This is an interesting question. No data to help you but just first-hand experience: No problem to mix viruses (AAV9, 2 types) but we do it the day of the injection. Or we mix and back to the -80 freezer until surgery day. Experiments work with viral mixes, one surgery one injection and off you go.

Will look forward to see what people answer to this question.

Hi John,

Like Roger, I have no data to help you but experience with a mix of 2 AAV (AAV9) we prepared the day of injection. I think it's better to prepare the mix the day of injection because you loose virus activity when you take it from the freezer and put it in back.

Good luck.

Hi John,

as with the previous replies, there is no problem in mixing the different virus and inject just once, this is actually very common when working with shRNAs, for example. AAV2 and 9 will both infect neurons however AAV9 has been reported to be more specific. Depending on the fluorophores you are using you might have some bleed through fluorescence that might complicate an accurate analysis. In this situation I would probably do bilateral injections with a different virus in each site; if you don't have them is quite easy to built double injectors.

Hope this helps,

Ezequiel

thanks for these replies.

Good to hear there have been no (un)reported complications in your hands (although of course if noone looked carefully some sort of interaction cannot be ruled out :) ).

I understand it's better to take AAV fresh from freezer, and mix on the day, but in this case that was unfortunately not done.. hopefully the virus activity is not too much reduced. Will report results in a couple of weeks :).

Dear John,

From what I understand, their receptor usage is different, although possibly not unique. Here are some references that may help with that question:

Shen S, Bryant K, Sun J, Brown S, Troupes A, Pulicherla N, and Asokan A. (2012) Glycan binding avidity determines the systemic fate of adeno-associated virus 9. J Virol

Nonnenmacher M, and Weber T. (2012) Intracellular transport of recombinant adeno-associated virus vectors. Gene Ther 19, 649-658

This reference describes the phylogenetic relationship between the viral archetypes and shows that AAV2 and 9 are not very closely related

Zinn E, Pacouret S, Khaychuk V, Turunen Heikki T, Carvalho Livia S, Andres-Mateos E, Shah S, Shelke R, Maurer Anna C, Plovie E, Xiao R, and Vandenberghe Luk H. (2015) In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector. Cell Reports 12, 1056-1068

BW, Simon

Hi John,

I agree with the rest!

I just wanted to add that it is more desirable to co-inject the same serotype of 2 different constructs, in order to raise the propability of hitting the same cells and the same amount.

Also, AAV9 has been reported to travel retrogradely (e.g. http://www.ncbi.nlm.nih.gov/pubmed/23600720) so you might expect to see fluorescence in other places in the brain, as well.

Hope it goes well!

Hi John,

Our lab has successfully injected two viruses, here's a reference: http://www.ncbi.nlm.nih.gov/pubmed/25195598

You can do it but need to control for two things. The first is multiplicity of infection as the two viruses will compete for the same receptors. The second is the competition for cellular synthetic resources to make proteins.  It is important not to compare a two virus treatment group with a single virus injection group. Including a second "control" virus expressing a irrelevant (or better yet mutated) protein will overcome these concerns

Hello Everyone:

Would you please describe how you mix the viruses? For example, add 1 ul of one virus to 1 ul of the other virus, then do you vertex, centrifuge......? Do you think they will mix evenly?

Thanks!

I agree with all of the above, however a note of caution should be taken when using two FLEX vectors delivered simultaneously as you can get recombination between the two FLEX'd genomes as they have compatible LoxP and Lox2272 sites. See Saunders et al 2012 Article Novel recombinant adeno-associated viruses for Cre activated...

Sa Li , how did you do it finally? I want to coinject an aav-gfp and an aav to overexpress one receptor, then I do not know if to reduce the concentration of the gfp as it is stronger than the other one. Both have same serotype and construction. if i inject 2 ul, I could eventually inject 1.8 ul of the aav to overexpress and 0.2 of the aav-gfp without reducing the concentration in which i checked i have optimal expression.

What do you think? Do you suggested me other way? Thank you