Shah Rashmi G, Ghodgaonkar Medini M, Affar El Bachir, Shah Girish M
Laboratory for Skin Cancer Research, CHUL Research Center (CHUQ), Faculty of Medicine, Laval University, Sainte-Foy, Que., Canada G1V 4G2.
Biochem Biophys Res Commun. 2005 May 27;331(1):167-74. doi: 10.1016/j.bbrc.2005.03.135.
RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO(4) protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2.
