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.

  • Article
  • Published:

A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening

Abstract

Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental design.
Figure 2: Regulation of the cNP cassette.
Figure 3: Isolation and characterization of homozygous mutants.
Figure 4: Phenotypic analyses of Dgcr8 and Ptpn11 homozygous mutant cells.
Figure 5: Phenotypic analyses of homozygous mutant ESC lines.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Birmingham, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006).

    Article  CAS  Google Scholar 

  2. Yusa, K. et al. Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom′s syndrome gene. Nature 429, 896–899 (2004).

    Article  CAS  Google Scholar 

  3. Guo, G., Wang, W. & Bradley, A. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 429, 891–895 (2004).

    Article  CAS  Google Scholar 

  4. Wang, W. & Bradley, A. A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells. Genome Biol. 8, R48 (2007).

    Article  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. USA 98, 6209–6214 (2001).

    Article  CAS  Google Scholar 

  7. Casanova, E. et al. ER-based double iCre fusion protein allows partial recombination in forebrain. Genesis 34, 208–214 (2002).

    Article  CAS  Google Scholar 

  8. Zambrowicz, B.P. et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).

    Article  CAS  Google Scholar 

  9. Kawakami, K. & Noda, T. Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166, 895–899 (2004).

    Article  CAS  Google Scholar 

  10. Chen, Y.T. & Bradley, A. A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 28, 31–35 (2000).

    Article  CAS  Google Scholar 

  11. Branda, C.S. & Dymecki, S.M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    Article  CAS  Google Scholar 

  12. Liu, X. et al. Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev. Dyn. 209, 85–91 (1997).

    Article  CAS  Google Scholar 

  13. Matsumura, H. et al. Targeted chromosome elimination from ES-somatic hybrid cells. Nat. Methods 4, 23–25 (2007).

    Article  CAS  Google Scholar 

  14. Lewandoski, M. & Martin, G.R. Cre-mediated chromosome loss in mice. Nat. Genet. 17, 223–225 (1997).

    Article  CAS  Google Scholar 

  15. Wang, Y., Medvid, R., Melton, C., Jaenisch, R. & Blelloch, R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39, 380–385 (2007).

    Article  CAS  Google Scholar 

  16. Wu, D. et al. A conserved mechanism for control of human and mouse embryonic stem cell pluripotency and differentiation by shp2 tyrosine phosphatase. PLoS ONE 4, e4914 (2009).

    Article  Google Scholar 

  17. Conti, L. et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 3, e283 (2005).

    Article  Google Scholar 

  18. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

    Article  CAS  Google Scholar 

  19. Buchou, T. et al. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol. Cell. Biol. 23, 908–915 (2003).

    Article  CAS  Google Scholar 

  20. Lu, R. et al. Systems-level dynamic analyses of fate change in murine embryonic stem cells. Nature 462, 358–362 (2009).

    Article  CAS  Google Scholar 

  21. Collins, F.S., Rossant, J. & Wurst, W. A mouse for all reasons. Cell 128, 9–13 (2007).

    Article  CAS  Google Scholar 

  22. Leeb, M. & Wutz, A. Derivation of haploid embryonic stem cells from mouse embryos. Nature advance online publication, doi:10.1038/nature10448 (7 September 2011).

  23. Ishida, Y. & Leder, P. RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells. Nucleic Acids Res. 27, e35 (1999).

    Article  CAS  Google Scholar 

  24. Liang, Q., Kong, J., Stalker, J. & Bradley, A. Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons. Genesis 47, 404–408 (2009).

    Article  CAS  Google Scholar 

  25. Chen, Y. et al. Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nat. Genet. 24, 314–317 (2000).

    Article  CAS  Google Scholar 

  26. Munroe, R.J. et al. Mouse mutants from chemically mutagenized embryonic stem cells. Nat. Genet. 24, 318–321 (2000).

    Article  CAS  Google Scholar 

  27. Goodwin, N.C. et al. DelBank: a mouse ES-cell resource for generating deletions. Nat. Genet. 28, 310–311 (2001).

    Article  CAS  Google Scholar 

  28. Guo, G., Huang, Y., Humphreys, P., Wang, X. & Smith, A.A. PiggyBac-based recessive screening method to identify pluripotency regulators. PLoS ONE 6, e18189 (2011).

    Article  CAS  Google Scholar 

  29. Huang, Y. et al. Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system. Nucleic Acids Res. (in the press).

  30. Ying, Q.L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639–649 (2006).

    Article  CAS  Google Scholar 

  33. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).

    Article  CAS  Google Scholar 

  34. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).

    Article  CAS  Google Scholar 

  35. Devon, R.S., Porteous, D.J. & Brookes, A.J. Splinkerettes—improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res. 23, 1644–1645 (1995).

    Article  CAS  Google Scholar 

  36. Thomas, P.D. et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Kawakami at Japan's National Institute of Genetics for providing the Tol2 transposon vector; E. Casanova at Ludwig Boltzmann Institute for Cancer Research for the ERT2-iCre fusion vectors; A.F. Stewart at Technische Universitaet Dresden for FLPe and FLPo expression vectors; F. Costantini at Columbia University for the generic Rosa26 targeting vector; T. Sudo and H. Akiyama for microarray analysis; Y. Esaki, Y. Koreeda and M. Okabe for assistance with the production of chimeric mice; M. Araki, K. Araki and K. Yamamura for deposition of ESC clones at the International Gene Trap Consortium; V. Keng for comments on the manuscript; and A. Yamanishi, M. Kawabata, M. Kouno, S. Tanaka, M. Tsunawaki and Y. Kuromi for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Precursory Research for Embryonic Science and Technology (PRESTO) program from the Japan Science and Technology Agency. This work was also supported in part by the Inamori Foundation, Takeda Science Foundation, Kato Memorial Bioscience Foundation and Mochida Memorial Foundation.

Author information

Authors and Affiliations

Authors

Contributions

K.H. designed experiments, constructed vectors, performed ESC culture and phenotypic analyses of mutant ESCs, and wrote the manuscript. C.K. performed bioinformatics analyses and contributed to writing the manuscript. J.Y. conducted vector construction and ESC culture. K.A. performed bioinformatics analyses and constructed the database. A.I. generated chimeric mice. A.O. conducted ESC culture. K.Y. performed gene targeting of ESCs. R.I. conducted ESC culture and PCR genotyping of mutant ESCs. Y.H. and A.B. contributed to the vector design for the selection of homozygous mutants. J.T. conducted vector construction and gene targeting of ESCs, and contributed to writing the manuscript.

Corresponding authors

Correspondence to Kyoji Horie or Junji Takeda.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1-5, Supplementary Tables 1-6 (PDF 1599 kb)

Supplementary Data

Vector insertion sites. (XLS 488 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Horie, K., Kokubu, C., Yoshida, J. et al. A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening. Nat Methods 8, 1071–1077 (2011). https://doi.org/10.1038/nmeth.1739

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1739

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing