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.

] Division of Medical Biotechnology Paul Ehrlich Institute Langen Germany [2]

] Friedrich Loeffler Institut Institut für Nutztiergenetik Neustadt Germany [2]

Biological Research Centre Hungarian Academy of Sciences Szeged Hungary

Department of Physiology Medical College of Wisconsin Milwaukee Wisconsin USA

Friedrich Loeffler Institut Institut für Nutztiergenetik Neustadt Germany

Institute of Laboratory Animal Science University of Veterinary Medicine Vienna Vienna Austria

Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

Max Delbrück Center for Molecular Medicine Berlin Germany

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FASEB J. 2013 Mar;27(3):930-41 PubMed

Gastroenterology. 2012 Nov;143(5):1173-1175.e7 PubMed

FASEB J. 2006 Jun;20(8):1200-2 PubMed

Hum Gene Ther. 2005 Nov;16(11):1241-6 PubMed

Nat Protoc. 2011 Sep 08;6(10):1521-35 PubMed

Stem Cells Dev. 2013 Jan 1;22(1):124-35 PubMed

Transgenic Res. 2013 Dec;22(6):1107-18 PubMed

Mob DNA. 2010 Dec 07;1(1):25 PubMed

Transgenic Res. 2011 Jun;20(3):533-45 PubMed

Elife. 2013 Jun 18;2:e00668 PubMed

Biol Reprod. 2011 Apr;84(4):723-33 PubMed

Physiol Genomics. 2007 Oct 22;31(2):159-73 PubMed

J Reprod Dev. 2007 Dec;53(6):1217-26 PubMed

Stem Cells Dev. 2013 Feb 1;22(3):473-82 PubMed

Nature. 1982 Aug 12;298(5875):623-8 PubMed

J Biol Chem. 2009 Jun 26;284(26):17634-40 PubMed

Mol Ther. 2006 Jan;13(1):59-66 PubMed

Nat Protoc. 2007;2(11):2910-7 PubMed

PLoS One. 2011;6(8):e23573 PubMed

Cell. 1997 Nov 14;91(4):501-10 PubMed

Cell Res. 2013 Jan;23(1):162-6 PubMed

Transgenic Res. 2012 Jun;21(3):605-18 PubMed

PLoS One. 2012;7(10):e46646 PubMed

PLoS One. 2011;6(11):e27563 PubMed

Mol Reprod Dev. 2011 Oct-Nov;78(10-11):879-91 PubMed

Cell Stem Cell. 2012 Jun 14;10(6):753-758 PubMed

Nat Protoc. 2014 Apr;9(4):794-809 PubMed

Mol Ther. 2011 Aug;19(8):1499-510 PubMed

Nat Protoc. 2014 Apr;9(4):773-93 PubMed

Biotechniques. 2009 Nov;47(5):959-68 PubMed

Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):10993-8 PubMed

Nucleic Acids Res. 2012 Aug;40(14):6693-712 PubMed

Transgenic Res. 2011 Oct;20(5):1125-37 PubMed

Nat Methods. 2009 Jun;6(6):415-22 PubMed

Hum Mol Genet. 2010 Oct 15;19(20):3983-94 PubMed

Stem Cells. 2011 Oct;29(10):1640-3 PubMed

J Cardiovasc Transl Res. 2011 Apr;4(2):121-30 PubMed

J Mol Cell Biol. 2009 Oct;1(1):46-54 PubMed

Mol Ther. 2004 Feb;9(2):292-304 PubMed

Science. 2008 Sep 26;321(5897):1837-41 PubMed

Nat Biotechnol. 1997 Oct;15(10):965-70 PubMed

Transgenic Res. 2009 Aug;18(4):545-58 PubMed

Mol Ther. 2010 Jun;18(6):1200-9 PubMed

Methods Mol Biol. 2012;859:229-40 PubMed

FEBS Lett. 2004 Jul 30;571(1-3):233-6 PubMed

Trends Biotechnol. 2012 Jul;30(7):386-93 PubMed

Nat Genet. 2009 Jun;41(6):753-61 PubMed

Reprod Fertil Dev. 2012;25(1):94-102 PubMed

Cell Stem Cell. 2013 Mar 7;12(3):271-4 PubMed

Biotechniques. 2012 May 01;52(5): PubMed

EMBO Rep. 2003 Nov;4(11):1054-60 PubMed

Nature. 2009 Apr 30;458(7242):1201-4 PubMed

Prev Vet Med. 2011 Nov 1;102(2):146-56 PubMed

FASEB J. 2011 Dec;25(12):4088-107 PubMed

Sci Transl Med. 2013 Jan 2;5(166):166ra1 PubMed

Mol Ther. 2012 Oct;20(10):1852-62 PubMed

Nucleic Acids Res. 2012 Oct;40(19):e150 PubMed

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