Mitosis pp 21-37

Part of the Methods in Molecular Biology book series (MIMB, volume 545)

Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells

  • Eli Berdougo
  • Marie-Emilie Terret
  • Prasad V. Jallepalli
Protocol

Abstract

With the human genome fully sequenced (1, 2), biologists continue to face the challenging task of evaluating the function of each of the ∼25,000 genes contained within it. Gene targeting in human cells provides a powerful and unique experimental tool in this regard (3–8). Although somewhat more involved than RNAi or pharmacological approaches, somatic cell gene targeting is a precise technique that avoids both incomplete knockdown and off-target effects, but is still much quicker than analogous manipulations in the mouse. Moreover, immortal knockout cell lines provide excellent platforms for both complementation analysis and biochemical purification of multiprotein complexes in native form. Here we present a detailed gene-targeting protocol that was recently applied to the mitotic regulator Polo-like kinase 1 (Plk1) (9).

Key words

Gene targeting rAAV human somatic cells mitosis Plk1 

References

  1. 1.
    Venter J.C., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.PubMedCrossRefGoogle Scholar
  2. 2.
    International Human Genome Sequencing Consortium. (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945.CrossRefGoogle Scholar
  3. 3.
    Hanson K.D. and Sedivy J.M. (1995) Analysis of biological selections for high-efficiency gene targeting. Mol Cell Biol. 15, 45–51.PubMedGoogle Scholar
  4. 4.
    Jallepalli P.V., Waizenegger I.C., Bunz F., Langer S., Speicher M.R., Peters J., Kinzler K.W., Vogelstein B. and Lengauer C. (2001) Securin is required for chromosomal stability in human cells. Cell 105, 445–457.PubMedCrossRefGoogle Scholar
  5. 5.
    Wang X., Zou L., Zheng H., Wei Q., Elledge S.J., Li L. (2003) Genomic instability and endoreduplication triggered by RAD17 deletion. Genes Dev. 17, 965–970.PubMedCrossRefGoogle Scholar
  6. 6.
    Jallepalli P.V., Lengauer C., Vogelstein B., Bunz F. (2003) The Chk2 tumor suppressor is not required for p53 responses in human cancer cells. J Biol Chem. 278, 20475–20479.PubMedCrossRefGoogle Scholar
  7. 7.
    Papi M., Berdougo E., Randall C.L., Ganguly S., Jallepalli P.V. (2005) Multiple roles for separase auto-cleavage during the G2/M transition. Nat Cell Biol 7, 1029–1035.PubMedCrossRefGoogle Scholar
  8. 8.
    Larochelle S., Merrick K.A., Terret M.E., Wohlbold L., Barboza N.M., Zhang C., Shokat K.M., Jallepalli P.V. and Fisher R.P. (2007) Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. Mol Cell 25, 839–850.PubMedCrossRefGoogle Scholar
  9. 9.
    Burkard M.E., Randall C.L., Larochelle S., Zhang C., Shokat K.M., Fisher R.P. and Jallepalli P.V. (2007) Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells. Proc Natl Acad Sci USA 104, 4383–4388.PubMedCrossRefGoogle Scholar
  10. 10.
    Hirata R., Chamberlain J., Dong R. and Russell D.W. (2002) Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat Biotechnol. 20, 735–738.PubMedCrossRefGoogle Scholar
  11. 11.
    Porteus M.H., Cathomen T., Weitzman M.D. and Baltimore D. (2003) Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol. 23, 3558–3565.PubMedCrossRefGoogle Scholar
  12. 12.
    Kohli M., Rago C., Lengauer C., Kinzler K.W. and Vogelstein B. (2004) Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 32, e3.PubMedCrossRefGoogle Scholar
  13. 13.
    Chamberlain J.R., Schwarze U., Wang P.R., Hirata R.K., Hankenson K.D., Pace J.M., Underwood R.A., Song K.M., Sussman M., Byers P.H., Russell D.W. (2004) Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198–1201.PubMedCrossRefGoogle Scholar
  14. 14.
    Liu X., Lei M., Erikson R.L. (2006) Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol Cell Biol. 26, 2093–2108.PubMedCrossRefGoogle Scholar
  15. 15.
    Sambrook J. and Russell D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition: Chapter 6, New York: CSHL Press, pp. 6.33–6.64.Google Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Eli Berdougo
    • 1
  • Marie-Emilie Terret
    • 1
  • Prasad V. Jallepalli
    • 1
  1. 1.Molecular Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA

Personalised recommendations