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Nat Protoc. 2011 Sep 8;6(10):1521-35. doi: 10.1038/nprot.2011.378.

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells.

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  • 1Max Delbrück Center for Molecular Medicine, Berlin, Germany. zivics@mdc-berlin.de

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.

PMID:
21959235
[PubMed - indexed for MEDLINE]
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