I am trying to transduce primary murine cerebellar granule cells using a lentiviral system. I tried transduction with polybrene, but it seems to be quite toxic for my cells. However, transduction efficiency without polybrene is quite low, both with and without spinfection. Does anyone have experience with less toxic alternatives to polybrene for transduction of neural cells or sensitive primary cells in general?
Thanks a lot in advance!
Hi Carolin,
In our experience, we also observe toxicity with polybrene and we no longer use it and design other strategies when we have to do complexe transductions.
Indeed primary cells are hard to transduce and you have to optimize all the parameters. First is to use very efficient and concentrated vectors to make possible the use of high MOI in small volumes of transudction.
What is the titer of your vectors?
Hi Nicolas,
Thanks a lot for your response!
My titer is around 8x10^7 IU/mL, but I guess I could optimize viral production and titers as I am still working on the protocol.
Our primary cells are quite small and are usually seeded at a high density of 1x10^6 per 24-well. This makes it difficult to apply high MOIs in small volumes. What range of MOIs would you recommend for transduction of primary cells?
For primary cells, we use MOIs between 20 and 100 with about 1E5 cells, depending the experimental context and the titer of our vectors. However, to do that you have to use vectors with a tier>1E9TU/mL
Hi Carolin,
you try spin-infection (@300 xg for 1h at 22C) for your small volume with MOI 5 to 10 worked for us.
Nicolas Grandchamp Nagarjun Kasaraneni
Thanks for your helpful advice!
I will try to increase my titer and test out your spinfection protocol.