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.

  • Protocol
  • Published:

Derivation and maintenance of mouse haploid embryonic stem cells

Nature Protocols volume 14, pages 1991–2014 (2019)Cite this article

Subjects

Abstract

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Representative flow cytometric analysis of the cell cycle profile.
Fig. 2: Qualitative control of the hESCs.
Fig. 3: Sorting of haploid cells.
Fig. 4: Workflow diagram of hESC derivation.
Fig. 5: Morphological selection during hESC derivation.
Fig. 6: Representative diagram of a transposon screen in hESCs.

Similar content being viewed by others

Data availability

All data presented in the article are available from the corresponding authors upon reasonable request.

References

  1. Maderspacher, F. Theodor Boveri and the natural experiment. Curr. Biol. 18, R279–R286 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Crosland, M. W. J. & Crozier, R. H. Myrmecia pilosula, an ant with only one pair of chromosomes. Science 231, 1278–1278 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. Swart, E. C. et al. The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol. 11, e1001473 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gallardo, M. H., Bickham, J. W., Honeycutt, R. L., Ojeda, R. A. & Köhler, N. Discovery of tetraploidy in a mammal. Nature 401, 341–341 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Marston, A. L. & Amon, A. Meiosis: cell-cycle controls shuffle and deal. Nat. Rev. Mol. Cell Biol. 5, 983–997 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2017).

    Article  CAS  Google Scholar 

  8. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Davoli, T. & de Lange, T. The causes and consequences of polyploidy in normal development and cancer. Ann. Rev. Cell Dev. Biol. 27, 585–610 (2011).

    Article  CAS  Google Scholar 

  10. Elling, U. et al. Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9, 563–574 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Leeb, M. & Wutz, A. Derivation of haploid embryonic stem cells from mouse embryos. Nature 479, 131–134 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sagi, I. et al. Derivation and differentiation of haploid human embryonic stem cells. Nature 532, 1–19 (2016).

    Article  Google Scholar 

  13. Zhong, C. et al. Generation of human haploid embryonic stem cells from parthenogenetic embryos obtained by microsurgical removal of male pronucleus. Cell Res. 26, 743–746 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Sagi, I. & Benvenisty, N. Haploidy in humans: an evolutionary and developmental perspective. Dev. Cell 41, 581–589 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Botstein, D. & Fink, G. R. Yeast: an experimental organism for 21st century biology. Genetics 189, 695–704 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cordes, S. P. N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express. Microbiol. Mol. Biol. Rev. 69, 426–439 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kotecki, M. Isolation and characterization of a near-haploid human cell line. Exp. Cell Res. 252, 273–280 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231–1235 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Carette, J. E. et al. Generation of iPSCs from cultured human malignant cells. Blood 115, 4039–4042 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pincus, G. Observations on the living eggs of the rabbit. Proc. R. Soc. Lond. B 107, 132–167 (1930).

    Article  Google Scholar 

  22. Tarkowski, A. K., Witkowska, A. & Nowicka, J. Experimental parthenogenesis in the mouse. Nature 226, 162–165 (1970).

    Article  CAS  PubMed  Google Scholar 

  23. Graham, C. F. Parthenogenetic mouse blastocysts. Nature 226, 165–167 (1970).

    Article  CAS  PubMed  Google Scholar 

  24. Kaufman, M. H. Early Mammalian Development: Parthenogenetic Studies (Cambridge Univ. Press, Cambridge, 1983).

  25. Fraser, L. R. Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res. 18, 363–374 (1987).

    Article  CAS  PubMed  Google Scholar 

  26. Kline, D. & Kline, J. T. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev. Biol. 149, 80–89 (1992).

    Article  CAS  PubMed  Google Scholar 

  27. Mikich, A. B. et al. Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes. Mol. Reprod. Dev. 41, 84–90 (1995).

    Article  Google Scholar 

  28. Cuthbertson, K. S. R., Whittingham, D. G. & Cobbold, P. H. Free Ca2+ increases in exponential phases during mouse oocyte activation. Nature 294, 754–757 (1981).

    Article  CAS  PubMed  Google Scholar 

  29. Stevens, L. C. & Varnum, D. S. The development of teratomas from parthenogenetically activated ovarian mouse eggs. Dev. Biol. 37, 369–380 (1974).

