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.

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…In order to assess copy numbers of integrated transposons and map the genomic integration sites, a ligation-mediated PCR procedure is applied 56 . The procedure consists of a restriction enzyme digest of the genomic DNA, ligation of an oligonucleotide adapter to the ends of the fragmented DNA, PCR amplification of a transgene/genomic DNA junction in two rounds of nested PCR with primers specific to the adapter and to the ITRs of the SB transposon, and sequencing of the junctions to map the insertion to the reference genome 57 .…”

Retake the Center Stage – New Development of Rat Genetics

“…The efficiency of SB transposon-mediated mutagenesis has been remarkably improved recently, by transfecting cultured rat SSCs with transposon constructs. The gene-trap clones can then be selected by antibiotic or reporter gene expression at the SSCs culture stage, so that only offspring with the desired genotypes are generated (Izsvák et al, 2010;Ivics et al, 2011aIvics et al, , 2011b.…”

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

“…In order to assess copy numbers of integrated transposons and map the genomic integration sites, a ligation-mediated PCR procedure is applied 40 . The procedure consists of a restriction enzyme digest of the genomic DNA, ligation of an oligonucleotide adapter to the ends of the fragmented DNA, PCR amplification of a transgene/genomic DNA junction in two rounds of nested PCR with primers specific to the adapter and to the ITRs of the SB transposon, and sequencing of the junctions to map the insertion to the reference genome 41 .…”