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Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Z. Ivics, L. Mátés, TY. Yau, V. Landa, V. Zidek, S. Bashir, OI. Hoffmann, L. Hiripi, W. Garrels, WA. Kues, Z. Bösze, A. Geurts, M. Pravenec, T. Rülicke, Z. Izsvák,

. 2014 ; 9 (4) : 773-93.

Jazyk angličtina Země Anglie, Velká Británie

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc15023543
E-zdroje Online Plný text

NLK ProQuest Central od 2006-06-01 do 2017-12-31
Medline Complete (EBSCOhost) od 2013-03-01 do 2015-12-31
Health & Medicine (ProQuest) od 2006-06-01 do 2017-12-31

Odkazy

PubMed 24625778
DOI 10.1038/nprot.2014.008
Knihovny.cz E-zdroje

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.

Citace poskytuje Crossref.org

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