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IRES constructs redux

I've received quite a few emails regarding my decision to no longer offer high titer viral stocks of the various IRES constructs via the UNC vector core.  And thought I'd clarify for those interested....

The constructs work beautifully in our hands (see for instance our recent paper where we used the CAMKII-hM4Di-IRES-mCitrene vector) and yield highly reproducible and high levels of transduction in mice.  

I have received a small number of emails from users who have not been able to reproducibly achieve high levels of transduction when the DIO-hSyn-DREADD-IRES-mCitrene constructs are used in combination with a Cre-driver line (even though they function well in our hands).

My sense is that there was some variability on the packaging on the part of the vector core and I directed them to take them off line.

The plasmids are/will be available from ADDGENE.

Bryan

Recommended strategy for anti-HA staining

We've characterized a number of antibodies and piloted many protocols for visualizing HA-tagged DREADDs.

Here's our recommended protocol (please note suppliers of antibodies for best results):

Immunofluorescence Staining Protocol (Anti-HA antibody)
1. Perfuse the mice with ice cold 4% paraformaldehyde (PFA; freshly prepared)

Cryosection procedure:
2. Fix the brain with 4%PFA @ 4°C overnight (O/N).
3. Transfer brain sample to 30% sucrose/1x phosphate buffered saline (PBS), allow sample sink @ 4°C for about 48hr.
4. Cryosection 40 uM

Staining procedure:
5. Permeabilize the tissue with 1xPBS/0.4% Triton x100 for 1hr at room temperature (RT).
6. Block the slide with 1% BSA 5% NGS (normal goat serum) in 1xPBS/0.4% Triton x100 at RT for 1hr.
7. Incubate the sections with primary antibody (1:500) in blocking buffer @ 4 O/N.
8. Wash the section with 1x PBS/0.4% Triton x100 three times at RT, 10min/each.
9. Incubate the sections with secondary antibody (1:250) in blocking buffer for 2hr a…

Recommended starting constructs for new DREADD users

For those of you using AAV-based delivery of DREADDs we recommend that you consider using the following constructs/viruses if you are using rodents.

Non-Cre-dependent

AAV8-hSyn-hM3Dq-mCherry
AAV8-hSyn-hM4Di-mCherry


Cre-dependent

AAV8-DIO-hSyn-hM3Dq-mCherry

AAV8-DIO-hSyn-hM4Di-mCherry

We've found these give excellent transduction in a variety of brain regions and high levels of expression in neurons.

Also, if you fix the tissue please consider using an anti-mCherry antibody rather than endogenous mCherry fluorescence to visualize the DREADD-mCherry fusion protein as considerable quenching of fluorescence occurs following fixation.

Bryan

IRES constructs

The Rothlab (http://pdsp.med.unc.edu/rothlab/) has received feedback from users regarding various DIO DREADD-based IRES constructs (e.g. DIO-hSyn-DREADD-IRES-YFP).  Our sense is that because of the large size of the insert, AAV-based packaging is suboptimal leading to low transduction efficiency.
Accordingly we are no longer offering AAV viral stocks of these vectors.  
If users wish to obtain the cDNAs they will soon be available via addgene:
https://www.addgene.org/Bryan_Roth/
Bryan
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This is a blog to provide feedback and comments from users regarding chemogenetic technologies.