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Nat Protoc. 2014 Apr;9(4):773-93. doi: 10.1038/nprot.2014.008. Epub 2014 Mar 13.

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons.

Author information

1
Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
2
Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
3
Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria.
4
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
5
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
6
Agricultural Biotechnology Center, Gödöllö, Hungary.
7
Friedrich Loeffler Institut, Institut für Nutztiergenetik, Neustadt, Germany.
8
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.

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.

PMID:
24625778
DOI:
10.1038/nprot.2014.008
[Indexed for MEDLINE]

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