Can I transfect my viral vector directly into cells, or do I need to make virus first?

Direct transfection of cells with the viral vector (rather than using live virus) may facilitate expression of your gene of interest (GOI), but there are a number of complications (see below). We therefore recommend that viral vectors be used for production of live virus, and not for direct transfection of cells.

Virus transduction can usually deliver DNA into target cells more efficiently than plasmid transfection. When using retrovirus such as lentivirus or MMLV, the viral genome can integrate into the host cell genome so that genes carried on virus can be stably expressed. By contrast, transfected vector plasmids only have transient expression in the cells since they do not integrate into the host genome. For retroviral vectors, comparing to virus transduction that has low copy number in the host genome, direct transfection of plasmids can often result in very high copy number in cells, which leads to very high expression levels of the genes carried on the vector. However, this can be very non-uniform (some cells can contain many copies while others carry very few or none).

Our lentiviral vector plasmids contain a strong RSV promoter within the 5' LTR, which is used to drive transcription of the viral RNA genome during virus packaging. After transduction of cells with the packaged lentivirus, the promoter activity of the 5’ LTR is inactivated, so it will not affect the expression of the user’s GOI present between the two LTRs in the viral vector. However, if the viral vector is used to directly transfect cells, the 5’ LTR promoter activity will remain active. This can have a number of effects, including activating, distorting, or even inhibiting expression of downstream gene(s) within the lentiviral vector.

Additionally, due to the presence of components necessary for virus production, viral vector tends to be significantly larger than regular plasmid containing the same expression cassette. As plasmid size increases, the efficiency of DNA preparation and plasmid transfection both decrease, which may result in very low efficiency for many viral vectors when being used in direct transfection. This matters a lot for adenoviral vectors which are more than 30 kb.

We recommend that you use VectorBuilder offered virus packaging services, which utilize a wide range of proprietary technologies to provide you with high-quality, high-titer viruses at lower cost and faster turnaround than what you can do on your own.