An Dong Sung, Xie Yiming, Mao Si Hua, Morizono Kouki, Kung Sam K P, Chen Irvin S Y
Department of Microbiology, Immunology, and Molecular Genetics and Medicine, UCLA AIDS Institute, David Geffen School of Medicine at UCLA, University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.
Hum Gene Ther. 2003 Aug 10;14(12):1207-12. doi: 10.1089/104303403322168037.
RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.
RNA干扰是一种进化上保守的基因沉默过程，在植物中它作为抵御外源病毒因子的天然防御机制。通过反向遗传学，RNA干扰正成为研究生物学过程的重要工具，并且在人类治疗应用方面具有潜力；然而，有效的递送仍然是一个主要问题。小分子干扰RNA（siRNA）和短发夹RNA（shRNA）已通过化学合成转染以及利用RNA聚合酶III转录的质粒盒进行RNA表达而被导入细胞。使用siRNA/shRNA进行基因敲除需要高效稳定的转染或转导过程。在此，我们报告了成功构建慢病毒载体以在人细胞中稳定表达shRNA。我们证明，在无药物筛选的情况下将表达靶向报告基因荧光素酶的siRNA的慢病毒载体稳定转导至人细胞后，这些载体能够保护细胞免受编码人源化萤火虫荧光素酶作为报告基因的慢病毒载体的感染。相对于用对照载体转导的细胞，我们观察到用shRNA载体转导的感染细胞中的基因表达降低了16至43倍。该模型系统证明了慢病毒载体作为细胞基因敲除工具以及作为抑制人细胞中外源感染因子（如病毒）的手段来稳定表达shRNA的实用性。
