Hi Sylvana,

I had the same problem and contacted the company from which I had bought the virus (Addgene). Their support has been very valuable, so here is a part of their reply: "LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo."

They have also developped an assay to measure the percentage of recombined viral particles in each prep. If you have bought a flex-AAV from Addgene, you can contact them and give them your lot number, so that they can tell you how much recombined viral particles there are in your lot (for me, it was estimated at 0.047%). These preps usually comes at high concentration, so if you don't dilute it, you'll increase the risk of having recombined particles infecting Cre-negative cells and express the transgene ectopically. The only way to solve this problem is to test different dilutions, in order to find an "optimal titer", i.e. a titer that will give you a nice expression of the transgene in Cre-positive cells, but without (or with minimal) non-specific expression in Cre-negative cells. This can be a quite tedious work to do, so good luck with that!

That being said, you can't completely rule out the possibility that you have as well an ectopic expression of the Cre recombinase (even at low levels, but sufficient to induce the recombination of the flex-virus). Doing and immunostaining for the Cre might be a good idea, to be sure there is not a huge leakage of Cre expression in your transgenic mice. I don't know on which organ you're working (brain I presume?), but for me, the rabbit anti-Cre monoclonal antibody #15036 from Cell Signaling is the only one that worked. I stain 30µm cryosections from perfused mice brains (fixation with 4% PFA ; 24h postfixation) using this antibody at 1/125 (yes, very concentrated), overnight @ 4°C. Then, if the signal is too weak, you should use an amplification such as Tyramide Signal Amplification: after incubating with an HRP-linked secondary antibody, add the TSA reagent (for me, 1/250 for 10min works well with the kits from Perkin Elmer). You should then be able to see the nuclear Cre staining and confirm its "specific" location.

Hope this helps, good luck with all that!

Hi Vincent,

Makes a lot of sense. Thanks for your extensive explanation and suggestions for overcoming this problem.

Best Sylvana