pAAV-hSyn-DIO-EGFP in Mouse (AAV Retrograde)
Addgene #50457-AAVrg

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.

Virus & Injection

Primary Virus
Promoter
Syn
Injected Titer
1.80E+13 GC/ml
Injected Volume
92 nl for inferior colliculus, 46 nl for medial geniculate
Injection Site
Brain parenchyma
Brain Region
Midbrain: Tegmentum
Injection Site Comments
We injected the inferior colliculus in some brains, and the medial geniculate body of the thalamus in others
View in Addgene Catalog

Subject

Species / Strain
Mouse ChAT-tdTomato reporter mouse (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
Stock Number
Jax mouse Stock No: 006410

Data Collection

Time After Injection
4 weeks
Detection Method
Direct fluorescence
Expression Site
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
Genetic Tool Outcome
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