Hybrid cytomegalovirus enhancer-h1 promoter-based plasmid and baculovirus vectors mediate effective RNA interference.
Plasmid and viral vectors harboring an RNA polymerase (Pol) III promoter would be useful in achieving sustained cellular expression of short interfering RNA (siRNA) to inhibit disease-associated genes. Given that transcription machineries directed by certain Pol II and III promoters may use common factors, we investigated whether the enhancer of the Pol II cytomegalovirus (CMV) immediate-early promoter could improve the efficacy of RNA interference mediated by the Pol III H1 promoter. We constructed a hybrid promoter by appending the CMV enhancer 5' to the H1 promoter. In the context of plasmid vectors, the hybrid promoter provided up to 50% greater inhibition of the expression of target genes than the unmodified H1 promoter and extended the silencing effect beyond that provided by the H1 promoter. Insect baculoviruses can infect a broad range of mammalian cell types. We constructed a baculovector expression cassette in which the synthesis of short hairpin RNA was under the control of the hybrid CMV enhancer-H1 promoter. This recombinant baculovirus vector was capable of suppressing expression of a target gene by 95% in cultured cells and by 82% in vivo in rat brain. These findings indicate that the hybrid CMV enhancer-H1 promoter can be used favorably for RNA interference.
Ong
Seow Theng
ST
Institute of Bioengineering and Nanotechnology, National University of Singapore, Singapore 138669.
Li
Feng
F
Du
Juan
J
Tan
Yu Wen
YW
Wang
Shu
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase; D003587:Cytomegalovirus / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D051381:Rats; D014162:Transfection / Q000379:methods
D015500:Chloramphenicol O-Acetyltransferase