title: Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons.
abstract: The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) 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: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Hiripi
authors.affiliation: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: Orsolya I
authors.initials: OI
authors.lastname: Hoffmann
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: Agricultural Biotechnology Center, Gödöllö, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Bösze
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; D005854:Germ Cells; D008297:Male; D008845:Microinjections; D011817:Rabbits; D013997:Time Factors; D019895:Transposases / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases