Gene disruption by regulated short interfering RNA expression, using a two-adenovirus system.
Specific gene ablation by RNA inference (RNAi) involves the binding of short interfering RNA (siRNA), 21 to 22 nucleotides long, to complementary mRNA sequences, leading to sequence-specific posttranslational gene silencing, thus providing a powerful tool for studying gene function with potential therapeutic applications. Here we describe the development of a two-vector adenovirus system for efficient, tightly controlled hairpin siRNA expression (shRNA). Regulated expression of the shRNA is conferred within an adenoviral vector by a modified RNA polymerase III promoter containing a Tet operator element adjacent to the transcription start site. In the presence of the tetracycline repressor protein (TetR), encoded in a second adenovirus, shRNA expression is repressed. Addition of tetracycline abolishes TetR binding, allowing shRNA transcription to proceed, and leading to reduced mRNA and protein expression. Here we establish the efficacy of this system by delivering siRNA targeted against the transcriptional coactivator p300. Our results show tetracycline-mediated inhibition of p300 mRNA and protein accumulation in the presence of both viruses, but no effect in the absence of antibiotic. Regulated adenoviral shRNA vectors offer the advantages of being able to infect a wide array of replicating and nonreplicating cells and of allowing temporal control of gene silencing.
Kuninger
David
D
Molecular Medicine Division, Oregon Health and Sciences University, Portland, OR 97239, USA.
Stauffer
Daniel
D
Eftekhari
Siavash
S
Wilson
Elizabeth
E
Thayer
Mathew
M
Rotwein
Peter
P
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D012313:RNA; D012320:RNA Polymerase III; D013752:Tetracycline