title: Hybrid cytomegalovirus enhancer-h1 promoter-based plasmid and baculovirus vectors mediate effective RNA interference.
abstract: 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.
authors.affiliation: Institute of Bioengineering and Nanotechnology, National University of Singapore, Singapore 138669.
authors.forename: Seow Theng
authors.initials: ST
authors.lastname: Ong
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.forename: Yu Wen
authors.initials: YW
authors.lastname: Tan
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
mesh_terms: 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
chemical_list: D015500:Chloramphenicol O-Acetyltransferase