Nucleofector 2 machine

Individual virus proteins were cloned into the pCAGGS vector containing a hemagglutinin tag at the N-terminus proximal to the insertion site (gift from Chris Basler) (44 (link)). The nsP and capsid proteolytic cleavage sites were chosen as the boundaries of the protein genes and a stop codon was added at the end of each gene. Viral genes were PCR-amplified from full-length cDNA clones using suitable primers with Not1 and Nhe1/Xma1 restriction sites added at the 5’ and 3’ ends respectively. Additionally, GFP was PCR-amplified from a previously constructed plasmid using suitable primers with Not1 and Nhe1 restriction sites added at the 5’ and 3’ ends respectively. PCR products and pCAGGS vector were digested and ligated, and individual clones were selected and confirmed by restriction digestion and sequencing. Huh7 and MEF cells were transiently transfected with plasmids (10 μg per plasmid) using the Nucleofector II machine and manufacturer’s protocols (Amaxa). Huh7 cells (1×10⁶ per reaction) were nucleofected using Kit V and protocol T-022. MEF cells (1×10⁶ per reaction) were nucleofected using Kit V and protocol T-020. Each transfection reaction was divided into two or three wells. A GFP-expressing plasmid was used as transfection control and reactions were used for experiments if GFP positive cells numbered >90%.

GIST-T1, GIST430, RMS176, LMS04 and EWS502 cell monolayers were disaggregated with trypsin and resuspended in Amaxa Nucleofector solution V (Amaxa Biosystems) at a concentration of 1 × 10⁶ cells per 100 μl. Nucleofection was performed using program T-030 on a Nucleofector II machine (Amaxa Biosystems). One microgram of pCR3.1-EGFP or pCR3.1-miniDMD plasmid was used for electroporation. Transfected cells were selected with G418 for 5 days before analyses.