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 Citations (Scopus)

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 2011

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Beauty
Mutagenesis
Stem cells
Rats
Stem Cells
Genes
Insertional Mutagenesis
Molecular Sequence Annotation
Microbial Drug Resistance
Germ Cells
Libraries
Transfection
Vertebrates
Testis
Cultured Cells
Plasmids
Clone Cells
Alleles
Economics
Genome

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells. / Ivics, Zoltán; Izsvák, Zsuzsanna; Medrano, Gerardo; Chapman, Karen M.; Hamra, F. Kent.

In: Nature Protocols, Vol. 6, No. 10, 10.2011, p. 1521-1535.

Research output: Contribution to journalArticle

Ivics, Zoltán ; Izsvák, Zsuzsanna ; Medrano, Gerardo ; Chapman, Karen M. ; Hamra, F. Kent. / Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells. In: Nature Protocols. 2011 ; Vol. 6, No. 10. pp. 1521-1535.
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