Simultaneous inhibition and subsequent re-expression of multiple genes

The use of RNAi technology to target the removal of mRNA expression has become a powerful tool in studying protein function (e.g., see references 1 & 2). RNAi technology has several constraints, however, and the more improvements that can be made in the procedure, the more powerful and useful the technique becomes. One drawback has been that RNAi technology has been used primarily to target the knockdown of a single gene. This technology was recently expanded to simultaneously target the knockdown of two or more genes in a variety of organisms by developing vectors for inserting multiple siRNA encoding sequences (3-6). Similarly, as described herein, we have developed a vector for simultaneously targeting the removal of multiple genes in mammalian systems. Another limitation in RNAi technology has been that it examined the role of proteins only by their loss preventing an in-depth analyses of overall function of the targeted protein. To overcome this limitation, several gene replacement strategies that circumvent the RNAi knockdown vector for restoring protein expression have been developed (7-16). Herein, we report that the targeted removal of multiple gene expression can also be complemented by replacement of one or more of the knockdown genes.