Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

Ivics, Zoltán and Hiripi, László and Hoffmann, Orsolya I. and Mátés, Lajos and Yau, Tien Yin and Bősze, Zsuzsanna and Izsvák, Zsuzsanna (2014) Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons. NATURE PROTOCOLS, 9 (4). pp. 794-809. ISSN 1754-2189

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

Abstract

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNARNARNA encoding the SB100X hyperactive transposase together with plasmid DNANA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNARNARNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ~2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) 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