Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

Zoltán Ivics, L. Mátés, Tien Yin Yau, Vladimír Landa, Vaclav Zidek, Sanum Bashir, Orsolya I. Hoffmann, László Hiripi, Wiebke Garrels, Wilfried A. Kues, Z. Bösze, Aron Geurts, Michal Pravenec, Thomas Rülicke, Zsuzsanna Izsvák

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)773-793
Number of pages21
JournalNature Protocols
Volume9
Issue number4
DOIs
Publication statusPublished - 2014

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Transposases
Beauty
Gene Transfer Techniques
Microinjections
Transgenes
Rodentia
Animals
Plasmids
Genetically Modified Animals
Messenger RNA
Toxicity
Rats
Circular DNA
Binding Sites
Protein Biosynthesis
DNA
Oocytes
Enzymes
Injections

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. / Ivics, Zoltán; Mátés, L.; Yau, Tien Yin; Landa, Vladimír; Zidek, Vaclav; Bashir, Sanum; Hoffmann, Orsolya I.; Hiripi, László; Garrels, Wiebke; Kues, Wilfried A.; Bösze, Z.; Geurts, Aron; Pravenec, Michal; Rülicke, Thomas; Izsvák, Zsuzsanna.

In: Nature Protocols, Vol. 9, No. 4, 2014, p. 773-793.

Research output: Contribution to journalArticle

Ivics, Z, Mátés, L, Yau, TY, Landa, V, Zidek, V, Bashir, S, Hoffmann, OI, Hiripi, L, Garrels, W, Kues, WA, Bösze, Z, Geurts, A, Pravenec, M, Rülicke, T & Izsvák, Z 2014, 'Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons', Nature Protocols, vol. 9, no. 4, pp. 773-793. https://doi.org/10.1038/nprot.2014.008
Ivics, Zoltán ; Mátés, L. ; Yau, Tien Yin ; Landa, Vladimír ; Zidek, Vaclav ; Bashir, Sanum ; Hoffmann, Orsolya I. ; Hiripi, László ; Garrels, Wiebke ; Kues, Wilfried A. ; Bösze, Z. ; Geurts, Aron ; Pravenec, Michal ; Rülicke, Thomas ; Izsvák, Zsuzsanna. / Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. In: Nature Protocols. 2014 ; Vol. 9, No. 4. pp. 773-793.
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