Faculty Collaboration Database

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 2014 Apr;9(4):794-809

Date

03/15/2014

Pubmed ID

24625779

DOI

10.1038/nprot.2014.009

Scopus ID

2-s2.0-84897143633 (requires institutional sign-in at Scopus site)   50 Citations

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.

Author List

Ivics Z, Hiripi L, Hoffmann OI, Mátés L, Yau TY, Bashir S, Zidek V, Landa V, Geurts A, Pravenec M, Rülicke T, Bösze Z, Izsvák Z

Author

Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Animals, Genetically Modified
DNA Transposable Elements
Female
Gene Transfer Techniques
Germ Cells
Male
Microinjections
Rabbits
Time Factors
Transposases