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

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 545:21-37. DOI: 10.1007/978-1-60327-993-2_2
Source: PubMed


With the human genome fully sequenced (1, 2), biologists continue to face the challenging task of evaluating the function of each of the approximately 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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Available from: Marie-Emilie Terret, Jan 30, 2015
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    • "Cells were seeded into 35-mm, poly-l-lysine–coated glass-bottom culture dishes (MatTek) and 24 h later transferred to CO 2 -independent media supplemented with 10% FBS, 100 U/ml penicillin, 100 U U/ml streptomycin, and 2 mM l-glutamine. For bulk population targeted clones was performed as described previously (Berdougo et al., 2009). "
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    ABSTRACT: Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.
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