RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-β-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
|Number of pages||6|
|Publication status||Published - 2004 May|
ASJC Scopus subject areas
- Cancer Research