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Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

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Item Type:Article
Title:Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons
Creators Name:Ivics, Z. and Hiripi, L. and Hoffmann, O.I. and Mátés, L. and Yau, T.Y. and Bashir, S. and Zidek, V. and Landa, V. and Geurts, A. and Pravenec, M. and Rülicke, T. and Bösze, Z. and Izsvák, Z.
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 mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA 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.
Keywords:DNA Transposable Elements, Gene Transfer Techniques, Genetically Modified Animals, Germ Cells, Microinjections, Time Factors, Transposases, Animals, Rabbits
Source:Nature Protocols
ISSN:1754-2189
Publisher:Nature Publishing Group (U.K.)
Volume:9
Number:4
Page Range:794-809
Date:April 2014
Official Publication:https://doi.org/10.1038/nprot.2014.009
PubMed:View item in PubMed

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