title: 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 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.
authors.affiliation: Department of Veterinary and Biomedical Sciences and Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, CMTC, 309 LSB, University Park, PA, 16802, USA.
authors.forename: Brett C
authors.initials: BC
authors.lastname: DiNatale
authors.forename: Gary H
authors.initials: GH
authors.lastname: Perdew
references.citation: Biophys J. 1992 Nov;63(5):1320-7
references.pmid: 1282374
references.citation: Proc Natl Acad Sci U S A. 1979 Jan;76(1):373-6
references.pmid: 106390
references.citation: Cancer Res. 2008 May 15;68(10):3609-17
references.pmid: 18483242
references.citation: Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2322-7
references.pmid: 8637871
references.citation: Annu Rev Pharmacol Toxicol. 1999;39:103-25
references.pmid: 10331078
references.citation: Anal Biochem. 2009 Mar 15;386(2):251-5
references.pmid: 19150324
references.citation: Lab Invest. 2009 Jun;89(6):695-707
references.pmid: 19333233
references.citation: Crit Rev Eukaryot Gene Expr. 2008;18(3):207-50
references.pmid: 18540824
references.citation: Nucleic Acids Res. 2006 Jan 23;34(2):e13
references.pmid: 16432257
references.citation: J Exp Med. 2009 Jan 16;206(1):43-9
references.pmid: 19114668
references.citation: J Biol Chem. 1996 Apr 5;271(14):7942-8
references.pmid: 8626473
references.citation: J Biomol Tech. 2008 Dec;19(5):328-34
references.pmid: 19183796
references.citation: Biophys J. 1991 Oct;60(4):804-11
references.pmid: 1660315