By intentionally targeting the above siRNA oligos to human and murine sequences that differed by several nucleotides, it was relatively straightforward to efficiently “knock down” p120 with the human-directed siRNA (pRS-h siRNA) and subsequently “knock up” p120 by infection with pLZRS-mp120, a retrovirus containing the murine p120 cDNA (Fig. 1 b). Restoring p120 levels by expressing murine p120 reversed the effects of the h siRNA and restored adhesion (Fig. 1, b and c). It is worth noting that this method is generally applicable to any protein. If a homologous gene is not available, a knock-up construct can be generated by making silent mutations in the region targeted by the siRNA. The method is a simple in vitro equivalent of transgenic knock-out and knock-in technology, and essentially solves the common dilemma associated with expressing mutant proteins in cells that already contain high levels of an endogenous counterpart. To our knowledge, this is the first example of this broadly applicable method.
