Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Ivics, Zoltán and Mátés, Lajos and Yau, Tien Yin and Landa, Vladimír and Zidek, Vaclav and Hoffmann, Orsolya I. and Hiripi, László and Bősze, Zsuzsanna and Izsvák, Zsuzsanna (2014) Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. NATURE PROTOCOLS, 9 (4). pp. 773-793. ISSN 1754-2189

Text IvicsZNatProtRodent.pdf Restricted to Repository staff only Download (1MB) | Request a copy

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 mRNARNARNA encoding the SB100X hyperactive transposase, together with circular plasmid DNANA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNARNARNA, 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.

Actions (login required)

Edit Item