Design and application of a shRNA-based gene replacement retrovirus.
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.
Zhang
Rugang
R
Fox Chase Cancer Center, Philadelphia, PA, USA.
Adams
Peter D
PD
Ye
Xiaofen
X
D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*
C491641:ASF1A protein, human; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D012313:RNA