T7 rna polymerase maxiscript kit

Recombinant luciferase-expressing VEEV plasmid (VEEV-3908-FLuc) and recombinant wild-type VEEV plasmid (VEEV-3908-WT) were obtained from Dr. Scott Weaver (University of Texas Medical Branch), and viruses were generated as follows. MluI linearized plasmids were purified by QIAprep Spin Miniprep Kit (Qiagen) and genomic RNA was transcribed in vitro using a T7 RNA Polymerase MAXIscript kit (Life Technologies). Vero E6 cells (4 × 10⁶ cells in 400 μL) (ATCC) were electroporated with genomic RNA using an ECM 630 electroporator (BTX Harvard Apparatus). Two pulses (450 V, 1200 Ω, and 150 μF) were administered. Forty-eight hours post electroporation virus was recovered from supernatant. EEEV-FL93-NLuc plasmid was obtained from Dr. William Klimstra (The University of Pittsburgh) and rescued as described (Sun et al., 2014 (link)). WEEV-McM-FLuc was constructed and generated as previously described (Phillips et al., 2016 (link)). All recombinant viruses were used without further passage after rescue. All viral stocks were titered by plaque assay on Vero cells as described previously (Mirchamsy and Rapp, 1968 (link)) and stored at −80° C.

Construction of recombinant WEEV (MacMillan strain) reporter viruses was previously described (Logue et al., 2009 (link); Phillips et al., 2013 ; Phillips et al., 2016 (link)). Briefly, a duplication of the subgenomic promoter (SGP) sequence (nucleotides 7341–7500 of viral genome) of the WEEV McMillan strain was used to express firefly luciferase or DsRed. Plasmids were purified by QIAprep Spin MiniPrep Kit (Qiagen, Valencia, CA USA) and RNA was transcribed in vitro using a T7 RNA polymerase (MAXIscript™ kit, Life Technologies, Grand Island, NY USA). BHK-21 cells (2×10⁷ in 400 μL) were transfected with 20 μL of total RNA using an ECM 630 electroporator (BTX Harvard Apparatus, Holliston, MA USA). The rescued virus was stored at −80°C before plaquing. All plaque titration assays were run in duplicate in Vero cells before experimental use as previously described (Liu et al., 1970 (link)).

In situ hybridization was conducted as described previously (Asok et al., 2013a (link); Asok et al., 2013b (link)). An antisense RNA probe (riboprobe) was transcribed from a plasmid containing a sense cDNA sequence coding for a 230 bp sequence of Egr-1 (gift from J. Milbrandt, Washington University, St. Louis, MO). The transcribed riboprobe incorporated a radioactively labeled ³⁵S UTP (approximately 1×10⁶ dpm) using a T7 RNA polymerase Maxiscript kit according to the manufacturer’s instructions (Life Technologies, Grand Island, NY). After hybridization and washing, the dry slides were exposed to Kodak Biomax MR Film for 2 days.