Hi Marsha,

What AAV serotype are you working with?  Usually using AAV2 or AAV5 you would see some GFP after 24 hrs.  

On a side note, why are you titering your vector using this type of assay?  The standard assays for titering AAV are qPCR for genomes and SDS-PAGE for total capsid.  

Good luck,

J

Hi Jonathan,

It's AAV serotype 2. I decided to give this protocol a go because other people have argued that qPCR based titration tends to overestimate viral titer. I will certainly give qPCR a try as well! Thank you

Hopefully you will see some expression after 48 hrs.  If not, I would try transfecting the plasmid that you used to make the vector to verify that it expresses GFP.  

Good luck

J

I would say that if you see AAV after 24h than your titer should be high. It could take 3 days to see the fluorescence under the microscope without staining, especially for high dilution. AAV is ssDNA virus, and there are 2 models at the moment how ssDNA AAV could express recombinant genes - it should form concatemers or replicate. Both processes take some time to generate enough FP to be visible under fluorescent microscope. Fluorescence level also depends on promoter strength. And even with strong promoter after 5 days of infection you could miss some weak cells. So, I always stain for the FP marker for precise calculation. Using infection of HEK, you will calculate the number of infectious AAV particles that will drive expression of your recombinant gene. If you use other methods, e.g. qPCR or ELISA, you will detect many damaged particles that are not infectious, but still contain viral DNA, or empty particles. Expect drop by a few orders of magnitude in the measured titer when you compare qPCR with HEK infection.

With respect to Konstantin's answer, I remember when I was working in the Adenovirus field and they had the same issues around how to titer, based on genome/particles or infectious units.  After some extensive work it was found that the problem with using an infectious titer assay is that it tends to underestimate the actual functional titer.  The infectious titer was found to change greatly once the transduction protocol was optimized.  If you will only be transducing HEK 293 cells, then doing an infectious titer makes sense, but keep in mind that the way that you perform the transduction (e.g. addition of dox and/or ponasterone A) will have a big effect on transduction efficiency.  What you might see is that as you optimize the transduction, those "damaged" and non functional genomes might not be so non-functional after all.  This will also mean that your infectious titer will be quite variable. 

Additionally, a part of the reason why AAV needs so much higher MOI's than other vectors is that it is very inefficient and many genomes are lost at each step of the transduction process (cell entry, endosomal release, conversion to stable ds genome).  Thus, it may take 1000 genomes in order to get the one or two that end up stably expressing.  This doesn't mean that the other 999 genomes are non functional, just that the process is inefficient. 

Dear all, I am currently facing some problem with AAV production. I have a construct that is expressing very well in HEK cells. When expressing with pHelper and AAV-DJ, I can see the the nice expression. I could see also a very high titer when qPCR was done against WPRE. But I could not see the infection when I used the viral particles to infect again the HEK cells. Could you please help me with trouble shooting. Thanks a lot.

http://www.cellbiolabs.com/sites/default/files/VPK-400-DJ-aav-helper-free-packaging-system.pdf

When we tried to titer our AAV's in HEK cells we didn't see very much GFP expression.  But when we used HT1080 cells, there was tons of GFP.  I'm not sure if HEK cells are the best cell line for testing titers, although I have seen others mention that they have used them.

@ Rahul Kaushik Did you get your problem solved? I have exactly the same issue. I am using AAV6 serotype from cellbiolabs. Tons of GFP expression after transfection, but no transduction at all in HT1080T cells with the crude lysate.

Thanks

I have found that it can take a long time to see transduction with GFP or mCherry in vitro, as long as 7 days for optimal viewing. Unfortunately, this is also the time when cells are starting to die off/become overgrown. I have had much better luck with alkaline phosphatase, which can be stained 24 to 48 hours post transduction. You might also want to try concentrating or purifying your virus.

For this purpose, we have developed some AAV purification kits that might be useful. Currently, we have 2 different scales for AAV purification: mini and midi. Our mini kit can purify from between 0.5 mL to 10 mL of cell culture media, cells, or the two mixed together. The midi kit can purify from 10 mL to 50 mL of similar input. I have found that as little as 0.5 mL of mixed cells/cell culture media input can lead to visible expression of an alkaline phosphatase transgene in vitro.  Only standard laboratory equipment ie, a swinging bucket centrifuge, microfuge is required, no ultracentrifugation necessary. The process is rapid (1 to 1.5 hours) and multiple purifications can be done in parallel to accommodate large screening experiments. We are also working on larger scale purification (50 mL to 1 liter or more).

Please send a message or visit our website for more information:

https://norgenbiotek.com/category/virus-purification

Good luck!

Hi Marsha

293T cells is not a good model to determine AAV titer because in vitro cells need very high MOI （multiplicity of infection）of AAV to get a visible GFP-indicated infection, nearly >10^5，with such a high MOI, there was no linear correlation between GFP positive ratio and titer at all. QPCR for WPRE or ITR sequece is a classical method.

Genemedi is experienced in AAV production, you could find more information on this website: www.genemedi.net/i/aav-packaging

Dear Researcher,

Your inquiry about observing GFP expression in HEK293 cells 24 hours post-transduction with unconcentrated AAV is a relevant question in the field of gene therapy and molecular biology. The success of AAV-mediated transduction and subsequent GFP expression can be influenced by several factors. Here are some key aspects to consider and optimize in your experimental setup:

In summary, successful GFP expression in HEK293 cells post-transduction with AAV involves optimizing various parameters such as AAV serotype, MOI, time post-transduction, cell culture conditions, and detection methods. Additionally, including proper controls will help in accurately interpreting the results of your transduction experiment.

Best regards,

Check out this protocol list; it might provide additional insights for resolving the issue.

As mentioned above, HEK cells are poor for testing AAV titer as they are pretty refractory to transduction (as are many common cell lines). HT1080 cells are a much better choice. They're available from Agilent as part of their AAV Helper Free system, but are also available from other places.