Protocol

Mitosis

Volume 545 of the series Methods in Molecular Biology pp 21-37

Date:

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

  • Eli BerdougoAffiliated withMolecular Biology Program, Memorial Sloan-Kettering Cancer Center
  • , Marie-Emilie TerretAffiliated withMolecular Biology Program, Memorial Sloan-Kettering Cancer Center
  • , Prasad V. JallepalliAffiliated withMolecular Biology Program, Memorial Sloan-Kettering Cancer Center

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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