Journal
NATURE PROTOCOLS
Volume 6, Issue 10, Pages 1521-1535Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.378
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Funding
- EU
- Deutsche Forschungsgemeinschaft [SPP1230]
- Bundesministerium fur Bildung und Forschung
- National Center for Research Resources, National Institutes of Health [R21RR023958]
- National Institute of Child Health and Human Development, National Institutes of Health [RO1HD036022, RO1HD053889, RO1HD061575]
- Cecil H. & Ida Green Center for Reproductive Biology Sciences at the University of Texas Southwestern Medical Center in Dallas
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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.
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