The biochemical approach of the Martinez group will be complemented by genetic approaches conducted by the Dickson group. This group is constructing a library of ~15,000 transgenic Drosophila strains, in which each strain carries a transgene that targets a single gene by RNAi. The transgene expresses a double-stranded "hairpin RNA", which is processed by Dicer2 to produce the siRNAs that trigger RNAi-mediated knock-down of the corresponding endogenous mRNA. This hairpin construct is normally silent, but can be activated by introducing in a simple genetic cross a second transgene that encodes the GAL4 transcriptional activator. Genes with either positive or negative roles in RNAi will then be subject to a more detailed genetic characterization in Drosophila. Biochemical analysis will be performed together with the Martinez and Peters groups. The aim of these experiments will be to determine the precise role of any newly identified factors in the RNAi machinery. These experiments are expected to lead to further insights into the basic mechanism of RNAi. Moreover, they should provide methods for greatly improved efficiency of RNAi in Drosophila in vivo, either by overexpression of positively-acting factors, or co-RNAi to target negatively-acting factors. As outlined below, the Peters group will determine whether expression or knock-down of the corresponding human genes similarly enhance RNAi efficiency in HeLa cells.
