I have some questions about using AAV in SH-SY5Y cultures:
- What kind of MOI / # genome copies per ml or similar would you start out with to test in cultures? I plan to use SH-SY5Y (neuroblastoma) cultures. I have read e.g. 1E4, does this mean 1E4 genome copies in a culture well (24-plate well?). Or do people actually mean that they add 1E4 = 10 000 gc per every cell in the culture dish? I plan to use 8-well chambered slides, where I can go as low as 300 ul in volume per well.
- We are also going to try to use the AAV-DJ serotype (Grimm et al.) as it has been claimed to work better in culture, perhaps this needs lower MOIs, does anyone have experience with this?
- Would you lower the serum concentration or fully remove the serum during transduction (and even 'wash' the cells before?). We normally grow the SH-SY5Y cells in F12:EMEM (1:1), 15% FBS, 1% non-essential amino acids, 1% L-glutamine, 1% penicillin/streptomycin.
Thanks for any help you may be able to provide!
Dear Josephine,
this post may seem to be a off-topic but I think it could be usefull ; very briefly I would like to present you the Magnetofection technology to enhance infection/transduction cells with viral particles. We have several magnetic beads dedicated either to lenti/retro or adenovirus, and recently AdenoMag was also demonstrated to enhance transduction/infection with AAV serotype 2 in fibroblasts.
- Adenomag : http://link.springer.com/article/10.1007/s11095-011-0629-9
- AAV2 + AdenoMag: https://repositories.tdl.org/uh-ir/handle/10657/ETD-UH-2012-05-418
I do know this will not directly answer your inquiry, but this may be of help. So please do not hesitate to contact me at [email protected] if you want to try this technology.
Good luck with your experiment,
Cedric
https://repositories.tdl.org/uh-ir/handle/10657/ETD-UH-2012-05-418
http://www.ozbiosciences.com/transduction-infection/29-adenomag-magnetofection-reagent-adenovirus-transduction.html
Yes, AAV-DJ is very good in culture - the best I have seen of all the AAV serotypes (some serotypes are just as good in vivo but DJ is the best in almost every type of cultured cell). I've used AAV-DJ in B35 neuroblastoma cells and it works great (pic attached).
I normally don't remove the serum, I just add the virus straight to the culture media. The MOI you use DOES depend on how the virus was titered. For example, you may have an infectious titre of 1e9 and a PCR titre of 1e13 - how was your virus titered? If you bombard the cells with massive quantities of AAV they do tend to die.
I also infect in the presence of etoposide - it really increases infection efficiency but it's toxic to the cells - change the media after 24h if possible.
One other thing - AAV doesn't integrate so as the cells divide, you will lose infected cells. The AAV may even be actively degraded so if your cells replicate quickly then infected cells will dilute out fairly rapidly.
Hope that helps!
Hi Jill,
Thank you for your response.
We have qPCR titres of 1e13. By 'infectious titre', you basically mean you try some different 1eNN until you get transduction in your cell type of interest? If so, what kind of scale do you use for a well slide with 1e5 cells seeded, how would you go about adding the AAV?
If you can give any indication on how much AAV-DJ or AAV of any other 'normal' serotype, that would be awesome. Also, have you only used scAAV or also ssAAV expressing your gene of interest?
Thanks!
/Josephine
PS I see you are in Radelaide, I did my PhD there. ;)
So infectious titre just means infecting cells and then counting positive cells by fluorescence. For AAV-DJ I usuallt get an infectious titre of about 1e9-1e10 and a QPCR titre of about 1e13.
It does depend on your virus, but for 1e5 cells I would do a serial dilution using 1ul, 0.1ul, 0.01ul and 0.001ul of virus (one dilution per well). Just add the virus directly to the normal culture media supplemented with 4uM Etoposide. I use etoposide from VWR (cat # 95037-118) dissolved in DMSO. AAV expression is fast so you can check after 24h and see how many fluorescent cells you have - if your cells are anything like B35s you'll see 100% infection in the 1ul well and positive cells in the 0.1 and 0.01ul wells.
I've only used conventional ssAAV.
Ah yes, Radelaide. Famous for wine, churches and possibly sharks :)
Hi Josephine,
coming back to your initial questions:
- MOI means vector genomes per cell. --> if they used a MOI of 10,000, you have to multiply this number by the amount of cells seeded per well... I would simply start with a dose of 100,000 MOI and do serial dilutions (2- or 3-fold). This usually works very well to identify a suitable dose. Determining the infectious titre can of course be done, but for your application seems superfluous.
- AAV-DJ: works great on a lot of cells, I am not aware about any experiments on SH-Sy5y, though.
- We usually do our AAV in vitro experiments in presence of serum and never saw any big differences / detrimental effects.
Hi!
Thanks for your recommendations, Benjamin! :)
All the best,
Josephine
PS I do know what MOI means ;) but it was so high that I doubted if the person talking about MOI actually meant that many particles per cell... :) )
Hello again everyone!
Anyone got experience in comparing ssAAV with scAAV (with same serotype) in culture? When using ssAAV, is it only 1) lower expression of the transgene 2) longer incubation times before achieving the same expression of the transgene or 3) both of the above?
Thank you
and all the best,
Josephine
Hi Josephine,
we usually see both, faster onset and higher expression levels with scAAVs.
Hi Jill,
Have you ever tried AAV-DJ/8 from cell biolabs. What is the transduction efficency about this type of AAV in cell culture. I am going to infect endothelial cells by this AAV, but not sure how much virus should I use.
Thank you,
Linzhang
Hi,
Does anyone use AAVDJ or any other serotype to transduce primary hepatocytes? If so, what efficiency and what amount needs to be added to the cells etc?
Cheers!
\Josephine
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