Article

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

ABSTRACT 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).

Download full-text

Full-text

Available from: Marie-Emilie Terret, Jan 30, 2015
0 Followers
 · 
107 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells.
    Radiotherapy and Oncology 07/2013; 108(3). DOI:10.1016/j.radonc.2013.06.013 · 4.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: As a mechanical system, the kinetochore can be viewed as a set of interacting springs, clutches and motors; the problem of kinetochore mechanism is now one of understanding how these functional modules assemble, disassemble and interact with one another to give rise to the emergent properties of the system. The sheer complexity of the kinetochore system points to a future requirement for data-driven mathematical modelling and statistical analysis based on quantitative empirical measurement of sister kinetochore trajectories. Here, we review existing models of chromosome motion in the context of recent advances in our understanding of kinetochore molecular biology.
    Chromosome Research 02/2011; 19(3):409-21. DOI:10.1007/s10577-011-9191-x · 2.69 Impact Factor