    Article  CAS  PubMed  Google Scholar 

  30. Linder, D., McCaw, B. K. & Hecht, F. Parthenogenic origin of benign ovarian teratomas. N. Engl. J. Med. 292, 63–66 (1975).

    Article  CAS  PubMed  Google Scholar 

  31. Leeb, M., Perry, A. C. F. & Wutz, A. Establishment and use of mouse haploid ES cells. Curr. Protoc. Mouse Biol. 5, 155–185 (2017).

    Article  Google Scholar 

  32. Shuai, L. et al. Generation of mammalian offspring by haploid embryonic stem cells microinjection. Curr. Protoc. Stem Cell Biol. 31, 1A.6.1–15 (2014).

    Article  Google Scholar 

  33. Balmus, G. et al. ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks. Nat Commun 10, 646 (2019).

    Article  Google Scholar 

  34. Yang, H. et al. Generation of haploid embryonic stem cells from Macaca fascicularis monkey parthenotes. Cell Res. 23, 1187–1200 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, X. et al. Generation and application of mouse-rat allodiploid embryonic stem cells. Cell 164, 279–292 (2016).

    Article  CAS  PubMed  Google Scholar 

  36. Yang, H. et al. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149, 605–617 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Li, W. et al. Androgenetic haploid embryonic stem cells produce live transgenic mice. Nature 490, 407–411 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Leeb, M. & Wutz, A. Germline potential of parthenogenetic haploid mouse embryonic stem cells. Development 139, 3301–3305 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Elling, U. & Penninger, J. M. Genome wide functional genetics in haploid cells. FEBS Lett. 588, 2415–2421 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Forment, J. V. et al. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells. Nat. Chem. Biol. 13, 12–14 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Herzog, M. et al. Detection of functional protein domains by unbiased genome-wide forward genetic screening. Sci. Rep. 8, 6161 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Elling, U. et al. A reversible haploid mouse embryonic stem cell biobank resource for functional genomics. Nature 550, 114 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gao, Q. et al. Derivation of haploid neural stem cell lines by selection for a Pax6-GFP reporter. Stem Cells Dev. 27, 479–487 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Olbrich, T. et al. A p53-dependent response limits the viability of mammalian haploid cells. Proc. Natl. Acad. Sci. 114, 9367–9372 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kim, Y. M., Lee, J.-Y., Xia, L., Mulvihill, J. J. & Li, S. Trisomy 8: a common finding in mouse embryonic stem (ES) cell lines. Mol. Cytogenet. 6, 3 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Morey, R. & Laurent, L. C. Getting off the ground state: X chromosome inactivation knocks down barriers to differentiation. Cell Stem Cell 14, 131–132 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Brownstein, D. G. Manipulating the Mouse Embryo: A Laboratory Manual, 4th edn (eds Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R.) Ch. 3, 85–107 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2014).

  49. Friedrich, M. J. et al. Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice. Nat. Protoc. 12, 289–309 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Rens, W., Fu, B., O’Brien, P. C. M. & Ferguson-Smith, M. Cross-species chromosome painting. Nat. Protoc. 1, 783–790 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, 2013).

  52. de Ridder, J., Uren, A., Kool, J., Reinders, M. & Wessels, L. Detecting statistically significant common insertion sites in retroviral insertional mutagenesis screens. PLoS Comput. Biol. 2, e166 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

M.W., B.D., B.F., B.L.N. and D.J.A. were supported by the Wellcome Trust through core funding to the Wellcome Trust Sanger Institute (WT098051). Research in the S.P.J. laboratory was funded by Cancer Research UK (grant C6/A18796) and a Wellcome Trust Investigator Award (206388/Z/17/Z). Core funding was provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). Research in the J.M.P. laboratory was funded by Advanced ERC and Era of Hope/DoD grants. Research in the G.B. laboratory was funded by a UK Dementia Research Institute fellowship (MC_PC_17111).

