TY - JOUR
T1 - Gene disruption by regulated short interfering RNA expression, using a two-adenovirus system
AU - Kuninger, David
AU - Stauffer, Daniel
AU - Eftekhari, Siavash
AU - Wilson, Elizabeth
AU - Thayer, Mathew
AU - Rotwein, Peter
PY - 2004/12
Y1 - 2004/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=11144224259&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=11144224259&partnerID=8YFLogxK
U2 - 10.1089/hum.2004.15.1287
DO - 10.1089/hum.2004.15.1287
M3 - Article
C2 - 15684704
AN - SCOPUS:11144224259
SN - 1043-0342
VL - 15
SP - 1287
EP - 1292
JO - Human Gene Therapy
JF - Human Gene Therapy
IS - 12
ER -