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Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

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Item Type:Article
Title:Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons
Creators Name:Ivics, Z. and Mátés, L. and Yau, T.Y. and Landa, V. and Zidek, V. and Bashir, S. and Hoffmann, O.I. and Hiripi, L. and Garrels, W. and Kues, W.A. and Boesze, Z. and Geurts, A. and Pravenec, M. and Rülicke, T. and Izsvák, Z.
Abstract:We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.
Keywords:Binding Sites, DNA Transposable Elements, Gene Transfer Techniques, Genetically Modified Animals, Germ Cells, Inbred C57BL Mice, Inbred F344 Rats, Microinjections, Transgenes, Transgenic Mice, Transgenic Rats, Transposases, Animals, Mice, Rats, Rodentia
Source:Nature Protocols
ISSN:1754-2189
Publisher:Nature Publishing Group (U.K.)
Volume:9
Number:4
Page Range:773-793
Date:April 2014
Official Publication:https://doi.org/10.1038/nprot.2014.008
PubMed:View item in PubMed

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