enhancement of rna interference effect in p19 ec cells by an rna-dependent rna polymerase

background: 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. methods: 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. results: 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. conclusion: 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.