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Nucleic Acids Research 2005 33(10):e94; doi:10.1093/nar/gni093
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Published online 8 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

A complementation method for functional analysis of mammalian genes

Juana Maria Gonzalez-Santos1,3, Huibi Cao1,3, Anan Wang1, David R. Koehler1,3, Bernard Martin1, Roya Navab1,3 and Jim Hu1,2,3,*

1Programme in Lung Biology Research and the Canadian Institutes of Health Research Group in Lung Development, Hospital for Sick Children Toronto, Canada M5G 1X8 and 2Department of Paediatrics, University of Toronto Toronto, Canada M5S 1A1 3Laboratory Medicine and Pathobiology, University of Toronto Toronto, Canada M5S 1A1

*To whom correspondence should be addressed. Tel: +1 416 813 6412; Fax: +1 416 813 5771; Email: jhu{at}sickkids.ca

Received April 6, 2005. Revised May 24, 2005. Accepted May 24, 2005.

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.


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