Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons
Jazyk angličtina Země Anglie, Velká Británie Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
24625779
DOI
10.1038/nprot.2014.009
PII: nprot.2014.009
Knihovny.cz E-zdroje
- MeSH
- časové faktory MeSH
- geneticky modifikovaná zvířata * MeSH
- králíci MeSH
- mikroinjekce MeSH
- technika přenosu genů * MeSH
- transposasy genetika MeSH
- transpozibilní elementy DNA * MeSH
- zárodečné buňky MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- transposasy MeSH
- transpozibilní elementy DNA * MeSH
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.
Agricultural Biotechnology Center Gödöllö Hungary
Biological Research Centre Hungarian Academy of Sciences Szeged Hungary
Department of Physiology Medical College of Wisconsin Milwaukee Wisconsin USA
Division of Medical Biotechnology Paul Ehrlich Institute Langen Germany
Institute of Laboratory Animal Science University of Veterinary Medicine Vienna Vienna Austria
Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic
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