A complementation method for functional analysis of mammalian genes.
Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.
Gonzalez-Santos
Juana Maria
JM
Programme in Lung Biology Research and the Canadian Institutes of Health Research Group in Lung Development, Hospital for Sick Children Toronto, Canada M5G 1X8.
Cao
Huibi
H
Wang
Anan
A
Koehler
David R
DR
Martin
Bernard
B
Navab
Roya
R
Hu
Jim
J
D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D005816:Genetic Complementation Test / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D034622:RNA Interference; D012326:RNA Splicing; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism
D009687:Nuclear Proteins; C108507:PRPF3 protein, human; D017414:Ribonucleoprotein, U4-U6 Small Nuclear