Coupling of RNAi-mediated target downregulation with gene replacement.
Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).
Kim
Dong-Ho
DH
Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Rossi
John J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D003062:Codon; D005786:Gene Expression Regulation; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014162:Transfection
D003062:Codon; D008164:Luminescent Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins