Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

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

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase, together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer (SCNT), 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. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in 1 d; generation of germline-transgenic lines by using this protocol takes ∼1 year.

Original languageEnglish
Pages (from-to)810-827
Number of pages18
JournalNature Protocols
Volume9
Issue number4
DOIs
Publication statusPublished - 2014

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Beauty
Gene Transfer Techniques
Microinjections
Transposases
Transgenes
Swine
Animals
Plasmids
Zygote
Genetically Modified Animals
Embryo Transfer
Toxicity
Genes
Binding Sites
Biomedical Research
Cytoplasm
Animal Models
Genome
Injections
Pharmaceutical Preparations

ASJC Scopus subject areas

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

Cite this

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. / Ivics, Zoltán; Garrels, Wiebke; Mátés, L.; Yau, Tien Yin; Bashir, Sanum; Zidek, Vaclav; Landa, Vladimír; Geurts, Aron; Pravenec, Michal; Rülicke, Thomas; Kues, Wilfried A.; Izsvák, Zsuzsanna.

In: Nature Protocols, Vol. 9, No. 4, 2014, p. 810-827.

Research output: Contribution to journalArticle

Ivics, Z, Garrels, W, Mátés, L, Yau, TY, Bashir, S, Zidek, V, Landa, V, Geurts, A, Pravenec, M, Rülicke, T, Kues, WA & Izsvák, Z 2014, 'Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons', Nature Protocols, vol. 9, no. 4, pp. 810-827. https://doi.org/10.1038/nprot.2014.010
Ivics, Zoltán ; Garrels, Wiebke ; Mátés, L. ; Yau, Tien Yin ; Bashir, Sanum ; Zidek, Vaclav ; Landa, Vladimír ; Geurts, Aron ; Pravenec, Michal ; Rülicke, Thomas ; Kues, Wilfried A. ; Izsvák, Zsuzsanna. / Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. In: Nature Protocols. 2014 ; Vol. 9, No. 4. pp. 810-827.
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