Mitosis pp 21-37
Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells
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 wordsGene targeting rAAV human somatic cells mitosis Plk1
- 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.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
- 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