Genes that will have been found to modulate RNAi either in vitro (by the Martinez group) or in vivo (by the Dickson group) will be analyzed for the effects on RNAi efficiency in human cultured cells. Based on the evolutionary conservation of known proteins in the RNAi machinery such as Dicer and members of the Argonaute family, it is expected that it will be possible to identify human orthologs of Drosophila genes through bioinformatic analyses. The expression of genes that decrease RNAi efficiency in vitro or in Drosophilain vivo will be suppressed through transfection with synthetic siRNAs, whereas the expression of genes that may be rate limiting for RNAi will be increased by overexpression. In both cases HeLa cells will be used because these can be easily cultured in large amounts, transfected with siRNAs or expression plasmids with higher than 90% efficiencies, can be used for high-throughput cellular screening, and because they contain a robust RNAi machinery. In the case of overexpression experiments, stable inducible cell lines using the "Tet on" system will be generated (Gossen and Bujard, 1992, PNAS 89:5547) in which expression levels of the transgene can be modulated. The generation of stable lines will be important to determine expression levels and the timing of expression which are optimal for RNAi efficiency without compromising cell viability, growth or proliferation, and such cell lines will represent renewable resources which could then widely be used for small scale or high throughput experiments in the academic and commercial research communities. Stable lines will also be established if proteins can be identified whose depletion increases the efficiency of RNAi. In this case we will generate cell lines that stably express short hairpin RNA constructs with which the expression of these proteins can be reduced.
