Ong Seow Theng, Li Feng, Du Juan, Tan Yu Wen, Wang Shu
Institute of Bioengineering and Nanotechnology, National University of Singapore, Singapore 138669.
Hum Gene Ther. 2005 Dec;16(12):1404-12. doi: 10.1089/hum.2005.16.1404.
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.
携带RNA聚合酶（Pol）III启动子的质粒和病毒载体，对于实现短干扰RNA（siRNA）的持续细胞表达以抑制疾病相关基因将是有用的。鉴于某些Pol II和III启动子指导的转录机制可能使用共同因子，我们研究了Pol II巨细胞病毒（CMV）立即早期启动子的增强子是否能提高由Pol III H1启动子介导的RNA干扰的效力。我们通过将CMV增强子附加到H1启动子的5'端构建了一个杂交启动子。在质粒载体的情况下，杂交启动子对靶基因表达的抑制作用比未修饰的H1启动子高50%，并且使沉默效果超出了H1启动子的作用范围。昆虫杆状病毒可以感染多种哺乳动物细胞类型。我们构建了一个杆状病毒表达盒，其中短发夹RNA的合成受杂交CMV增强子-H1启动子的控制。这种重组杆状病毒载体能够在培养细胞中抑制靶基因表达95%，在大鼠脑内抑制82%。这些发现表明，杂交CMV增强子-H1启动子可有效地用于RNA干扰。
