title: Efficient lentiviral vectors for short hairpin RNA delivery into human cells.
abstract: 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.
authors.affiliation: 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.
authors.forename: Dong Sung
authors.initials: DS
authors.lastname: An
authors.forename: Yiming
authors.initials: Y
authors.lastname: Xie
authors.forename: Si Hua
authors.initials: SH
authors.lastname: Mao
authors.forename: Kouki
authors.initials: K
authors.lastname: Morizono
authors.forename: Sam K P
authors.initials: SK
authors.lastname: Kung
authors.forename: Irvin S Y
authors.initials: IS
authors.lastname: Chen
mesh_terms: D002460:Cell Line; D005095:Exoribonucleases / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D034741:RNA, Small Interfering; D008156:Luciferases; D012320:RNA Polymerase III; D005095:Exoribonucleases; C103661:U6 nuclease