Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

被引：47

作者：

Ivics, Zoltan ^{[1
]}

Hiripi, Laszlo ^{[2
]}

Hoffmann, Orsolya I. ^{[2
]}

Mates, Lajos ^{[3
]}

Yau, Tien Yin ^{[4
]}

Bashir, Sanum ^{[5
]}

Zidek, Vaclav ^{[6
]}

Landa, Vladimir ^{[6
]}

Geurts, Aron ^{[7
]}

Pravenec, Michal ^{[6
]}

Ruelicke, Thomas ^{[4
]}

Boesze, Zsuzsanna ^{[2
]}

Izsvak, Zsuzsanna ^{[5
]}

机构：

[1] Paul Ehrlich Inst, Div Med Biotechnol, Langen, Germany

[2] Agr Biotechnol Ctr, H-2101 Godollo, Hungary

[3] Hungarian Acad Sci, Biol Res Ctr, H-6701 Szeged, Hungary

[4] Univ Vet Med Vienna, Inst Lab Anim Sci, Vienna, Austria

[5] Max Delbruck Ctr Mol Med, Berlin, Germany

[6] Acad Sci Czech Republ, Inst Physiol, Prague, Czech Republic

[7] Med Coll Wisconsin, Dept Physiol, Milwaukee, WI 53226 USA

来源：

NATURE PROTOCOLS | 2014年 / 9卷 / 04期

关键词：

NUCLEAR TRANSFER; HUMAN-CELLS; MEDIATED TRANSGENESIS; LENTIVIRAL VECTORS; STEM-CELLS; EMBRYOS; GENE; VERTEBRATES; INSERTIONS; EXPRESSION;

D O I：

10.1038/nprot.2014.009

中图分类号：

Q5 [生物化学];

学科分类号：

070307 [化学生物学];

摘要：

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 similar to 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.

引用

页码：794 / 809

页数：16

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