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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    • "In fact, tagged protein may have a different sub-cellular localization or perturb cell metabolism and/or DNA repair complex. Thus, the knock-in replacement of the endogenous protein may address this question [97], although partly. An elegant alternative may be the use of an indirect approach, with split-luciferase (or split-GFP) or through intracellular antibodies. "
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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.
    Molecular biology of the cell 03/2012; 23(10):1838-45. DOI:10.1091/mbc.E11-12-1043 · 4.47 Impact Factor
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    • "We set out to develop an approach to avoid these drawbacks. The method is based on the use of rAAV vectors [2], [14] that feature yellow fluorescent protein (EYFP) as the only foreign DNA to be introduced into target cells flanked by short ∼1 kb homology arms. EYFP is included as a promoter-less and ATG-less (ORF-trap) construct which will result in expression only upon in-frame integration within an expressed gene. "
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    ABSTRACT: Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.
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