# Intersectional transgenic targeting of discrete neuronal and glial subtypes

> **NIH NIH RF1** · JOHNS HOPKINS UNIVERSITY · 2021 · $1,921,258

## Abstract

PROJECT SUMMARY
Tools for exclusively targeting neuronal and glial subtypes are needed to advance our understanding of the brain.
“Intersectional” systems improve targeting by restricting “reporter/effector” transgenes to a subdomain defined
by the expression overlap between two activating factors. “Split-driver” systems have enhanced targeting
precision in flies and are operable in fish, but have yet to be systematically deployed in vertebrate systems. Our
goal is to improve transgene expression precision in the vertebrate nervous system by creating a series of
intersectional vectors designed to enable exclusive targeting of discrete neuronal and glial cell subtypes.
Binary systems, such as Gal4/UAS, separate transgene expression into “drivers” and “reporter/effectors”. The
combinatorial nature ensures versatility; however, most driver lines fail to target specific cell types. In turn, this
can compromise the integrity of reporter/effector-based manipulations. To enhance expression specificity,
drivers have been split into two “hemidriver” components: a DNA-binding domain (DBD) and transactivation
domain (AD). Hemidrivers can only be assembled where DBD and AD expression overlaps, thus restricting
driver-dependent reporter/effectors to the “intersect”. DBD-AD and reporter/effector activity can be further
refined by expressing repressor proteins in non-targeted domains. Moreover, efficient knock-in methods and
single-cell transcriptomics now afford an unprecedented level of transgene expression fidelity and targeting
precision. We propose to leverage these advances to create a series of DBD hemidriver, AD hemidriver, and
repressor toolsets for targeting discrete neuronal and glial subtypes. To further enhance targeting precision,
repressor resources will developed to inhibit effector activity in non-targeted cells. Given its utility in dissecting
cell function, we propose to identify genetic repressors of the nitroreductase (NTR) system of inducible targeted
cell ablation. While the utility of these resources will be validated in the zebrafish, universal vectors will be created
to facilitate adaptation to any other vertebrate model amenable to transgenesis. Three aims are proposed:
Aim 1: Create and validate tools for labeling and functionally dissecting discrete neuronal cell subtypes.
Aim 2: Create and validate tools for labeling and functionally dissecting discrete glial cell subtypes.
Aim 3: Create and validate tools for repressing effector activity non-targeted brain regions/cells.
Funding will allow us to create toolsets facilitating transgenic targeting at unparalleled levels of cellular and circuit
precision in the brain. We anticipate creating ~25 DBD, ~50 AD, and ~25 repressor vectors/lines, allowing
targeting of thousands of unique neuronal and glial subtypes. In combination with existing reporter/effectors
expressing optogenetic, cell ablation, transsynaptic and other tools for monitoring and manipulating cells and
circuits, the propo...

## Key facts

- **NIH application ID:** 10259997
- **Project number:** 1RF1MH126731-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** JEFFREY MUMM
- **Activity code:** RF1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,921,258
- **Award type:** 1
- **Project period:** 2021-08-27 → 2025-08-26

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10259997

## Citation

> US National Institutes of Health, RePORTER application 10259997, Intersectional transgenic targeting of discrete neuronal and glial subtypes (1RF1MH126731-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10259997. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*