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Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

Item Type:Article
Title:Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells
Creators Name:Ivics, Z. and Izsvak, Z. and Medrano, G. and 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 (U.K.)
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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