Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

Zoltán Ivics, Zsuzsanna Izsvák, Gerardo Medrano, Karen M. Chapman, F. Kent Hamra

Research output: Contribution to journalArticle

24 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1521-1535
Number of pages15
JournalNature Protocols
Volume6
Issue number10
DOIs
StatePublished - Oct 1 2011

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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