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Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. Nat Protoc 2014 Apr;9(4):810-27

Date

03/15/2014

Pubmed ID

24625780

DOI

10.1038/nprot.2014.010

Scopus ID

2-s2.0-84897143787   52 Citations

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.

Author List

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

Author

Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Animals, Genetically Modified
DNA Transposable Elements
Female
Gene Transfer Techniques
Genetic Vectors
Genome
Germ Cells
Male
Microinjections
Swine
Transposases