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Small hairpin RNA

About: Small hairpin RNA is a research topic. Over the lifetime, 9279 publications have been published within this topic receiving 285471 citations.


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Journal ArticleDOI
Bryan R. Cullen1
TL;DR: A set of design criteria, experimental steps and expression vectors that can facilitate the effective knock-down of almost any vertebrate gene product in cultured cells or in experimental animals are described.
Abstract: Over the last years, RNA interference (RNAi) has become a widely used technique that permits the knock-down, and hence functional analysis, of individual genes in vertebrate cells. However, the high failure rate of the RNA molecules used in RNAi experiments continues to be a problem. In this paper, I describe a set of design criteria, experimental steps and expression vectors that can facilitate the effective knock-down of almost any vertebrate gene product in cultured cells or in experimental animals.Gene Therapy (2006) 13, 503-508. doi:10.1038/sj.gt.3302656; published online 29 September 2005.

84 citations

Journal ArticleDOI
TL;DR: The potential of post-transcriptional gene silencing using RNA interference for the selective down-regulation of pathogenic mutant AChR subunits is investigated, finding that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence.
Abstract: Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125 I-abungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of aS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.

84 citations

Journal ArticleDOI
TL;DR: Less common chemical modifications of the RNA nucleobases have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.
Abstract: Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely, those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.

84 citations

Journal ArticleDOI
TL;DR: Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity.
Abstract: Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

84 citations

Journal ArticleDOI
TL;DR: It is suggested that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling through peroxisome proliferator–activated receptor γ, and may have efficacy in the treatment of NSCLC.
Abstract: Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in non-small cell lung cancer (NSCLC). We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared with a nontransformed cell line, suggesting that Spry4 has tumor-suppressing activity. When Spry4 was stably transfected into H157 and H2122 NSCLC cell lines, decreased migration and invasion were observed. Matrix metalloproteinase-9 activity was decreased, and the expression of matrix metalloproteinase inhibitors TIMP1 and CD82 were increased. Stable expression of Spry4 led to reduced cell growth and reduced anchorage-independent growth in NSCLC cell lines, along with upregulation of tumor suppressors p53 and p21. Changes in epithelial and mesenchymal markers indicated that Spry4 expression induces a reversal of the epithelial to mesenchymal transition characteristic of tumor cells. Treatment of a nontransformed lung epithelial cell line with short hairpin RNA to Spry4 led to the decreased expression of epithelial markers and increased cell growth, supporting the concept of Spry4 acting as a tumor suppressor. We showed that the activity of the Spry4 promoter is increased by Wnt7A/Fzd9 signaling through peroxisome proliferator-activated receptor gamma. These data present previously undescribed targets of Spry4 and suggest that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling. Spry4 may have efficacy in the treatment of NSCLC.

84 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023804
2022477
2021384
2020454
2019541
2018518