Protein function analysis: Rapid, cell-based SiRNA-mediated ablation of endogenous expression with simultaneous ectopic replacement

Abstract

Current methods for determining and dissecting the function of a specific protein within a cell are laborious and limiting. We have developed a method by which endogenous protein levels are rapidly ablated and simultaneous expression of a designed, inserted variant takes place in the native setting. Through optimized electroporation, siRNA oligonucleotides and codon-optimized coding sequence containing vectors can be co-transfected, leading to expression of ectopic mRNA not targeted by siRNA. Using the commonly encountered MCF-7 breast cancer cell line, we were able to reach 90% transfection efficiency. Under these conditions, siRNA oligonucleotides were transfected simultaneously with a codon-optimized, cDNA containing vector encoding the AHR protein. Thus, endogenous protein was ablated while the designed protein was fully expressed in the native environment. The codonoptimized AHR was shown to be fully functional in its ability to induce CYP1A1 transcription and to rescue a B[a]P-susceptible phenotype. © Springer Science+Business Media B.V. 2010.