Genome-wide forward genetic screens in mouse ES cells
- PMID: 20699144
- DOI: 10.1016/S0076-6879(10)77012-9
Genome-wide forward genetic screens in mouse ES cells
Abstract
Mouse embryonic stem (ES) cells are an attractive model system for investigating mammalian biology. Their relatively stable genome and high amenability to genome modification enables the generation of large-scale mutant libraries, which can be subsequently used for phenotype-driven genetic screens. While retroviral vectors have traditionally been used to generate insertional mutations in ES cells, their severe distribution-bias in the mammalian genome substantially limits genome-wide mutagenesis. The recent development of the DNA transposon piggyBac offers an efficient and highly versatile alternative for achieving more unbiased mutagenesis. Furthermore, heterozygous mutations created by insertional mutagens can be converted in parallel to homozygosity by using Blm-deficient ES cells, allowing genome-wide loss-of-function screens to be conducted. In this chapter, we describe the principles underpinning genetic screens in mouse ES cells with examples of previously successful screens. Protocols are provided for piggyBac transposon-mediated mutagenesis, production of the corresponding homozygous mutants in a Blm-deficient genetic background, and methods for mapping and validation of mutations recovered from screens of such libraries.
2010 Elsevier Inc. All rights reserved.
Similar articles
-
Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.Nature. 2004 Jun 24;429(6994):891-5. doi: 10.1038/nature02653. Nature. 2004. PMID: 15215866
-
piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.Methods Mol Biol. 2015;1239:15-28. doi: 10.1007/978-1-4939-1862-1_2. Methods Mol Biol. 2015. PMID: 25408399
-
Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene.Nature. 2004 Jun 24;429(6994):896-9. doi: 10.1038/nature02646. Nature. 2004. PMID: 15215867
-
Insertional mutagenesis in mice: new perspectives and tools.Nat Rev Genet. 2005 Jul;6(7):568-80. doi: 10.1038/nrg1638. Nat Rev Genet. 2005. PMID: 15995698 Review.
-
Generation of radiation-induced deletion complexes in the mouse genome using embryonic stem cells.Methods. 1997 Dec;13(4):409-21. doi: 10.1006/meth.1997.0547. Methods. 1997. PMID: 9480785 Review.
Cited by
-
Identification of genes that promote PI3K pathway activation and prostate tumour formation.Oncogene. 2024 Jun;43(24):1824-1835. doi: 10.1038/s41388-024-03028-x. Epub 2024 Apr 23. Oncogene. 2024. PMID: 38654106 Free PMC article.
-
Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status.Sci Adv. 2023 Dec 22;9(51):eadi5683. doi: 10.1126/sciadv.adi5683. Epub 2023 Dec 20. Sci Adv. 2023. PMID: 38117886 Free PMC article.
-
Applications of piggyBac Transposons for Genome Manipulation in Stem Cells.Stem Cells Int. 2021 Sep 14;2021:3829286. doi: 10.1155/2021/3829286. eCollection 2021. Stem Cells Int. 2021. PMID: 34567130 Free PMC article. Review.
-
TrapSeq: An RNA Sequencing-Based Pipeline for the Identification of Gene-Trap Insertions in Mammalian Cells.J Mol Biol. 2017 Sep 1;429(18):2780-2789. doi: 10.1016/j.jmb.2017.07.020. Epub 2017 Aug 4. J Mol Biol. 2017. PMID: 28782559 Free PMC article.
-
Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells.Sci Data. 2017 Mar 1;4:170020. doi: 10.1038/sdata.2017.20. Sci Data. 2017. PMID: 28248920 Free PMC article.
Publication types
MeSH terms
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
