Enhancement of RNA interference effect in P19 EC cells by an RNA-dependent RNA polymerase.
RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can also function as a primer converting mRNA into dsRNA that are further cleaved to produce more siRNA. This activity involves the enzyme RNA-dependent RNA polymerase (RdRP). There are no known RdRP involved in RNAi in mammals. By using an RdRP from Caenorhabditis elegance named ego-1, investigators intend to enhance RNAi effect in mammalian cells. The aims of this project were: 1) to investigate the efficiency of siRNA to enhanced green fluorescent protein (eGFP) gene silencing and 2) to enhance the RNAi effect.



We used a vector-based siRNA to target eGFP. Also we used a vector expressing ego-1 to test for a possible amplification effect of RNAi. The expression of eGFP in the cells was detected by using fluorescent microscopy, flowcytometry and Western-blotting.



Transfection of the plasmid into P19 cells significantly decreased eGFP fluorescence. In addition, eGFP protein was reduced. Preliminary data suggested that the presence of ego-1 enhanced the RNAi effect.



The results indicated that use of hairpin siRNA expression vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells. Also, introducing RdRP enzyme to mammalian cells might amplify the RNAi effect in the cells.
Esmaeili
Fariba
F
Ottawa Regional Cancer (Orcc), Ottawa, Canada.|Dept. of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran.
D000818:Animals; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000201:enzymology; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D018236:Carcinoma, Embryonal / Q000235:genetics*; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D034622:RNA Interference; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; C406772:EGO-1 protein, C elegans; D012324:RNA-Dependent RNA Polymerase