Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

 

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells
Zoltán Ivics,1, 2 Zsuzsanna Izsvák,1, 2 Gerardo Medrano,3, 4 Karen M Chapman3, 4 & F Kent Hamra3, 4

Nature Protocols Volume: 6, Pages: 1521�1535 Year published: (2011) DOI: doi:10.1038/nprot.2011.378
Published online 08 September 2011

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 F1 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 F1 offspring that harbor genomic SB gene-trap insertions takes 5�6 months.

原文：http://www.nature.com/nprot/journal/v6/n10/abs/nprot.2011.378.html