Plasmid driven reverse genetics:

293 T cells human embryonic kidney cells were transfected with 17 plasmids( 8 constructs for the 8 RNA segments and 9 constructs for 9 structural proteins), each encoding a viral RNA of influenza virus A except the M gene, flanked by human RNA polymerase I promoter and mouse RNA polymerase I terminator sequences. Transcription of these viral genes by cellular RNA polymerase I yielded all eight influenza viral RNAs. Cotransfection with protein expression plasmids for the nucleoprotein and polymerase proteins resulted in the generation of >10⁷ infectious viruses per ml of supernatant.

  Only the M genes of the transfected virus was sequenced to confirm the presence of the intended mutations.