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Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

Nature Protocols volume 6, pages 15211535 (2011) | Download Citation

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.

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Acknowledgements

Work in the authors' laboratories has been supported by EU FP6 (INTHER) and EU FP7 (PERSIST and InduStem), and grants from the Deutsche Forschungsgemeinschaft SPP1230 'Mechanisms of gene vector entry and persistence', and from the Bundesministerium fur Bildung und Forschung (NGFN-2, NGFNplus—ENGINE). Methods for experimental manipulation of rat spermatogonia in culture and for production of mutant rats using spermatogonia were supported by National Institutes of Health grants R21RR023958 from the National Center for Research Resources and RO1HD036022, RO1HD053889, RO1HD061575 from the National Institute of Child Health and Human Development to F.K. Hamra; and by the Cecil H. & Ida Green Center for Reproductive Biology Sciences at the University of Texas Southwestern Medical Center in Dallas.

Author information

Affiliations

  1. Max Delbrück Center for Molecular Medicine, Berlin, Germany.

    • Zoltán Ivics
    •  & Zsuzsanna Izsvák
  2. University of Debrecen, Debrecen, Hungary.

    • Zoltán Ivics
    •  & Zsuzsanna Izsvák
  3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Gerardo Medrano
    • , Karen M Chapman
    •  & F Kent Hamra
  4. Cecil H. & Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

    • Gerardo Medrano
    • , Karen M Chapman
    •  & F Kent Hamra

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Contributions

F.K.H. and K.M.C. provided concepts on applications of mutant SSC libraries and the use of DAZL-deficient rats as recipient founders; established methods for in vitro culture, gene delivery, clonal selection and transplantation of rat SSC lines for production of mutant rats; supervised the project; and wrote the paper. G.M. produced and edited the movie of F.K.H. showing spermatogonial transplantation procedure. Z. Ivics and Z. Izsvák established SB gene-trap mutagenesis in cultured cells, provided the concept of applying transposon mutagenesis in SSCs, supervised the project and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Zoltán Ivics or Zsuzsanna Izsvák or F Kent Hamra.

Supplementary information

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    Supplementary Video 1

    Injection of transfected spermatogonial stem cells into the seminiferous tubules.

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DOI

https://doi.org/10.1038/nprot.2011.378

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