title: Construction and identification of an RNA interference lentiviral vector targeting the mouse TNF-α gene.
abstract: The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-α (TNF-α) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-α gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-α mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNF-α protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)-1β and IL-6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2×106TU/µl. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-α gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for thein vivostudy of RNAi gene therapy targeting TNF-α.
authors.affiliation: Department of Rheumatology and Clinical Immunology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Jibo
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Rheumatology and Clinical Immunology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Hongda
authors.initials: H
authors.lastname: Liang
authors.affiliation: Department of Rheumatology and Clinical Immunology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Central Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Xiangping
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Central Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.affiliation: Department of Central Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Sui
keywords: RNA interference; lentiviral vector; macrophages; tumor necrosis factor-α