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| Item Type: | Article |
|---|---|
| Title: | Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells |
| Creators Name: | Ivics, Z., Izsvak, Z., Medrano, G., Chapman, K.M. and Hamra, F.K. |
| Abstract: | We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months. |
| Keywords: | DNA Transposable Elements, Gene Dosage, Genotyping Techniques, Insertional Mutagenesis, Reporter Genes, Spermatogonia, Transfection, Animals, Rats |
| Source: | Nature Protocols |
| ISSN: | 1745-2473 |
| Publisher: | Nature Publishing Group |
| Volume: | 6 |
| Number: | 10 |
| Page Range: | 1521-1535 |
| Date: | October 2011 |
| Official Publication: | https://doi.org/10.1038/nprot.2011.378 |
| PubMed: | View item in PubMed |
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