Author information

Author notes

  1. These authors contributed equally: Ulrich Elling, Michael Woods.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria

    Ulrich Elling & Josef M. Penninger

  2. UK Dementia Research Institute at University of Cambridge and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK

    Michael Woods, Jose R. Vicente & Gabriel Balmus

  3. Wellcome Trust Sanger Institute, Cambridge, UK

    Michael Woods, Beiyuan Fu, Fengtang Yang, Bee Ling Ng, David J. Adams & Brendan Doe

  4. DNA Damage Response Biology, Oncology Innovative Medicines, AstraZeneca, Cambridge, UK

    Josep V. Forment

  5. The Wellcome Trust CRUK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

    Stephen P. Jackson

  6. Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC, Canada

    Josef M. Penninger

Authors
  1. Ulrich Elling
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Woods
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Josep V. Forment
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beiyuan Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fengtang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bee Ling Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jose R. Vicente
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David J. Adams
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Brendan Doe
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stephen P. Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Josef M. Penninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Gabriel Balmus
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

U.E., M.W. and G.B. performed experimental analysis and procedures throughout and wrote the manuscript. B.D. and D.J.A. helped M.W. with setup of blastocyst work. B.L.N. assisted with flow cytometry, with help from J.V.F. and S.P.J. B.F. and F.Y. performed the FISH and karyotyped the cell lines. J.R.V. wrote the transposon-induced mutagenesis protocol with help from U.E. G.B. and J.M.P. conceived the idea of this article. All authors commented on the manuscript.

Corresponding authors

Correspondence to Josef M. Penninger or Gabriel Balmus.

Ethics declarations

Competing interests

J.M.P. and U.E. are shareholders of JLP Health.

Additional information

Journal peer review information: Nature Protocols thanks Ling Shuai and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Elling, U. et al. Cell Stem Cell 9, 563–574 (2011): http://www.sciencedirect.com/science/article/pii/S1934590911004929

Elling, U. et al. Nature 550, 114–118 (2017): https://www.nature.com/articles/nature24027

Balmus, G. et al. Nat. Commun. 10, 87 (2019): https://www.nature.com/articles/s41467-018-07729-2

Key data used in this protocol

Elling, U. et al. Nature 550, 114–118 (2017): https://www.nature.com/articles/nature24027

Balmus, G. et al. Nat. Commun. 10, 87 (2019): https://www.nature.com/articles/s41467-018-07729-2

Integrated supplementary information

Supplementary Figure 1 Gene trap vectors and library preparation.

(a) Schematic representation of the gene trap vectors as presented in Elling et al. 201742 and on the Haplobank website (www.haplobank.at). Retroviral enhanced gene trap (Retro-EGT; related sequence provided as Supplementary Data file 1). Lentiviral enhanced gene trap (Lenti-EGT; related sequence provided as Supplementary Data file 2). Tol2 autonomous transposon enhanced gene trap (Tol2-EGT; related sequence provided as Supplementary Data file 3). Tol2 autonomous transposon polyadenylation enhanced gene trap (Tol2-polyA-EGT; related sequence provided as Supplementary Data file 4). Abbreviations used: LTR, long terminal repeat; 6xOPE, six osteopontin enhancer elements; FRT/F3, heterotypic improved flippase target sequences; LoxP/Lox5171, heterotypic target sequences for the Cre-recombinase; SA, splice acceptor; βgal, β-galactosidase; NeoR, neomycin phosphotransferase fusion gene; polyA, bovine growth hormone polyadenylation sequence; L200/R175, left and right Tol2 transposon elements; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein; RPB1, DNA-directed RNA polymerase II subunit rpb1; SD, splice donor. (b) Schematic representation of library preparation of gene trap vectors integration site. Tol2 – EGT is shown as example. Following fragmentation of the genome with enzyme 1 (E1, NlaIII in the example), the gene trap end containing the barcode and a genomic DNA portion is circularized (ring ligation). Prior to PCR amplification, linearization with enzyme 2 (E2, PaqI in the example) is needed. Each integration site can be mapped by using two different E1 enzymes. The genomic region is then amplified by PCR using US and DS primers.

Supplementary information

Supplementary Information

Supplementary Figure 1

Reporting Summary

Supplementary Data 1–4

Four sequences for gene trap cassettes harboring disruptive splice acceptor sites.

Supplementary Video 1

Removing cumulus oocyte complex from ampulla.

Supplementary Video 2

Identification and isolation of subviable (pathogenic) embryos.

Supplementary Video 3

Zona pellucida removal (denuding), first 20 s.

Supplementary Video 4

Zona pellucida removal (denuding), making sure zona is gone (up to 70 s).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elling, U., Woods, M., Forment, J.V. et al. Derivation and maintenance of mouse haploid embryonic stem cells. Nat Protoc 14, 1991–2014 (2019). https://doi.org/10.1038/s41596-019-0169-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0169-z

This article is cited by

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

Advanced 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