title: Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells.
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.
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin, Germany. zivics@mdc-berlin.de
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
authors.forename: Gerardo
authors.initials: G
authors.lastname: Medrano
authors.forename: Karen M
authors.initials: KM
authors.lastname: Chapman
authors.forename: F Kent
authors.initials: FK
authors.lastname: Hamra
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D018628:Gene Dosage; D017930:Genes, Reporter; D060005:Genotyping Techniques; D008297:Male; D016254:Mutagenesis, Insertional / Q000379:methods*; D051381:Rats; D013093:Spermatogonia / Q000166:cytology*; D014162:Transfection / Q000379:methods
chemical_list: D004251:DNA Transposable Elements