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

摘要

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 codon-optimized AHR was shown to be fully functional in its ability to induce CYP1A1 transcription and to rescue a B[a]P-susceptible phenotype.