Design and application of a shRNA-based gene replacement retrovirus.
Methods Mol Biol
; 408: 211-21, 2007.
Article
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| MEDLINE
| ID: mdl-18314585
ABSTRACT
To perform structure/function analyses of a protein in vivo, ideally one should be able to simultaneously abolish expression of the endogenous wild-type protein, substitute it with a form of the protein containing a targeted mutation, and analyze the functional consequences. Until recently, this was a highly challenging and/or laborious approach in mammalian systems, requiring a targeted gene knockin in a human cell line or mouse. Herein is described a RNA interference (RNAi)-based approach to achieve this much more simply in mammalian cells. A single retrovirus has been constructed, which directs expression of a short hairpin RNA (shRNA) to knockdown expression of the endogenous protein of interest; a cDNA coding for a wild-type or mutant version of the same protein that also contains "silent mutations" that do not affect the protein sequence, but do make the mRNA resistant to the shRNA; and a puromycin-resistance gene to allow rapid drug selection of the virus-infected cells. Using this virus, expression of the endogenous Anti-Silencing Function 1a (ASF1a) histone chaperone has been efficiently replaced in primary human cells, by an ectopically expressed epitope-tagged version. Moreover, the virus is designed so that other shRNA and shRNA-resistant cDNA cassettes can easily be substituted, making the approach readily applicable to other protein targets.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Retroviridae
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ARN
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Interferencia de ARN
Límite:
Animals
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Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2007
Tipo del documento:
Article
País de afiliación:
Estados Unidos