Inhibition of Gene Expression by RNA Interference (RNAi) in P19 Embryonal Carcinoma Cell Line

 Introduction: RNA interference (RNAi) is a phenomenon of gene silencing that uses double-stranded RNA (dsRNA), specifically inhibits gene expression by degrading mRNA efficiently. The mediators of degradation are 21- to 23-nt small interfering RNAs (siRNA). The use of siRNAs as inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells.  Aim: To investigate the efficiency of siRNAs to eGFP gene silencing in P19 embryonal carcinoma (EC) stem cell.  Materials & Methods: Here, we used a vector-based siRNA expression system, pSUPER that can induce RNAi in mammalian cells (P19 line of murine embryonal carcinoma stem cell). The vector containing a small hairpin RNA (shRNA) to target exogenous reporter gene, enhanced green fluorescent protein (eGFP). The expression of eGFP in the cells was detected by using fluorescent microscopy and flow cytometry. Results: Using eGFP as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector can specifically inhibit gene expression. Ttransfection of the plasmid into P19 cells significantly decreased the number of eGFP-expressing cells and overall eGFP fluorescence. The RNA interfering effect was successfully observed in both transient and stable transfected cells.  Conclusion: The results indicate that use of hairpin siRNA expressing vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells.