Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 2014 Apr;9(4):773-93
Date
03/15/2014Pubmed ID
24625778DOI
10.1038/nprot.2014.008Scopus ID
2-s2.0-84897127002 (requires institutional sign-in at Scopus site) 55 CitationsAbstract
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.
Author List
Ivics Z, Mátés L, Yau TY, Landa V, Zidek V, Bashir S, Hoffmann OI, Hiripi L, Garrels W, Kues WA, Bösze Z, Geurts A, Pravenec M, Rülicke T, Izsvák ZAuthor
Aron Geurts PhD Professor in the Physiology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAnimals, Genetically Modified
Binding Sites
DNA Transposable Elements
Female
Gene Transfer Techniques
Germ Cells
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Microinjections
Rats
Rats, Inbred F344
Rats, Transgenic
Rodentia
Transgenes
Transposases
