Hi all,
I am producing AAV vector-containing shRNA to knockdown my target protein.
Three pDNAs (transfer plasmid, pHelper, RepCap) are transfected into HEK293A (80% confluency). I can get a very high yield of transfected cells by checking GFP (AAV vector that I use contains CMV promoter). AAV particle will be purified 72hrs post-transfection using this kit ( https://www.takarabio.com/assets/documents/User%20Manual/6235_e.v1509Da.pdf ). As stated in the product manual, the AAV particles extracted by this kit are suitable for in-vitro experiment.
However, I have tried to use this AAV (extracted from that kit) to infect the some cell line that I have. The infection efficiency is very low (by checking GFP)
I would to ask if the AAV particles extracted by that kit is cleaned enough to use for in-vitro?
I found this paper ( https://www.nature.com/articles/s41598-019-49624-w ). The paper mentioned that AAV particles were purified by PEG/NaCl precipation and Chloroform extraction before using in in-vitro experiments
Depending on your recipient cells, AAV doesn't need to be clean at all. What serotype are you making, and what are your recipient cells?
Some AAV serotypes don't work well in vitro. You can try 4uM Etoposide to enhance transduction but remove it after 6-12 hours as it's quite toxic
A couple of things - this paper used scAAV, which might perform differently from ssAAV. Also, they used an MOI of 100 000 - that's very high! Do you know the titer of your preps?
which plasmid do you use to make your standard curve using ITR? not all plasmids giving consistent results relying on the ITR-containing sequence.
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