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Nat Protoc. 2019 Jul;14(7):1991-2014. doi: 10.1038/s41596-019-0169-z. Epub 2019 Jun 3.

Derivation and maintenance of mouse haploid embryonic stem cells.

Author information

1
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria.
2
UK Dementia Research Institute at University of Cambridge and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.
3
Wellcome Trust Sanger Institute, Cambridge, UK.
4
DNA Damage Response Biology, Oncology Innovative Medicines, AstraZeneca, Cambridge, UK.
5
The Wellcome Trust CRUK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
6
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria. josef.penninger@ubc.ca.
7
Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC, Canada. josef.penninger@ubc.ca.
8
UK Dementia Research Institute at University of Cambridge and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. gb318@cam.ac.uk.

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.

PMID:
31160788
DOI:
10.1038/s41596-019-0169-z

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