Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-04

AUTHORS

Zoltán Ivics, László Hiripi, Orsolya I Hoffmann, Lajos Mátés, Tien Yin Yau, Sanum Bashir, Vaclav Zidek, Vladimír Landa, Aron Geurts, Michal Pravenec, Thomas Rülicke, Zsuzsanna Bösze, Zsuzsanna Izsvák

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. More... »

PAGES

794-809

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2014.009

DOI

http://dx.doi.org/10.1038/nprot.2014.009

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1043144608

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24625779


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