Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Experimental pipeline for the generation of mutant rats by transposon mutagenesis in spermatogonial stem cells.
Figure 2: Transposon vector system for stable gene delivery.
Figure 3: Mutagenesis of genes by gene-trap transposon insertions.
Figure 4: Diagram summarizing key aspects of rat testicular anatomy and surgical materials used to transplant spermatogonial cell suspensions.
Figure 5: Generation of mutant rats by transplantation of spermatogonial stem cells harboring transposon insertions into the testes of recipient males.

References

  1. 1

    Jacob, H.J., Lazar, J., Dwinell, M.R., Moreno, C. & Geurts, A.M. Gene targeting in the rat: advances and opportunities. Trends Genet. 26, 510–518 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Izsvák, Z. et al. Generating knockout rats by transposon mutagenesis in spermatogonial stem cells. Nat. Methods 7, 443–445 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Ivics, Z. et al. Transposon-mediated genome manipulation in vertebrates. Nat. Methods 6, 415–422 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Ivics, Z. & Izsvak, Z. The expanding universe of transposon technologies for gene and cell engineering. Mob. DNA 1, 25 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Miskey, C., Izsvak, Z., Plasterk, R.H. & Ivics, Z. The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Res. 31, 6873–6881 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Zagoraiou, L. et al. In vivo transposition of Minos, a Drosophila mobile element, in mammalian tissues. Proc. Natl. Acad. Sci. USA 98, 11474–11478 (2001).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    de Wit, T. et al. Tagged mutagenesis by efficient Minos-based germ line transposition. Mol. Cell Biol. 30, 68–77 (2010).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Ding, S. et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473–483 (2005).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Wilson, M.H., Coates, C.J. & George, A.L. Jr. PiggyBac transposon-mediated gene transfer in human cells. Mol. Ther. 15, 139–145 (2007).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Kawakami, K., Shima, A. & Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl. Acad. Sci. USA 97, 11403–11408 (2000).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Dupuy, A.J., Fritz, S. & Largaespada, D.A. Transposition and gene disruption in the male germline of the mouse. Genesis 30, 82–88 (2001).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Horie, K. et al. Efficient chromosomal transposition of a Tc1/mariner-like transposon Sleeping Beauty in mice. Proc. Natl. Acad. Sci. USA 98, 9191–9196 (2001).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Keng, V.W. et al. Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat. Methods 2, 763–769 (2005).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Suzuki, N. & Withers, H.R. Exponential decrease during aging and random lifetime of mouse spermatogonial stem cells. Science 202, 1214–1215 (1978).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Hamra, F.K. et al. Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc. Natl. Acad. Sci. USA 102, 17430–17435 (2005).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Ryu, B.Y., Kubota, H., Avarbock, M.R. & Brinster, R.L. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc. Natl. Acad. Sci. USA 102, 14302–14307 (2005).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Buehr, M. et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 (2008).

    CAS  Article  PubMed  Google Scholar 

  18. 18

    Li, P. et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299–1310 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Hamra, F.K. et al. Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc. Natl. Acad. Sci. USA 99, 14931–14936 (2002).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Kanatsu-Shinohara, M. et al. Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells. Biol. Reprod. 79, 1121–1128 (2008).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Ryu, B.Y. et al. Efficient generation of transgenic rats through the male germline using lentiviral transduction and transplantation of spermatogonial stem cells. J. Androl. 28, 353–360 (2007).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Kanatsu-Shinohara, M. et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616 (2003).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Kanatsu-Shinohara, M., Toyokuni, S. & Shinohara, T. Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. Biol. Reprod. 72, 236–240 (2005).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Kanatsu-Shinohara, M. et al. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 103, 8018–8023 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Brinster, R.L. & Avarbock, M.R. Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. USA 91, 11303–11307 (1994).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Hamra, F.K. et al. Defining the spermatogonial stem cell. Dev. Biol. 269, 393–410 (2004).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Russell, W.L. et al. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc. Natl. Acad. Sci. USA 76, 5818–5819 (1979).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Driever, W. et al. A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37–46 (1996).

    CAS  PubMed  Google Scholar 

  29. 29

    Haffter, P. et al. The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36 (1996).

