Functional dissection of mitotic regulators through gene targeting in human somatic cells.
Methods Mol Biol
; 545: 21-37, 2009.
Article
em En
| MEDLINE
| ID: mdl-19475380
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).
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Proto-Oncogênicas
/
Proteínas Serina-Treonina Quinases
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Marcação de Genes
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Proteínas de Ciclo Celular
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Mitose
Limite:
Humans
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article