title: Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons.
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.
authors.affiliation: 1] Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany. [2].
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
authors.affiliation: 1] Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Neustadt, Germany. [2].
authors.forename: Wiebke
authors.initials: W
authors.lastname: Garrels
authors.affiliation: Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Lajos
authors.initials: L
authors.lastname: Mátés
authors.affiliation: Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Tien Yin
authors.initials: TY
authors.lastname: Yau
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Sanum
authors.initials: S
authors.lastname: Bashir
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Vaclav
authors.initials: V
authors.lastname: Zidek
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Landa
authors.affiliation: Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: Aron
authors.initials: A
authors.lastname: Geurts
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Pravenec
authors.affiliation: Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: Thomas
authors.identifier: 0000000221219496
authors.initials: T
authors.lastname: Rülicke
authors.affiliation: Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Neustadt, Germany.
authors.forename: Wilfried A
authors.initials: WA
authors.lastname: Kues
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004251:DNA Transposable Elements; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016678:Genome; D005854:Germ Cells; D008297:Male; D008845:Microinjections; D013552:Swine / Q000235:genetics*; D019895:Transposases
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases