Hi everyone, I have been using the AAVpro® Helper Free System from TAKARA to generate AAV2 particles with HEK293T cells. However, after the AAV extraction step, the titer is only ~3x10e5 vg/ul using qRT-PCR for titration, which is much lower that it is should be (based on the TAKARA protocol, the titer after extraction is ~10e8-9 vg/ul). The TAKARA suggested me to follow the protocol, though I have followed it strictly. Does anyone have the experience or advice about it? What should I do to improve the titer? Does it really help if I change the cell line from 297T to 293T-17, which is said to generate more AAV particles?
Many thanks!
You can please refer to the links below to obtain certain interesting informations:-
1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308604/
2. http://www.biomedcentral.com/1471-2202/15/28
That titre is really low, so there's definitely something fundamentally wrong. Have you checked and sequenced your AAV plasmid? What transfection method did you use? If you're using a fluorescent protein, what do you see after transfection?
I used specific restrictive enzymes to identify the AAV plasmid, and it seemed to be correct. I used the calcium phosphate method with the CalPhos™ Mammalian Transfection Kit (TAKARA). The fluorescent protein is mCherry. I added the extracted AAV into HEK293T (MOI=10e3), and the fluorescent was fine and the infection efficiency was ~80-90%. I am not sure which step goes wrong. Thank you all for helping me with this predicament...
While that is not always the case, we have found it much easier to produce high titers of AAV5 and some of the engineered serotypes such as DJ and DJ8. We use a slightly different process as referenced above, but we generally produce minimum titers more in the range of 1-3 e12. Our purification process uses a ultracentrifugation step to separate the virus from lysate, though, which is very different from the TAKARA kit. We have not seen a large divergence in titers using either AAV293 or FT293 cells, so unless you have issues with the viability of your cell line I don't think that that's the issue, especially given that you are seeing stable transduction from your reporter protein. Are you sure that your standards used to titer are reliable?
Ah OK, so when you add AAV to 293Ts at an MOI of 1000 you get plenty of infection. Can you do a serial dilution to infect multiple wells (say 1ul, 0.1ul, 0.01ul...), until the infection rate is low enough for you to count individual cells. This way you can accurately determine your infectious titre.
The infectious titre will always be lower than the QPCR titre, and that's also very much serotype dependent (eg AAV-DJ is great in culture, AAV-8 isn't good at all but in vivo they both work well). However, if you get an infectious titre of about 1e7 or above then your packaging is working fine, and it's just the QPCR that's at fault.
Thank you all for these valuable suggestions. I really really appreciate it!
I will try to modify these procedures. Many, many thanks!
I am also facing the similar problem with takara AAV purification kit, I have tranfected 300 ug of pAAV-CMV ,, still I am getting 10^7 vg/ul ..can anybody help me?@ Tianci Chu have you solved the problem? I will be immensely grateful if you kindly share the trouble shooting procedure.
Increasing the titer of Adeno-Associated Virus serotype 2 (AAV2) can be important for various applications in gene therapy, gene delivery, and molecular biology. A higher titer allows for more efficient transduction of target cells. Here are some strategies to increase the titer of AAV2:
Remember that optimizing AAV2 production can be a complex process, and the strategies may need to be tailored to your specific experimental setup and goals. Additionally, always follow biosafety guidelines and ethical considerations when working with viral vectors like AAV2.
l This list of protocols might help us better address the issue.