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pAAV-hSyn-DIO-EGFP in Mouse (AAV Retrograde)

Addgene #50457-AAVrg

Data Submitted By
Nichole Beebe and Brett Schofield
Submission Date
February 08, 2021
Publication Date
February 08, 2021 (modified May 16, 2023)
Abstract
Not provided
On the left, a cholinergic cell group (the PMT) can be seen to express a cre-dependent tdTomato gene (magenta), indicating cre expression in these cells. On the right, EGFP expression (green) can be seen in cells of the inferior colliculus, where an injection of virus was made. These cells do not express cre recombinase as indicated by the lack of tdTomato expression. The tdTomato-labeled cholinergic cell group ventral to the inferior colliculus is both cre-expressing and is known to make projections into the injected area, however exhibits no EGFP expression.
Caption: On the left, a cholinergic cell group (the PMT) can be seen to express a cre-dependent tdTomato gene (magenta), indicating cre expression in these cells. On the right, EGFP expression (green) can be seen in cells of the inferior colliculus, where an injection of virus was made. These cells do not express cre recombinase as indicated by the lack of tdTomato expression. The tdTomato-labeled cholinergic cell group ventral to the inferior colliculus is both cre-expressing and is known to make projections into the injected area, however exhibits no EGFP expression.

Any further use of this image requires permission from the lab who submitted the data.

Vector Used
pAAV-hSyn-DIO-EGFP
Addgene #50457-AAVrg

Virus & Injection

Virus

Virus Name
pAAV-hSyn-DIO-EGFP
Serotype
AAV Retrograde
Source
Addgene
Catalog Number
50457-AAVrg
Promoter
Synapsin
Cargo Type
Control
Injection Titer / Dose
1.80E+13 GC/mL
Injection Volume
92 nL for inferior colliculus, 46 nL for medial geniculate
Injection Rate
Not provided
Injector Material
Not provided
Injection Site / Route
Inferior colliculus, NeuroNames ID: 476
Additional Details
We injected the inferior colliculus in some brains, and the medial geniculate body of the thalamus in others.
View in Addgene Catalog

Other Details

Species
Mouse
Strain or Cell Line
ChAT-tdTomato reporter mouse
Source
Jackson Labs
Stock Number
006410
Other Information
offspring of a cross between an Ai14 reporter mouse and a mixed background ChAT-Cre cdh23 wt mouse, which is a hearing-corrected version of the ChAT Cre mouse 006410 from Jackson Labs; background is mixed CBA/CaJ and B6; cholinergic cells express tdTomato in these mice
Age at Injection
5-8 months
Time After Injection
4 weeks
Detection Method
Direct fluorescence
Assay & Results
In each case, only cells at the injection site expressed fluorescent protein. These brain regions contain local synaptic connections so the virus may have been taken up at terminals, however no other brain regions, including those known to make projections into the injection site, exhibited fluorescent label.
Functional Testing
The fluorescent protein gene in the virus should be cre-dependent. The animals were reporters, and expressed tdTomato in cre-expressing cells. We saw tdTomato expression in the expected nuclei, however EGFP expression (virally mediated) only occurred in non-cre-expressing cells, indicating that it must be spurious.
Tips & Comments
It seems that these vectors don't work as intended in this model. It could be an issue with infection of cholinergic cells specifically. Additionally, the amount of spurious label at the injection site makes the virus less than ideal for experiments seeking to look at collaterals of cells making projections into the injections site.