Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Zoltán Ivics, Lajos Mátés, Tien Yin Yau, Vladimír Landa, Vaclav Zidek, Sanum Bashir, Orsolya I. Hoffmann, László Hiripi, Wiebke Garrels, Wilfried A. Kues, Zsuzsanna Bösze, Aron Geurts, Michal Pravenec, Thomas Rülicke, Zsuzsanna Izsvák

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39 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)773-793
Number of pages21
JournalNature Protocols
Volume9
Issue number4
DOIs
Publication statusPublished - Apr 2014

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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    Ivics, Z., Mátés, L., Yau, T. Y., Landa, V., Zidek, V., Bashir, S., Hoffmann, O. I., Hiripi, L., Garrels, W., Kues, W. A., Bösze, Z., Geurts, A., Pravenec, M., Rülicke, T., & Izsvák, Z. (2014). Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. Nature Protocols, 9(4), 773-793. https://doi.org/10.1038/nprot.2014.008