What are the main criteria for best transfection/infection? - (May/21/2007 )
Hi friends, I am doing transfections using retroviral and lentiviral vectors on primary human epithelial cells. The lentiviral transfections on HEK 293T cells are excellent(always >>95%) but retroviral transfection is never >40% and infection never > 20%!!. WHat should be the reason for this? I have tried using just the empty vector(pMIG) but efficiency is too poor. Which criteria should I follow to achive maximum infection? What are your comments on cell status, passage number, confluency, DNA quality and quantity, duration, transfection reagent etc!
Virus production varied just because of the plasmid backbone. We have tried different vectors, some don't give us good productin infact poor like you described. You have to change some things with the vector or the helper plasmids or transfection. A lab next door, use fugene and use the 3 plasmid system and get good titres. But when we tried the same with lipofectamine, we dont get decent titres at all. But with our plasmids we get high titres even with a complete different plasmid from a second lab, it works.
cell passage number can matter.
In our lab, we always transfect with Fugene and get pretty impressive virus production.
As for the infection efficiency, you have to remember that retroviruses can only infect dividing cells. Lentiviruses can infect dividing or non dividing cells, and are therefore more powerful than the retro.
We only get good viral titer by doing calcium phosphate transfection. Lipofectamine never worked. In case of low virus titer we use magnetofection for better infection rate Ozbioscience. Then the titer is not so important.