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.
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Acknowledgements
The research of M.P. and V.L. was supported by grant no. TA02010013 from Technological Agency of the Czech Republic (TACR) and grants LH12061 and LL1204 (in the European Research Council (ERC) CZ program) from the Ministry of Education, Youth and Sports of the Czech Republic. Financial support from the Deutsche Forschungsgemeinschaft (grants KU 1586/2-1 and IV 21/6-1) to W.A.K. and Z. Ivics, and from the Austrian Genome Research Programme GEN-AU II and III (Austromouse) to T.R. is gratefully acknowledged.
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Design of study: T.R., M.P., A.G., Z. Ivics, Z. Izsvák, L.M.; performance of experiments: L.M., T.Y.Y., S.B., V.Z., V.L., A.G.; evaluation of data: W.A.K., Z. Ivics, W.G., O.I.H., L.H. and Z.B.; writing of manuscript: Z. Ivics, T.R., M.P., L.M., V.L. and Z. Izsvák.
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Ivics, Z., Mátés, L., Yau, T. et al. Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 9, 773–793 (2014). https://doi.org/10.1038/nprot.2014.008
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DOI: https://doi.org/10.1038/nprot.2014.008
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