shRNA driven by Pol II/T7 dual-promoter system effectively induce cell-specific RNA interference in mammalian cells

Peng, Ying; Lü, Jianxin; Shen, Xinfeng

doi:10.1016/j.bbrc.2007.06.100

Cited by 12 publications

(6 citation statements)

References 22 publications

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“…48 The shRNAs can be designed to exhibit promoter-specific expression, 49 and can be genetically integrated to generate stable RNAi regulators. 50 The use of plasmid-based shRNA for gene suppression has been successfully applied in a variety of mammalian cell systems, [51][52][53][54] and T7RNAp-driven shRNAs have been reported for the regulation of gene expression in live mammalian cells 55,56 and zebrafish embryos. 57 Here, we coupled our developed photochemical gene regulation system with an Eg5 shRNA component as a proof of principle study in the photochemical regulation of RNA interference.…”

Section: Resultsmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

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Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the sitespecific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

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Section: Resultsmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

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“…Although these are not yet popular techniques, they do have a lot of potential. Another way to induce tissuespecific expression of shRNAs was presented by Peng et al [174]. They expressed the shRNA cassette under control of a T7 promoter.…”

Section: Recent Inducible Systemsmentioning

confidence: 99%

In Search of the Most Suitable Lentiviral shRNA System

Bos

Bruyne

Heirman³

et al. 2009

CGT

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A decade after its discovery, RNA interference has proven to be an instant success both in fundamental research and clinical applications. Lentiviral delivery of shRNAs is one of the most popular approaches to study gene functionalities in both developmental biology and disorders. During the past 10 years, several adaptations and novel techniques have emerged to improve (conditional) transgene expression and to meet researchers' needs. However, due to this magnitude of diversity, it is sometimes difficult to select the most suitable approach for a specific experimental setup. Here, we summarize the different systems and techniques available for every step in the generation of shRNA-bearing lentiviruses. The most crucial point is inevitably the selection of the target sequence itself. A good shRNA design is indispensable and determines almost completely the success of the experiments. In addition, an adequate promoter that drives the shRNA expression has to be chosen depending on its strength, inducibility, tissue-specificity, At this point, the researcher has also to decide whether the expression of the shRNA should be inducible or not. Another point one has to keep in mind is the choice of lentiviral vector in which the silencing cassette will be incorporated; single- or double-copy vectors are available. The last 2 years, shRNA multiplex approaches in which several targets are silenced with one vector have emerged and have shown a lot of potential in complex studies (like HIV-1). Finally, in the last section, we will discuss the possible induction of an immune response by short dsRNA molecules.

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“…To validate the ability of the zebrafish U6 and H1 promoters to express functional shRNAs, the putative sequences were used to construct six vectors pZFU6-1shEGFP, pZFU6-2 shEGFP,pZFU6-3 shEGFP, pZFU6-4v1shEGFP, pZFU6-4v2shEGFP, and, pZF-H1shEGFP, in which the pol III promoter mediates expression of a shRNA molecule targeting EGFP. Additional constructs using chicken and mouse promoters (pCH-U6shEGFP and pM-U6shEGFP, respectively) have previously been shown to strongly express shRNAs in a wide variety of cells, 3,5,22,31 the construct pFUGU-shEGFP has been shown to be an effective silencer of EGFP in Bluefin gill (BF-2) cells, 22 and more recently Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells 17 were used as controls. Two irrelevant control vectors pZFU6-2 Irrelevant and pCHU6-4 NP were used to express an irrelevant shRNA as negative controls in ZF-4 and Vero cells, respectively.…”

Section: The Zebrafish U6 and H1 Promoters Express Shrnasmentioning

confidence: 99%

“…28,29 DNA vectors transcribing short hairpin RNAi precursor molecules (shRNAs) have been used to demonstrate specific RNAi silencing, in numerous cell lines 1,3,5,10,20,30 and animals [24][25][26] with greater efficiency and long-term effects than siRNAs. Furthermore, DNA-delivered RNAi allow the use promoters that are tissue specific, 31,32 inducible, 33 or modified for a particular application. Shorthairpin RNA molecules are transcribed from inverted complementary sequences separated by a loop sequence of between 4-25 nt.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Zebrafish Polymerase III Promoters for the Expression of Short-Hairpin RNA Interference Molecules

et al. 2013

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RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.

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shRNA driven by Pol II/T7 dual-promoter system effectively induce cell-specific RNA interference in mammalian cells

Cited by 12 publications

References 22 publications

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

In Search of the Most Suitable Lentiviral shRNA System

Characterization of Zebrafish Polymerase III Promoters for the Expression of Short-Hairpin RNA Interference Molecules

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