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.

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References

  1. 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
  2. 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
  3. Ledermann, B. (2000) Embryonic stem cells and gene targeting. Exp. Physiol. 85, 603–613.
    Article CAS PubMed Google Scholar
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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

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Author information

Authors and Affiliations

  1. Markey Cancer Center, University of Kentucky, Lexington, KY
    Andrew J. Pierce
  2. Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, NY
    Maria Jasin

Authors

  1. Andrew J. Pierce
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  2. Maria Jasin
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Editor information

Editors and Affiliations

  1. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA
    Phouthone Keohavong & Stephen G. Grant &

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© 2005 Humana Press Inc.

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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

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