Abstract
The chemical mutagenesis of mouse embryonic stem (ES) cells is an approach complementary to chemical mutagenesis of spermatogonia in whole animals. It has great potential to contribute significantly to the generation of a comprehensive collection of multiple alleles for most mammalian genes and to facilitate the progression from gene sequence to gene functional analyses. The general strategy includes the treatment of ES cells with a chemical mutagen and the isolation of individual mutagenized clones that are then cultured in duplicate. Whereas one set of samples is cryopreserved for archiving and subsequent generation of germline chimeras by means of blastocyst injection, the second set is used for genotypeor phenotype-driven screens to identify mutant alleles.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Munroe R. J., Bergstrom R. A., Zheng Q. Y., et al. (2000) Mouse mutants from chemically mutagenized embryonic stem cells. Nat. Genet. 24, 318–321.
Chen Y., Schimenti J., and Magnuson T. (2000) Toward the yeastification of mouse genetics: chemical mutagenesis of embryonic stem cells. Mamm. Genome 11, 598–602.
Coghill E. L., Hugill A., Parkinson N., et al. (2002) A gene-driven approach to the identifiation of ENU mutants in the mouse. Nat. Genet. 30, 255–256.
Chen Y., Yee D., Dains K., et al. (2000) Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nat. Genet. 24, 314–317.
Browning V. L., Chaudhry S. S., Planchart A., Dixon M. J., and Schimenti J. C. (2001) Mutations of the mouse Twist and sy (fibrillin 2) genes induced by chemical mutagenesis of ES cells. Genomics 73, 291–298.
Ashburner M. (1989) Drosophila: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Liu L. X., Spoerke J. M., Mulligan E. L., et al. (1999) High-throughput isolation of Caenorhabditis elegans deletion mutants. Genome Res. 9, 859–867.
Vivian J. L., Chen Y., Yee D., Schneider E., and Magnuson T. (2002) An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N-ethyl-Nnitrosourea-mutagenized mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 15,542–15,547.
Weinstein M., Yang X., and Deng C. (2000) Functions of mammalian Smad genes as revealed by targeted gene disruption in mice. Cytokine Growth Factor Rev. 11, 49–58.
Littlefield J. W. (1989) Compaction-defective embryonal carcinoma cell variants. Dev. Genet. 10, 292–297.
Beckers J., Hoheisel J. D., Mewes H. W., Vingron M., and Hrabe de Angelis M. (2002) Molecular phenotyping of mouse mutant resources by RNA expression profiling. Curr. Genomics 3, 121–129.
Beier D. R. (2000) Sequence-based analysis of mutagenized mice. Mamm. Genome 11, 594–597.
Andersen P. S., Jespersgaard C., Vuust J., Christiansen M., and Larsen L. A. (2003) Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening. Hum. Mutat. 21, 455–465.
Lilleberg S. L. (2003) In-depth mutation and SNP discovery using DHPLC gene scanning. Curr. Opin. Drug Discov. Dev. 6, 237–252.
Chen Y., Vivian J. L., and Magnuson T. (2003) Gene-based chemical mutagenesis in mouse embryonic stem cells. Methods Enzymol. 365, 406–415.
Li Q., Liu Z., Monroe H., and Culiat C. T. (2002) Integrated platform for detection of DNA sequence variants using capillary array electrophoresis. Electrophoresis 23, 1499–1511.
Zhu L., Lee H. K., Lin B., and Yeung E. S. (2001) Spatial temperature gradient capillary electrophoresis for DNA mutation detection. Electrophoresis 22, 3683–3687.
Murphy K., Hafez M., Philips J., Yarnell K., Gutshall K., and Berg K. (2003) Evaluation of temperature gradient capillary electrophoresis for detection of the Factor V Leiden mutation: coincident identification of a novel polymorphism in Factor V. Mol. Diagn. 7, 35–40.
Soewarto D., Blanquet V., and Hrabe de Angelis M. (2003) Random ENU mutagenesis. Methods Mol. Biol. 209, 249–266.
Shibuya T. and Morimoto K. (1993) A review of the genotoxicity of 1-ethyl-1-nitrosourea. Mutat. Res. 297, 3–38.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Becker, S., de Angelis, M.H., Beckers, J. (2006). Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 329. Humana Press. https://doi.org/10.1385/1-59745-037-5:397
Download citation
DOI: https://doi.org/10.1385/1-59745-037-5:397
Publisher Name: Humana Press
Print ISBN: 978-1-58829-498-2
Online ISBN: 978-1-59745-037-9
eBook Packages: Springer Protocols