeric URA3/5FOA system reflect changes to gene silencing and not nucleotide metabolism

    Directory of Open Access Journals (Sweden)

    Betty Po Kei Poon

    2012-08-01

    Full Text Available Telomeres are repetitive DNA sequences that protect the ends of linear chromosomes. Telomeres also recruit histone deacetylase complexes that can then spread along chromosome arms and repress the expression of subtelomeric genes in a process known as telomere position effect (TPE. In the budding yeast Saccharomyces cerevisiae, association of telomeres with the nuclear envelope is thought to promote TPE by increasing the local concentration of histone deacetylase complexes at chromosome ends. Importantly, our understanding of TPE stems primarily from studies that employed marker genes inserted within yeast subtelomeres. In particular, the prototrophic marker URA3 is commonly used to assay TPE by negative selection on media supplemented with 5-fluoro-orotic acid (5FOA. Recent findings suggested that decreased growth on 5FOA-containing media may not always indicate increased expression of a telomeric URA3 reporter, but can rather reflect an increase in ribonucleotide reductase (RNR function and nucleotide metabolism. Thus, we set out to test if the 5FOA sensitivity of subtelomeric URA3-harbouring cells in which we deleted various factors implicated in perinuclear telomere tethering reflects changes to TPE and/or RNR. We report that RNR inhibition restores 5FOA resistance to cells lacking RNR regulatory factors but not any of the major telomere tethering and silencing factors, including Sir2, Cohibin, Mps3, Heh1, and Esc1. In addition, we find that the disruption of tethering pathways in which these factors participate increases the level of URA3 transcripts originating from the telomeric reporter gene and abrogates silencing of subtelomeric HIS3 reporter genes without altering RNR gene expression. Thus, increased 5FOA sensitivity of telomeric URA3-harbouring cells deficient in telomere tethers reflects the dysregulation of TPE but not RNR. This is key to understanding relationships between telomere positioning, chromatin silencing, and lifespan.

  4. Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

    Science.gov (United States)

    Li, J L; Cui, J; Cheng, D Y

    2015-08-07

    Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

  5. Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions

    Science.gov (United States)

    Teterina, Natalya L.; Liu, Guangping; Maximova, Olga A.; Pletnev, Alexander G.

    2014-01-01

    In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. Here, using a neurotropic flavivirus as a model, we demonstrate that simultaneous miRNA targeting of the viral genome in the open reading frame and 3′-noncoding regions for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. This miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing. PMID:24889244

  6. The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

    Science.gov (United States)

    Chen, W; Gao, B; Hao, L; Zhu, G; Jules, J; MacDougall, M J; Wang, J; Han, X; Zhou, X; Li, Y-P

    2016-10-01

    Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Discovering Host Genes Involved in the Infection by the Tomato Yellow Leaf Curl Virus Complex and in the Establishment of Resistance to the Virus Using Tobacco Rattle Virus-based Post Transcriptional Gene Silencing

    Science.gov (United States)

    Czosnek, Henryk; Eybishtz, Assaf; Sade, Dagan; Gorovits, Rena; Sobol, Iris; Bejarano, Eduardo; Rosas-Díaz, Tábata; Lozano-Durán, Rosa

    2013-01-01

    The development of high-throughput technologies allows for evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infection of susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, which over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty-nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the 25 genes studied so far, the silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicates that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular

  8. Methylation of polycomb target genes in intestinal cancer is mediated by inflammation.

    Science.gov (United States)

    Hahn, Maria A; Hahn, Torsten; Lee, Dong-Hyun; Esworthy, R Steven; Kim, Byung-Wook; Riggs, Arthur D; Chu, Fong-Fong; Pfeifer, Gerd P

    2008-12-15

    Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

  9. Gene therapy of cancer and development of therapeutic target gene

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Min; Kwon, Hee Chung

    1998-04-01

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

  10. Gene therapy of cancer and development of therapeutic target gene

    International Nuclear Information System (INIS)