Hi Alek,

thanks for your answer.

I am producing the viruses in HEK293T cells and it works super fine for me. Let me know if you have any questions about culturing etc.

What I am looking for is a neuronal cell line that I can use instead of primary cells to test the ready-to-use AAV before they are used in vivo.

Dear Tanja,

thanks for your comment.

The readout for my transduction is GFP expression as well (or any other detectable reporter signal). It takes about 3-5 days for AAV to transduce primary neurons - this is the time frame where you should see a first signal. This signal increases with time. In the neuronal cell lines, that I´ve tested so far, there was still no signal after 7-10 days. My produced AAV´s have titers around 1011-1013gc/ml.

The promoters I am using are either ubiquitous or neuronal promoters.

Hi Tanja,

thanks a lot for your reply. Yes, I did test the MOI (Multiplicity of infection) by trying different concentrations of the viruses to obtain the optimal amount for infection without being toxic. Unfortunately the transduction didn´t work in adhaerent cells (it works great in primary neurons). Are you using replication deficient AAV or replication active AAV?

Hi Tanja,

the transduction efficiency is absolutely depending on the serotype of the AAV and on the cell type they are used for. The paper you indicated describes the transduction with AAV8 in N2A cells - this is unfortunately not comparable with AAV1/2 in the same cell type.

Due to this it doesn´t necessarily mean, that you get more signal, if you add more virus.

Your conclusion is correct: You need lower MOI for primary neurons than for any other immortalized cell type, because primaries are not dividing anymore.

Best,

Maria

Hi Catherine,

the MOI depends on the virus and the serotype that is being used. Normally you would determine the MOI for each virus separately before starting the actual experiment. in vitro.

I used MOIs between 1x10_3 and 1x10_4 for primary cells.

I would recommend to determine the MOI prior to the actual experiment.

I hope this is of help.

Best,

Maria