Luciferase t7 control dna plasmid

Plasmids pSGR JFH-1 Fluc WT and pSGR JFH-1 Fluc GND encode sub-genomic JFH-1-derived HCV replicons with a firefly luciferase reporter; the GND contains an inactivating mutation in the viral polymerase [57] (link), [58] (link). Plasmids pSGR S1+S2:p3 Fluc WT and pSGR S1+S2:p3 Fluc GND have C to G mutations at position 3 in both miR-122 seed binding sites in the HCV 5′ UTR and were described in [26] (link). Plasmids pJ6/JFH-1(p7-Rluc2a) “FL WT” and pJ6/JFH-1(p7-Rluc2a) GNN “FL GNN” bear full-length viral sequences derived from the J6 (structural proteins) and JFH-1 (non-structural proteins) isolates of HCV, and a Renilla luciferase reporter, with the GNN having inactivating mutations in the viral polymerase [59] (link). Firefly luciferase control mRNA was transcribed from Luciferase T7 Control DNA plasmid (Promega; Nepean, ON, Canada), while Renilla luciferase control mRNA was transcribed from the pRL-TK plasmid (Promega). Plasmid templates for viral RNA and mRNA were prepared and in vitro transcribed with the MEGAScript T7 High Yield Transcription Kit and mMessage mMachine T7 Transcription Kit (Life Technologies; Burlington, ON, Canada), respectively, as described in [26] (link).

The pJFH-1_T plasmid, encoding a cell culture-adapted Japanese Fulminant Hepatitis (JFH-1; HCV genotype 2a) with three adaptive mutations that increase viral titers in cell culture, was provided by Rodney Russell (Memorial University of Newfoundland, St. John’s, NL, Canada) [17 (link)]. The pJ6/JFH1 plasmid bears a full-length viral sequence derived from the J6 (structural genes through first transmembrane domain of NS2) and JFH-1 (remaining NS genes) isolates of HCV, and the pJ6/JFH-1 FL RLuc GNN (“RLuc-GNN”) viral sequence also includes a Renilla luciferase (RLuc) reporter gene inserted between p7 and NS2 and an inactivating GNN mutation within the NS5B RNA polymerase active site [4 (link),18 (link)]. The pJ6/JFH-1 mono RLuc-NS2 plasmid (“Δcore-p7”), a truncated version of the Renilla reporter virus with a deletion of the structural genes through p7, was provided by Joyce Wilson (University of Saskatchewan, Saskatoon, SK, Canada) [19 (link)].
To make full-length uncapped viral RNAs, all plasmid templates were linearized and in vitro transcribed as previously described [20 (link)]. The firefly luciferase (FLuc) mRNA was transcribed from the Luciferase T7 Control DNA plasmid (Promega, Madison, WI, USA) linearized using XmnI and in vitro transcribed using the mMessage mMachine T7 Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.