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| Item Type: | Article |
|---|---|
| Title: | Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons |
| Creators Name: | Ivics, Z., Mátés, L., Yau, T.Y., Landa, V., Zidek, V., Bashir, S., Hoffmann, O.I., Hiripi, L., Garrels, W., Kues, W.A., Boesze, Z., Geurts, A., Pravenec, M., Rülicke, T. and Izsvák, Z. |
| 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. |
| Keywords: | Binding Sites, DNA Transposable Elements, Gene Transfer Techniques, Genetically Modified Animals, Germ Cells, Inbred C57BL Mice, Inbred F344 Rats, Microinjections, Transgenes, Transgenic Mice, Transgenic Rats, Transposases, Animals, Mice, Rats, Rodentia |
| Source: | Nature Protocols |
| ISSN: | 1754-2189 |
| Publisher: | Nature Publishing Group |
| Volume: | 9 |
| Number: | 4 |
| Page Range: | 773-793 |
| Date: | April 2014 |
| Official Publication: | https://doi.org/10.1038/nprot.2014.008 |
| PubMed: | View item in PubMed |
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