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).
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References
Venter J.C., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.
International Human Genome Sequencing Consortium. (2004) Finishing the euchromatic sequence of the human genome. Nature 431, 931–945.
Hanson K.D. and Sedivy J.M. (1995) Analysis of biological selections for high-efficiency gene targeting. Mol Cell Biol. 15, 45–51.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Liu X., Lei M., Erikson R.L. (2006) Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol Cell Biol. 26, 2093–2108.
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.
Acknowledgments
The authors thank Catherine Randall for generously providing the primary data used in Fig. 2.3. Work in the laboratory of P.V.J. is supported by grants from the National Institutes of Health (CA 107342) and the American Cancer Society (RSG-08-093-01-CCG). P.V.J. is a Pew Scholar in the Biomedical Sciences.
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Berdougo, E., Terret, ME., Jallepalli, P.V. (2009). Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_2
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DOI: https://doi.org/10.1007/978-1-60327-993-2_2
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