AAV quality control for DREADDs--request input

Occasionally we get emails from users regarding AAV serotypes/lots which they believe are problematic.  We normally forward these to the UNC vector core but would greatly appreciate having a 'running total' of those lots/serotypes/constructs which are problematic.  That way we can take that information back to the vector core and help them to institute rigorous (and recommended) quality control above and beyond what they typically do.

So here's what you should do:

Simply reply to this listing the AAV lot/serotype/construct which works as expected or which does not work as expected.

Example:  Lot#666/8/DEADDREADD:  works fine--all cells exploded upon contact with CNO as predicted.

Comments

  1. Lot#AV4500E/Serotype2/AAV-hSyn-DIO-hM4D(Gi)-mCherry: Off target expression, even in wildtype mice.

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  2. Lot# AV4618c / Serotype5 / AAV-CamKIIa-HA-hM3D(Gq)-IRES-mCitrine: No expression observed in cortex at 2,3, and 6 weeks post-injection. ~6*10^9 viral copies were injected per animal.

    Lot# AV4614f/ Serotype 5 / AAV-GFAP-HA-hM3D(Gq)-IRES-mCitrine. Off-target expression in neurons. May be infecting RGCs in the SVZ? Weak expression at 2 weeks post-injection. Strong expression at 6 weeks post-injection.

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    2. We also had no expression with Lot# AV4618c / Serotype5 / AAV-CamKIIa-HA-hM3D(Gq)-IRES-mCitrine in vivo (mice cell culture) and in vitro (rats after 4 weeks).

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  3. We have used rAAV2 hSYN DIO hM4D mCherry Lot no #AV4500f and rAAV2 hSYN DIO hM3D mCherry lot no #4499f2. Initially we found off-target expression in a well-characterized Cre-expressing mouse. We then injected into a Sprague Dawley rats and found expression after 2 weeks in the medulla oblongata, suggesting non Cre dependant expression. Expression was greater using rAAV2 hSYN DIO hM4D mCherry.

    We then tested that the viral DNA was in the DIO orientation, as suggested by Dr Justin English. ssDNA was extracted from the purified viruses, PCR was performed using primers within and outside the loxP flanked regions, and the PCR products were sequenced. These results confirmed that the virus was in the correct DIO orientation, and that they were not non DIO viruses given by accident by UNC. We are at a loss to explain this. We have not seen this with other DIO viruses expressing ChR2.

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