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AAV-EF1a-BbTagBY in Rat (AAV9)

Addgene #45185-AAV9

Data Submitted By
Nichole Beebe and Brett Schofield
Submission Date
August 14, 2019
Publication Date
August 14, 2019 (modified May 16, 2023)
Abstract
Not Provided
This is an image taken at 63X on a Zeiss AxioImager microscope. The tissue is paraformaldehyde-fixed, frozen-sectioned, then mounted onto a glass slide and coverslipped with Prolong Glass antifade mountant from Thermo-Fisher. The cyan neuron mainly expresses mTFP (via the other virus that was co-injected), while the yellow axons originate from a neuron mainly expressing EYFP. This image was taken in the superior colliculus of a VGAT-Cre rat; neurons expressing fluorescent protein are believed to be GABAergic. The inset shows an EYFP-expressing axon making presumptive contact with a dendrite of the mTFP-expressing cell.
Caption: This is an image taken at 63X on a Zeiss AxioImager microscope. The tissue is paraformaldehyde-fixed, frozen-sectioned, then mounted onto a glass slide and coverslipped with Prolong Glass antifade mountant from Thermo-Fisher. The cyan neuron mainly expresses mTFP (via the other virus that was co-injected), while the yellow axons originate from a neuron mainly expressing EYFP. This image was taken in the superior colliculus of a VGAT-Cre rat; neurons expressing fluorescent protein are believed to be GABAergic. The inset shows an EYFP-expressing axon making presumptive contact with a dendrite of the mTFP-expressing cell.
Image attribution: Taken by Nichole Beebe

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

Vectors Used
AAV-EF1a-BbTagBY
Addgene #45185-AAV9
AAV-EF1a-BbChT
Addgene #45186-AAV9

Virus & Injection

Virus 1

Virus Name
AAV-EF1a-BbTagBY
Serotype
AAV9
Source
Addgene
Catalog Number
45185-AAV9
Promoter
EF1a
Cargo Type
Control
Injection Titer / Dose
3.40E+13 GC/mL
Injection Volume
0.03 μL to 0.15 μL
Injection Rate
10 nL/minute
Injector Material
Not Provided
Injection Site / Route
Inferior colliculus, NeuroNames ID: 476
Additional Details
We were attempting to achieve Brainbow labeling of GABAergic cells in the inferior colliculus of VGAT-Cre rats in order to reconstruct the projections of these neurons (multiple volumes were compared across several cases). In one case, an injection was accidentally placed in caudal superior colliculus.
View in Addgene Catalog

Virus 2

Virus Name
AAV-EF1a-BbChT
Serotype
AAV9
Source
Addgene
Catalog Number
45186-AAV9
Promoter
EF1a
Injection Titer / Dose
1.70E+13 GC/mL
Injection Volume
The two viruses were mixed and co-injected.
Injection Rate
10 nL/min
Injector Material
Not Provided
Injection Site / Route
Inferior colliculus, NeuroNames ID: 476
Additional Details
We co-injected the two viruses in an attempt to achieve Brainbow labeling in GABAergic cells in the rat inferior colliculus. The viruses were mixed before being aliquoted and were injected in the same syringe.
View in Addgene Catalog

Other Details

Species
Rat
Strain or Cell Line
VGAT-Cre rat on a Long-Evans background: LEH-Vgat-Cretm1sage
Source
Horizon Discovery Group.
Stock Number
TGRL9310
Age at Injection
approximately 3-6 months
Time After Injection
8 weeks
Detection Method
Direct fluorescence
Assay & Results
Based on location and morphology, we felt confident that fluorescent cells in the inferior and superior colliculus were GABAergic, which was the cell class expressing Cre-recombinase in the animal model. In each case where an injection was made, fluorescent cells were present in the injected nucleus.
Functional Testing
The tool operated correctly. Cre-based fluorescent protein expression should have been restricted to GABAergic cells in the midbrain of this animal model, and that appeared to be true.
Other Information
With greater injection volumes (ex. Three 0.05 μL injections), more of an effect of inflammation or toxicity seemed present. This seemed less apparent with our smallest volume (0.03 μL).
Tips & Comments
For the Brainbow viruses, we have decided to try to minimize our injection volume and maximize our survival time. The best success in fluorescent protein expression was achieved with an injection of 0.03 μL and a survival time of 8 weeks.