Abstract
The technique described in this chapter—gene targeting in cultured human cancer cells—brings a powerful tool to scientists studying the function of cell cycle control genes (1). This technology allows scientists to knock out genes in cultured human cells in an analogous fashion to the creation of knockout mice. This approach brings the power of genetics (the comparison of cells or organisms that are genetically identical except for a single, well-defined mutation) to the study of human genes in cultured human cells. Gene targeting is a particularly valuable approach for the study of cell cycle control genes because ectopic expression of these genes frequently results in cell cycle arrest or apoptosis. To date, several cell cycle control genes have been studied using human somatic-cell gene targeting, including p21WAF1/CIP1, p53, 14-3-3σ, and (ataxia-telangiectasia and Rad3 [ATR]) (2–5).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sedivy, J. M. and Dutriaux, A. (1999) Gene targeting and somatic cell genetics—a rebirth or a coming of age? Trends Genet. 15, 88–90.
Waldman, T., Kinzler, K. W., and Vogelstein, B. (1995) p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 55, 5187–5190.
Bunz, F., Dutriaux, A., Lengauer, C., et al. (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1501.
Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1999) 14-3-3σ is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616–620.
Cortez, D., Guntuku, S., Qin, J., and Elledge, S. J. (2001) ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716.
Hanson, K. D. and Sedivy, J. M. (1995) Analysis of biological selections for high-efficiency gene targeting. Mol. Cell. Biol. 15, 45–51.
Brattain, M. G., Fine, W. D., Khaled, F. M., Thompson, J., and Brattain, D. E. (1981) Heterogeneity of malignant cells from a human colonic carcinoma. Cancer Res. 41, 1751–1756.
Dexter, D. L., Barbosa, J. A., and Calabresi, P. (1979) N,N-dimethylformamide-induced alteration of cell culture characteristics and loss of tumorigenicity in cultured human colon carcinoma cells. Cancer Res. 39, 1020–1025.
Shirasawa, S., Furuse, M., Yokoyamo, N., and Sasazuki, T. (1993) Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 260, 85–88.
Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998) Genetic instabilities in human cancers. Nature 396, 643–649.
Brown, J. P., Wei, W., and Sedivy, J. M. (1997) Bypass of senescence after disruption of p21WAF1/CIP1 gene in normal diploid human fibroblasts. Science 277, 831–834.
Storck, T., Kruth, U., Kolhekar, R., Sprengel, R., and Seeburg, P. (1996) Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse. Nucleic Acids Res. 24, 4594–4596.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Waldman, T.A. (2004). Gene Targeting in Cultured Human Cells. In: Lieberman, H.B. (eds) Cell Cycle Checkpoint Control Protocols. Methods in Molecular Biology™, vol 241. Humana Press. https://doi.org/10.1385/1-59259-646-0:163
Download citation
DOI: https://doi.org/10.1385/1-59259-646-0:163
Publisher Name: Humana Press
Print ISBN: 978-1-58829-115-8
Online ISBN: 978-1-59259-646-1
eBook Packages: Springer Protocols