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Characterization of an enhancer-AAV aimed at targeting distinct interneuron subtypes

Addgene #213944-PHPeB

Data Submitted By
Elisabetta Furlanis , Min Dai , Brenda Leyva Garcia , Sara Wills , Jordane Dimidschstein , Gordon Fishell and Yating Wang
Lab Name
Gordon Fishell, Harvard Medical School
Submission Date
September 25, 2025
Publication Date
January 26, 2026 (modified January 26, 2026)
DOI
https://doi.org/10.57733/addgene.nrfk9v
Abstract
We injected interneuron enhancer, BiSSTe4, tagged with a dTomato reporter through retro-orbital injection routes into mice. After waiting 3 weeks to allow for infection, we processed the brains and looked at endogenous dTomato reporter expression to assess enhancer activity in the primary somatosensory cortex. This project took place as part of the Brain Initiative and we have uploaded our results here.
Citation
Characterization of an enhancer-AAV aimed at targeting distinct interneuron subtypes. Furlanis E, Dai M, Leyva Garcia B, Wills S, Dimidschstein J, Fishell G, Wang Y 2026. Addgene Report, https://doi.org/10.57733/addgene.nrfk9v
pAAV_BiSSTe4_dTomato_nlsdTomato (AAV PHP.eB) replicate 1 was injected into the retro-orbital sinus of a 4 week old mouse. Coronal section showing clear native fluorescent reporter expression (red) indicating enhancer activity, particularly in S1 deeper layers. Imaged on ZEISS Axioscan 7 Slidescanner at 20x magnification.
Caption: pAAV_BiSSTe4_dTomato_nlsdTomato (AAV PHP.eB) replicate 1 was injected into the retro-orbital sinus of a 4 week old mouse. Coronal section showing clear native fluorescent reporter expression (red) indicating enhancer activity, particularly in S1 deeper layers. Imaged on ZEISS Axioscan 7 Slidescanner at 20x magnification.
pAAV_BiSSTe4_dTomato_nlsdTomato (AAV PHP.eB) replicate 1 was injected into the retro-orbital sinus of a 4 week old Cre-dependent transgenic mouse selectively expressing GFP in SST interneurons. Coronal section showing results from IHC with an antibody for GFP to label SST interneurons (green). Clear native fluorescent reporter expression (red) indicating enhancer activity, and high level of colocalization with SST cells (orange/yellow cells). Imaged on ZEISS Axioscan 7 Slidescanner at 20x magnification.
Caption: pAAV_BiSSTe4_dTomato_nlsdTomato (AAV PHP.eB) replicate 1 was injected into the retro-orbital sinus of a 4 week old Cre-dependent transgenic mouse selectively expressing GFP in SST interneurons. Coronal section showing results from IHC with an antibody for GFP to label SST interneurons (green). Clear native fluorescent reporter expression (red) indicating enhancer activity, and high level of colocalization with SST cells (orange/yellow cells). Imaged on ZEISS Axioscan 7 Slidescanner at 20x magnification.

This report is made available under the Creative Commons Attribution 4.0 International License.

Vector Used
pAAV_BiSSTe4_dTomato_nlsdTomato
Addgene #213944-PHPeB

Virus & Injection

Virus

Virus Name
pAAV_BiSSTe4_dTomato_nlsdTomato
Serotype
PHP.eB
Source
Addgene
Catalog Number
213944-PHPeB
Promoter
Other
Other Promoter
BiSSTe4 (enhancer)
Cargo Type
Control
Injection Titer / Dose
2.00E+11 vg/animal
Injection Volume
80 µL
Injection Rate
N/A
Injector Material
BD Insulin Syringe 0.3 mL
Injection Time
N/A
Injection Site / Route
Retro-Orbital (RO) injection into the retro-orbital sinus of 4 week old mouse for systemic expression across the brain. Note: This is different than the optimal injection route for this enhancer, ICV, as mentioned in our paper.
Additional Details
Original titer of 6.56E+12 vg/mL. Diluted with PBS1X to achieve 2.00E+11 vg/animal in an 80 µL injection
View in Addgene Catalog

Other Details

Species
Mouse
Strain or Cell Line
For mouse injected with BiSSTe4 enhancer: SST-Cre x INTACT (CAG-Sun1/sfGFP)
Source
JAX
Other Information
SST-Cre x INTACT mouse line used was obtained by breeding lines JAX:008199 (Sst-IRES-Cre) and JAX:021039 (INTACT).
Age at Injection
4 weeks
Sex
Male
Time After Injection
3 weeks before perfusion
Detection Method
Native fluorescence, IHC
Assay & Results
1 RO 4 weeks BiSSTe4 replicate showing clear labeling of SST cells, particularly in deeper cortical layers. One image shows native dTomato reporter flourescence (red/violet), and the other shows native dTomato reporter flourescence (red/violet) with a PV IHC stain (green).
Tips & Comments
Please see the original publication for more details: Furlanis, E., Dai, M., Leyva Garcia, B., Tran, T., Vergara, J., Pereira, A., Gorissen, B. L., Wills, S., Vlachos, A., Hairston, A., Dwivedi, D., Du, S., McMahon, J., Huang, S., Morabito, A., Vazquez, A., Kim, S., Lee, A. T., Chang, E. F., Razzaq, T., … Wang, Y. (2025). An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes. Neuron, 113(10), 1525–1547.e15. https://doi.org/10.1016/j.neuron.2025.05.002