title: A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system.
abstract: RNA interference technology is a widely used tool for the regulation of gene expression at the post-transcriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs.
authors.affiliation: Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China; State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.affiliation: Department of Cell Biology, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China.
authors.forename: Qiangguo
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Zongming
authors.initials: Z
authors.lastname: Dong
authors.affiliation: Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Tengfei
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China.
authors.forename: Xinyin
authors.initials: X
authors.lastname: Guan
authors.affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China. Electronic address: hellojjx@126.com.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Jiang
mesh_terms: D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D050839:Interferon Regulatory Factor-7 / Q000235:genetics; D016086:Lentivirus; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: C494234:IRF7 protein, human; D050839:Interferon Regulatory Factor-7; D034741:RNA, Small Interfering
keywords: Dual luciferase assay; Lentivirus delivery; siRNA screening