Single copy shRNA configuration for ubiquitous gene knockdown in mice

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Abstract

RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo. However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo. Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches.

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Most cited references 29

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Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.

Patrick Paddison, Amy Caudy, Emily Bernstein … (2002)

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

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Cre-lox-regulated conditional RNA interference from transgenes.

Andrea Ventura, Alexander Meissner, Christopher P. Dillon … (2004)

We have generated two lentiviral vectors for conditional, Cre-lox-regulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals.

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Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

Philippe Soriano, Klaus Friedrich (1991)

A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.

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Author and article information

Journal

Journal ID (nlm-ta): Nucleic Acids Res

Journal ID (publisher-id): Nucleic Acids Research

Title: Nucleic Acids Research

Publisher: Oxford University Press

ISSN (Print): 0305-1048

ISSN (Electronic): 1362-4962

Publication date Collection: 2005

Publication date (Print): 2005

Publication date (Electronic): 14 April 2005

Volume: 33

Issue: 7

Page: e67

Affiliations

¹Artemis Pharmaceuticals GmbH Neurather Ring 1, 51063 Cologne, Germany

²Department of Mouse Genetics and Metabolism and Center for Molecular Medicine (CMMC), Institute for Genetics Germany

³Klinik II and Poliklinik für Innere Medizin, University of Cologne Germany

⁴Department of Applied Natural Sciences, University of Applied Science Gelsenkirchen August-Schmidt-Ring 10, 45665 Recklinghausen, Germany

Author notes

^*To whom correspondence should be addressed. Tel: +49 221 9645342; Fax: +49 221 964 5321; Email: j.seibler@ 123456artemispharma.de

Present address: Ralf Kühn, GSF Research Center, Institute for Developmental Genetics, Neuherberg/Munich, Germany

Article

DOI: 10.1093/nar/gni065

PMC ID: 1079974

PubMed ID: 15831785

SO-VID: 4b9979cf-df2e-48ab-9673-80f76d1c6b40

License:

The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@ 123456oupjournals.org

History

Date received : 24 January 2005

Date revision received : 18 March 2005

Date accepted : 18 March 2005

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