. I am trying to produce Enterovirus virions by transfection. At first I tried to transfect the transcribed viral RNA into HeLa cells with lipofectamine 2000, but I did not get any infectious particles in the supernatant, and I couldn't determine the cause, my transfection system works well (validated by GFP plasmid transfection)... So i thought if i maybe used the cDNA to transfect the viral genome, a more stable form of genetic material i would have some better results. My question is which promoter should i use in front of my 5'UTR of the viral cDNA genome to be able to get viral RNA in cDNA transfected cells... Does the T7 promoter works?? if not what promoter should i use?? I appreciate your help! Thanks
Hi Michel yes, A T7 promoter and poly (A) tail should be added to the 5’ and 3’ends, respectively
Thank you all for your answers. I had some doubt about the viral cDNA i had, because i could see viral proteins when i transfected extracted infectious viral genome. So i received a new plasmid containing the genome of my virus, and now i am running my tests with this one, i hope it works! I can keep you up to date if you want... Thanks again, greetings!!
Hello again,
So i got my new plasmid, i transformed, extracted and purified it. After the digestion, i transcribed with T7 polymerase, that i transfected along with the plasmid (containing CMV promoter). At 24h i was able to observe infected cells, and the capsid viral protein by immunofluorescence, at 48h CPE was very clear in the transfected wells. This week i verified the presence of infectious paricles in the supernatant by adding it to cells, after 48h i had an CPE. => So i concluded that the previous viral cDNA had a mutation!! :S
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