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WIREs Dev Biol
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RNAi in the mouse: rapid and affordable gene function studies in a vertebrate system

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The addition of RNA interference (RNAi) to the mammalian genomic toolbox has significantly expanded our ability to use higher‐order models in studies of development and disease. The mouse, in particular, has benefited most from RNAi technology. Unique combinations of RNAi vectors and delivery methods now offer a broad platform for gene silencing in transgenic mice, enabling the design of new physiologically relevant models. The era of RNAi mice has accelerated the pace of genetic study and made high‐throughput screens not only feasible but also affordable. WIREs Dev Biol 2015, 4:45–57. doi: 10.1002/wdev.164 This article is categorized under: Technologies > Perturbing Genes and Generating Modified Animals
Pooled in vivo RNAi screens are a newly available approach for functional genetic studies. Experimental groups may be designed to compare different genetic models (i.e., wildtype vs. mutant) or the same model at different time points. If the study consists of exactly two experimental groups, microarray analysis is a quantitative option. However, for two or more experimental groups, deep sequencing analysis is required. This sophisticated technique has become the new gold standard for thorough transgene identification, particularly in negative‐selection screens where the constructs of interest may be depleted.
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RNAi constructs for plasmid‐ and viral‐based gene knockdown are highly modular, consisting of a primary promoter driving shRNA expression with the option for additional reporter or selection genes and regulatory elements for spatio‐temporal control. Vector backbones are available through public repositories (Addgene) or commercial vendors for quick assembly with specific shRNA sequences. Generally, a wider variety of construct options are available for Pol III‐based expression since these promoters have been utilized the longest (and were the basis of first‐generation shRNA libraries). Plasmids can either be delivered directly or further packaged into viral particles.
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Germline RNAi can be achieved using viral‐ or plasmid‐based vectors. Here, early embryos are transduced or transfected in vitro before transplantation into a pseudopregnant surrogate. While ESC‐based transgenesis results in mosaic progeny, pronuclear and perivitelline space injections most commonly result in hemizygotes carrying a distribution of transgene copy numbers. Somatic RNAi can be achieved in both late‐stage embryos (e9.5 or later) and the adult mouse. These approaches are most suitable for targeted or tissue‐specific rather than ubiquitous knockdown.
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The endogenous RNAi pathway can be co‐opted at several different levels by exogenously delivered constructs. Choosing which construct to deliver will determine the efficiency and longevity of gene knockdown. Pol III shRNA transcription enables highly potent knockdown in a wide variety of cell types, but its prolific transcription can oversaturate the exportin‐mediated traffic, resulting in cytotoxicity. Unlike Pol III shRNA, Pol II shRNA transcription is subject to regulation by the Drosha‐DGC8 complex, which mitigates the risk of excess transcripts oversaturating nuclear export. In addition, Pol II‐based constructs can be designed with cell‐specific promoters for spatial control since they utilize endogenous RNAi machinery. Both Pol II and Pol III‐based shRNA constructs have been successfully used for long‐term stable knockdown. Synthetic siRNA, on the other hand, enables transient knockdown and is typically found in drug‐delivery based applications as a therapeutic agent.
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