How long does it take for a high titre AAV to start expressing in HEK293 cell line? Mine hasn't shown any expression even after 48hrs.
Dear Pranav,
It is good to know that you have a batch of AAV particles with high titre.
However, in your case it is crucial to know the AAV serotype and the MOI used to transduce the HEK cells.
Provided your AAVs belong to AAV2 or AAV6 serotype, contain a CMV (or any other strong ubiquitous promoter that can be expressed in HEK cells) and the HEK cells have been transduced with an MOI of 10^4 (or similar) then you should be able to see expression latest within 48-72 hours.
When I have transduced HEK cells with AAV6 GFP, I can see GFP positive HEK cells as early as 24 hours which increases gradually from 48h to 72h. By 48hours AAV6 GFP can transduce above 80% of the HEK cells.
If you are using other AAV serotypes (for eg AAV9) then it will not work efficiently for cells in vitro. AAV9 works really well for in vivo set up with high tropism towards liver and heart.
Hope this helps you with troubleshooting.
Good luck!
Best wishes
Shambhabi
Thank you Shambhabi for the quick reply. I think this makes sense. Regards
Shambhabi Chatterjee
Hi
Do you have any reference or hands on experience with AAV9? Actually, I am struggling with in vitro transduction of AAV9 GFP in Hela or Hek293 cells.
Regards
Sajib
Abdul Mohin Sajib
I did in the end manage to get in vitro transduction in my HEK293 cells.
For me it took about 8 days for them to start expressing.
Dear Abdul Mohin Sajib
We normally produce AAV6 GFP for transducing HEK / in vitro cells and it works really well (AAV2 has also worked really well in vitro for us).
We test AAV9 constructs for in vivo experiments in our lab and do not use them in vitro for cells.
In general if your experiments are aimed for infecting cell lines with AAV particles in vitro, then I will recommend to use AAV2 or AAV6 serotype.
Otherwise as suggested by Pranav Seth you might wait for 8 days after AAV9 transduction and see if the HEK cells turn green.
The AAV serotype of choice primarily depends on your target cell type in vitro / organ in vivo.
The Table 1 in this publication provides a good overview of the tropism of different AAV serotypes (in vivo and in vitro)- Article Optimization of AAV vectors to target persistent viral reservoirs
Hope this will be helpful.
best wishes
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