title: Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons.
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.
authors.affiliation: Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
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: Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Landa
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: Max Delbrück Center for Molecular Medicine, Berlin, Germany.
authors.forename: Sanum
authors.initials: S
authors.lastname: Bashir
authors.affiliation: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: Orsolya I
authors.initials: OI
authors.lastname: Hoffmann
authors.affiliation: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Hiripi
authors.affiliation: Friedrich Loeffler Institut, Institut für Nutztiergenetik, Neustadt, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Garrels
authors.affiliation: Friedrich Loeffler Institut, Institut für Nutztiergenetik, Neustadt, Germany.
authors.forename: Wilfried A
authors.initials: WA
authors.lastname: Kues
authors.affiliation: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Bösze
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: 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; D001665:Binding Sites; D004251:DNA Transposable Elements; D005260:Female; D018014:Gene Transfer Techniques; D005854:Germ Cells; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008845:Microinjections; D051381:Rats; D011916:Rats, Inbred F344; D055647:Rats, Transgenic; D012377:Rodentia / Q000235:genetics*; D019076:Transgenes; D019895:Transposases / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases