Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

Lambeth, Luke S.; Wise, Terry G.; Moore, Robert J.; Muralitharan, Morley; Doran, Timothy

doi:10.1111/j.1365-2052.2006.01468.x

Cited by 24 publications

(31 citation statements)

References 11 publications

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“…These nine loci are dispersed throughout the genome and fi ve were found to have associated promoter regions, each with different transcriptional activities. These fi ndings spurred the characterization and comparison of U6 promoters for shRNA transcription from a variety of other species including bovine ( 69,70 ) , chicken ( 71,72 ) , fugu ( 73 ) , zebra fi sh ( 74 ) , sheep ( 75 ) , and pig ( 76 ) .…”

Section: Promoters For Shrna Transcriptionmentioning

confidence: 98%

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: Promoters For Shrna Transcriptionmentioning

confidence: 98%

Short Hairpin RNA-Mediated Gene Silencing

Lambeth

Smith

2012

Methods in Molecular Biology

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“…sequence element (PSE), a TATA like element and an enhancer region consisting of an octamer motif (OCT) and a SPH element (7)(8)(9). Recently, various U6 promoters for shRNA expression were reported in several species including chicken (10, 11), bovine (12,13), and pig (14). Here, we have cloned two sheep U6 promoters and analyzed their potential use as shRNA expression vectors.…”

Section: Cloning and Functional Analysis Of Sheep U6 Promotersmentioning

confidence: 98%

Cloning and Functional Analysis of Sheep U6 Promoters

Hazi

et al. 2011

Animal Biotechnology

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Gene silencing mediated by small interfering RNA has become a powerful biological tool for the regulation of gene expression. In order to develop an effective short hairpin RNA (shRNA) expression vector, specifically for use in sheep species, we have identified two sheep U6 promoters based on the highly conserved polymerase III promoter elements. Promoter activity was measured by U6 promoter-driven shRNA to suppress enhanced green fluorescent protein (EGFP) expression. The knock down assay demonstrated that the two sheep U6 promoters and mouse U6 promoter induced a similar level of EGFP knockdown. These results suggest that the two sheep U6 promoters could efficiently drive shRNA expression for gene silencing and may have applications in RNAi-based sheep research.

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“…However, the human H1 promoter is usable in both human and mouse cells, while the mouse U6 promoter is restricted to mouse cells [80]. Lambeth et al [81] identified a bovine U6 promoter that was more potent than its mouse variant. To a lesser extent, the human 7SK promoter is gaining a lot of popularity due to its stronger nature compared to U6 [82].…”

Section: Rna Polymerase III Type 3 Promotersmentioning

confidence: 98%

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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Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

Cited by 24 publications

References 11 publications

Short Hairpin RNA-Mediated Gene Silencing

Short Hairpin RNA-Mediated Gene Silencing

Cloning and Functional Analysis of Sheep U6 Promoters

In Search of the Most Suitable Lentiviral shRNA System

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