Tuning silence: conditional systems for RNA interference

Wiznerowicz, Maciej; Szulc, Jolanta; Trono, Didier

doi:10.1038/nmeth914

Cited by 110 publications

(90 citation statements)

References 40 publications

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“…Previous shRNA mir designs demonstrated that inclusion of spacer sequence between the upstream promoter and the miRNA cassette greatly increased RNAi-based transcript depletion. 4,45,55 This increase cannot simply be a factor of distance, as CMV GIN possessed the greater distance between transcriptional start site and shRNA mir cassette, whereas the bidirectional CMV design produced more substantive RNA depletion. Within bicistronic expression systems, IRES sequences have well-characterized inhibitory effects on protein production.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3][4] Because of their ability to stably transduce both dividing progenitor and non-dividing terminally differentiated cells, lentiviral vectors have proven an efficient means of gene delivery both in vitro and in vivo. [5][6][7][8] As such, a diverse range of lentiviral tools have been developed for genetic modification of mammalian cells, animal models of disease, agricultural livestock and human patients.…”

Section: Introductionmentioning

confidence: 99%

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A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells

Golding

Mann

2011

Gene Ther

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The two main challenges facing retroviral transgenesis are variable expression and epigenetic silencing. Although modern lentiviral vectors incorporate several elements to increase transgene expression and reduce position effect variegation and silencing, therapeutic research in stem cells, as well as production of transgenic animals, is still hampered by these two key problems. On the basis of recent studies demonstrating the chromatin insulating properties of divergent promoters, we sought to develop a bidirectional lentiviral vector with which to conduct RNA interference (RNAi)-based genetic screens in embryonic and extraembryonic stem cells. To this end, we designed and tested a series of synthetic bidirectional promoters, combining the mouse phosphoglycerate kinase 1 (Pgk1) promoter with other strong mammalian and viral promoters. Here, we demonstrate that a back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters provided a substantive increase in both transgene expression and RNAi-based transcript depletion as compared with previous designs and other promoter combinations. Using this vector, we were able to achieve stable and robust depletion of a transfected luciferase reporter, as well as an endogenous non-coding RNA. The described constructs are an improved transgene delivery system capable of conducting RNAi screens in stem cells at single copy.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells

Golding

Mann

2011

Gene Ther

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“…The methods now available range from direct transfection of synthetic siRNAs to the use of plasmid, retroviral, lentiviral, and adenoviral vectors to introduce genes encoding shRNAs or microRNA precursors under the control of RNA polymerase III or RNA polymerase II promoters. Specific strategies for knocking down the expression of a gene by RNAi are beyond the scope of this article, but they have been the topic of hundreds of articles (for review, see Elbashir et al 2002;Amarzguioui et al 2006;Cullen 2006;Pei and Tuschl 2006;Sen and Blau 2006;Snøve and Rossi 2006;Wiznerowicz et al 2006;Kim and Rossi 2007;Svoboda 2007;Paddison 2008).…”

Section: Loss-of-function Studies By Gene Disruption or Rna Interferencementioning

confidence: 99%

Confirming the Functional Importance of a Protein–DNA Interaction

Carey¹,

Peterson²,

Smale³

2012

Cold Spring Harb Protoc

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Identifying DNA-binding proteins that interact with a control region of interest has become quite straightforward. However, the functional relevance of a given protein-DNA interaction is difficult to establish. The hypothesis that an interaction is relevant can be tested by several different experiments, 12 of which are outlined in this article. It must be remembered that none of these experiments by itself is conclusive. The information gained from each approach is described and explanations are given for why each yields useful but inconclusive results. The approaches vary widely with respect to the amount of effort required and the quality of information obtained.

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“…However, the high and constitutive transcriptional activity of polIII promoters can lead to the saturation of the endogenous miRNA processing factors, such as RNA induced silencing complex (RISC) and exportin-5, that are required for efficient shRNA processing, which in turn can lead to cytotoxicity and tissue damage. 42 Although additional regulatory elements can be combined with polIII promoter elements to restrict their transcriptional abundance, 43 such approaches typically rely on the coexpression of additional protein elements that may well be recognized as foreign by the ultimate host.…”

Section: Integrating Vectors May Offer Advantages For Stem Cell Targementioning

confidence: 99%