    CAS  PubMed  Google Scholar 

  30. 30

    Kile, B.T. et al. Functional genetic analysis of mouse chromosome 11. Nature 425, 81–86 (2003).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Smits, B.M. & Cuppen, E. Rat genetics: the next episode. Trends Genet. 22, 232–240 (2006).

    CAS  Article  PubMed  Google Scholar 

  32. 32

    Bushman, F.D. Targeting survival: integration site selection by retroviruses and LTR-retrotransposons. Cell 115, 135–138 (2003).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Jahner, D. et al. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298, 623–628 (1982).

    CAS  Article  Google Scholar 

  34. 34

    Jahner, D. & Jaenisch, R. Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity. Nature 315, 594–597 (1985).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Carlson, C.M. et al. Transposon mutagenesis of the mouse germline. Genetics 165, 243–256 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Fischer, S.E., Wienholds, E. & Plasterk, R.H. Regulated transposition of a fish transposon in the mouse germ line. Proc. Natl. Acad. Sci. USA 98, 6759–6764 (2001).

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Horie, K. et al. Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol. Cell Biol. 23, 9189–9207 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Yae, K. et al. Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth. Mol. Cell Biol. 26, 6185–6196 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Geurts, A.M. et al. Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet. 2, e156 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Kitada, K. et al. Transposon-tagged mutagenesis in the rat. Nat. Methods 4, 131–133 (2007).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Lu, B. et al. Generation of rat mutants using a coat color-tagged Sleeping Beauty transposon system. Mamm. Genome 18, 338–346 (2007).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Wang, W., Bradley, A. & Huang, Y. A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells. Genome Res. 19, 667–673 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Tong, C., Li, P., Wu, N.L., Yan, Y. & Ying, Q.L. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467, 211–213 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44

    Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    CAS  Article  Google Scholar 

  45. 45

    Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Hansen, G.M. et al. Large-scale gene trapping in C57BL/6N mouse embryonic stem cells. Genome Res. 18, 1670–1679 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48

    Stanford, W.L., Cohn, J.B. & Cordes, S.P. Gene-trap mutagenesis: past, present and beyond. Nat. Rev. Genet. 2, 756–768 (2001).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Friedrich, G. & Soriano, P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523 (1991).

    CAS  Article  PubMed  Google Scholar 

  50. 50

    Zambrowicz, B.P. et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392, 608–611 (1998).

    CAS  Article  PubMed  Google Scholar 

  51. 51

    Skarnes, W.C. et al. A public gene trap resource for mouse functional genomics. Nat. Genet. 36, 543–544 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52

    Ivics, Z., Izsvak, Z., Chapman, K.M. & Hamra, F.K. Sleeping Beauty transposon mutagenesis of the rat genome in spermatogonial stem cells. Methods 53, 356–365 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Chapman, K.M. et al. Rat spermatogonial stem cell mediated gene transfer. in Advanced Protocols for Animal Transgenesis Vol. XVI (eds. Pease, S. & Saunders, T.L.) (Humana Press, 2011).

  54. 54

    Grabundzija, I. et al. Comparative analysis of transposable element vector systems in human cells. Mol. Ther. 18, 1200–1209 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55

    Richardson, T.E., Chapman, K.M., Tenenhaus Dann, C., Hammer, R.E. & Hamra, F.K. Sterile testis complementation with spermatogonial lines restores fertility to DAZL-deficient rats and maximizes donor germline transmission. PLoS One 4, e6308 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  56. 56

    Wu, Z. et al. Spermatogonial culture medium: an effective and efficient nutrient mixture for culturing rat spermatogonial stem cells. Biol. Reprod. 81, 77–86 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Hamra, F.K. Gene targeting: enter the rat. Nature 467, 161–163 (2010).

    CAS  Article  PubMed  Google Scholar 

Download references

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

Authors

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Injection of transfected spermatogonial stem cells into the seminiferous tubules. (MOV 8220 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ivics, Z., Izsvák, Z., Medrano, G. et al. Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells. Nat Protoc 6, 1521–1535 (2011). https://doi.org/10.1038/nprot.2011.378

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links