Derivation and maintenance of mouse haploid embryonic stem cells

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.

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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.

Data availability

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

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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

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.

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).

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