DNA vector-based RNAi approach for stable depletion of poly(ADP-ribose) polymerase-1.
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.
Shah
Rashmi G
RG
Laboratory for Skin Cancer Research, CHUL Research Center (CHUQ), Faculty of Medicine, Laval University, Sainte-Foy, Que., Canada G1V 4G2.
Ghodgaonkar
Medini M
MM
Affar
El Bachir
el B
Shah
Girish M
GM
D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D018390:Gene Targeting; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis / Q000235:genetics*; D034622:RNA Interference; D012867:Skin / Q000166:cytology
D004247:DNA; C499320:PARP1 protein, human; C483041:PARP2 protein, human; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C459820:Parp2 protein, mouse