Psuper retro

Luciferase reporter plasmids containing an IFN-sensitive response element (ISRE), NF-κB, or IFN-β promoter conjugated to the firefly luciferase reporter gene and mammalian expression vectors expressing RLR signaling pathway components, including RIG-I and its mutant RIG-I-N (deleted C-terminal repressor domain and DECH-box helicase domain), MAVS, TBK1, IKKε, IRF3 and its point mutant IRF3-5D (active form of IRF3), and ubiquitin and its mutant K48 or K63 ubiquitin, were prepared as described previously [7 (link),27 (link)]. Human THOC7 (hTHOC7) was cloned into a cytomegalovirus promoter-based mammalian expression vector along with a sequence encoding an N-terminal HA, FLAG, or Myc tag using standard molecular biology techniques. THOC7-specific siRNA constructs were generated by cloning double-stranded oligonucleotides corresponding to the hTHOC7 target sequence into an RNA interference (RNAi) vector pSuper.retro (OligoEngine, Seattle, WA, USA) according to the manufacturer’s protocol. The following target sequences were designed for hTHOC7 cDNA: THOC7-RNAi#1, 5′-GGAAACAGTTTCATGTTCT-3′; THOC7-RNAi#2, 5′-CCAGACAGGCATGAGACAT-3′; and THOC7-RNAi#3, 5′-TCTTCTTAGTACCATCCAT-3′.

Plasmids expressing ISGs were cloned by standard molecular biology techniques. IFN-β-luc reporter, pTK-renilla, Flag-tagged RIG-I, MDA5, VISA, TBK1 and IKKε were generous gifts from Dr. Hongbing Shu (Wuhan University, China). The plasmids expressing shRNAs were generated by annealing pairs of oligonucleotides and cloning into pSuper-Retro (OligoEngine). The target sequences are as follows: Ctrli: 5′-GCGCGCTTTGTAGGATTCG-3′; shPIM1: 5′-CCATCCATGGATGCAAGAT-3′. SeV was kindly provided by Zhengfan Jiang (Peking University, China).