Measuring Recombination Proficiency in Mouse Embryonic Stem Cells (original) (raw)
Abstract
A method is presented to measure homologous recombination in mouse embryonic stem cells by both gene targeting and short-tract gene conversion of a double-strand break. A fluorescence-based reporter is first gene targeted to the Hprt locus in a quantifiable way. A homing endonuclease expression vector is then introduced to generate a double-strand break, the repair of which is also quantifiable.
Similar content being viewed by others
References
- Liang, F., Han, M., Romanienko, P. J., and Jasin, M. (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. USA 95, 5172–5177.
Article CAS PubMed Google Scholar - Rouet, P., Smih, F., and Jasin, M. (1994) Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106.
CAS PubMed Google Scholar - Ledermann, B. (2000) Embryonic stem cells and gene targeting. Exp. Physiol. 85, 603–613.
Article CAS PubMed Google Scholar - Pierce, A. J., Hu, P., Han, M., Ellis, N., and Jasin, M. (2001) Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev. 15, 3237–3242.
Article CAS PubMed Google Scholar - Richardson, C., Moynahan, M. E., and Jasin, M. (1998) Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev. 12, 3831–3842.
Article CAS PubMed Google Scholar - Donoho, G., Jasin, M., and Berg, P. (1998) Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol. Cell. Biol. 18, 4070–4078.
CAS PubMed Google Scholar - Pierce, A. J., Johnson, R. D., Thompson, L. H., and Jasin, M. (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13, 2633–2638.
Article CAS PubMed Google Scholar - Johnson, R. D. and Jasin, M. (2000) Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells. EMBO J. 19, 3398–3407.
Article CAS PubMed Google Scholar - Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Article CAS PubMed Google Scholar - Aljanabi, S. M. and Martinez, I. (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 25, 4692–4693.
Article CAS PubMed Google Scholar
Author information
Authors and Affiliations
- Markey Cancer Center, University of Kentucky, Lexington, KY
Andrew J. Pierce - Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY
Maria Jasin
Authors
- Andrew J. Pierce
You can also search for this author inPubMed Google Scholar - Maria Jasin
You can also search for this author inPubMed Google Scholar
Editor information
Editors and Affiliations
- Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA
Phouthone Keohavong & Stephen G. Grant &
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Pierce, A.J., Jasin, M. (2005). Measuring Recombination Proficiency in Mouse Embryonic Stem Cells. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:373
Download citation
- .RIS
- .ENW
- .BIB
- DOI: https://doi.org/10.1385/1-59259-840-4:373
- Publisher Name: Humana Press
- Print ISBN: 978-1-58829-084-7
- Online ISBN: 978-1-59259-840-3
- eBook Packages: Springer Protocols