Multiple shRNA expressing vector enhances efficiency of gene silencing

Song, Jun; Giang, An Hoa; Lu, Yingchun; Pang, Shen; Chiu, Robert

doi:10.5483/bmbrep.2008.41.5.358

Cited by 25 publications

(20 citation statements)

References 9 publications

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“…4 ). Recent studies have included the use of shRNA cassettes in combinations of two ( 133 ) , three ( 132,134,135 ) , four ( 5,136,137 ) , six ( 138,139 ) , and in one study a cloning strategy for the production of up to seven was described but not validated ( 140 ) . Results consistently show that such approaches provide an additive effect for single-and multiple-gene knockdown as demonstrated on a variety of host and viral gene targets.…”

Section: Potential Toxicity Of Shrnas In Vivomentioning

confidence: 99%

Short Hairpin RNA-Mediated Gene Silencing

Lambeth

Smith

2012

Methods in Molecular Biology

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Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficult-to-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different effector molecule formats, promoters, and vector types, has meant that experiments can be tailored to target specific cell types and minimize cellular toxicities. Through the application of combinatorial RNAi (co-RNAi), multiple shRNA delivery strategies can improve gene knockdown, permit multiple transcripts to be targeted simultaneously, and curtail the emergence of viral escape mutants. This chapter reviews the history, cellular processing, and various applications of shRNAs in mammalian systems, including options for effector molecule design, vector and promoter types, and methods for multiple shRNA delivery.

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Section: Potential Toxicity Of Shrnas In Vivomentioning

confidence: 99%

Short Hairpin RNA-Mediated Gene Silencing

Lambeth

Smith

2012

Methods in Molecular Biology

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“…SiRNA was widely found in plants and animals and, by complementary binding to its homologous sequences, silences the targeted gene [21] . Nowadays, RNAi technology, as a simple and effective gene, has been extensively used in the gene engineering [22] .…”

Section: Discussionmentioning

confidence: 99%

Impact of silencing MMP9 gene on the biological behaviors of trophoblasts

Luo

Qiao

Yin

2011

J. Huazhong Univ. Sci. Technol. [Med. Sci.]

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This study examined the effect of MMP9 gene on the biological behaviors of trophoblasts and explore the relation between MMP9 gene and the "superficial implantation of placenta". In vitro cultured trophoblasts (TEV-1 cells) were transfected with synthesized double-stranded MMP9 RNA (siRNA) by using lipofectamine2000™ technique and the expressions of MMP9 mRNA and protein and the growth and invasiveness of the TEV-1 cells were determined. Our results showed that siRNA transfection could significantly inhibit the expression of MMP9 gene in the TEV-1 cells and the growth and invasiveness of the TEV-1 cells transfected RNA was significantly reduced (P<0.01). We are led to conclude that silencing of MMP9 gene with siRNA can inhibit the growth and invasiveness of trophoblasts and increasing the expression of MMP9 might help prevent and treat preeclampsia.

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“…RNAi has been successfully used for gene silencing and is highly specific and efficient, easy to manipulate, controlabe, time-saving, versatile and inexpensive. RNAi has been extensively used in the research on gene functions, tumors and antiviral therapy [17][18][19][20][21] . RNAi can also be employed for inhibiting the expression of OPN.…”

Section: Discussionmentioning

confidence: 99%

Influence of osteopontin short hairpin RNA on the proliferation and activity of rat vascular smooth muscle cells

Sun

Bie

et al. 2009

J. Huazhong Univ. Sci. Technol. [Med. Sci.]

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To investigate the influence of osteopontin (OPN) short hairpin RNA (shRNA) on the proliferation and activity of rat vascular smooth muscle cells (VSMCs), the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 (PG1), pGenesil-1/OPNshRNA2 (PG2) and pGenesil-1/OPNshRNAHK (PGH) were transfected into the cultured rat VSMC by Lipofectamine 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and III collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% (P<0.01) by PG1, 73% and 52% (P<0.01) by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type I and III collagen. It is concluded that recombinant plasmid can be successfully transfected into VSMCs by Lipofectamine 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type I and III collagen is inhibited after knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention (PCI) by RNA interference (RNAi) technology.

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Multiple shRNA expressing vector enhances efficiency of gene silencing

Cited by 25 publications

References 9 publications

Short Hairpin RNA-Mediated Gene Silencing

Short Hairpin RNA-Mediated Gene Silencing

Impact of silencing MMP9 gene on the biological behaviors of trophoblasts

Influence of osteopontin short hairpin RNA on the proliferation and activity of rat vascular smooth muscle cells

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