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[Research Tool Highlight] Featured in Cell! Brain Case launches new product — Enhancer AAV Vectors, a breakthrough for research Tool !

Time：2025-08-26 16:59:09

The mammalian brain, as a highly complex organ, contains a cortex composed of hundreds of distinct cell types. These cells can be classified in multiple dimensions based on shared morphological features, functional properties, and molecular expression profiles. Defining the contribution of each cell type to cortical physiological processes is critical for understanding their roles in both health and disease.

On May 29, 2025, a major milestone of the U.S. NIH BRAIN Initiative was published in Cell. The paper, titled “A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types”, reports that researchers utilized transcriptomic and epigenomic classifications of mouse and human cortical cell types to define marker genes and putative enhancers. They created a large toolkit comprising 15 transgenic driver mouse lines, 2 reporter lines, and over 1,000 enhancer AAV vectors designed for specific targeting of cortical subpopulations, with some tools achieving over 90% specificity.

https://www.cell.com/cell/fulltext/S0092-8674(25)00513-6

All tools have been made available through Addgene and Jackson Labs. Experimental data related to SYFP2 expression vectors in this study can be accessed via the Genetic Tools Atlas network portal — a new public resource developed by the Allen Institute (RRID:SCR_025643; https://portal.brain-map.org/genetic-tools/genetic-tools-atlas) and BioFileFinder (BFF) (RRID:SCR_026930; https://bff.allencell.org/), which also includes datasets from individual scRNA-seq experiments.

Figure 1. Two strategies for generating and characterizing genetic tools.

Enhancer AAV Vector Screening and Optimization

Researchers built a cross-species (mouse/human) transcriptomic and epigenomic taxonomy at sub-class resolution by integrating historical genomic datasets with newly generated single-nucleus multiomic data (snMultiome). Mouse data were obtained from the primary somatosensory area (SSp), primary motor area (MOp), and primary visual area (VISp), while human data were collected from the middle temporal gyrus (MTG).

These approaches enabled the measurement of tens of thousands of molecular features in single cells, classification into molecularly defined cell types, and identification of marker genes and genomic enhancers consistent with specific cell types. Based on these findings, researchers designed and tested 1,164 unique enhancer AAV vectors, including 802 candidate sequences (643 mouse-derived and 159 human-derived). Approximately 40% (313 vectors) demonstrated targeting specificity.

Each putative enhancer sequence was assigned a unique ID: AiE = enhancers discovered by the Allen Institute. ExE = sequences based on coordinates reported by other research groups. The IDs are followed by four digits and a species identifier (“m” for mouse, “h” for human). Candidate enhancer sequences were amplified from their respective genomes and cloned upstream of a minimal promoter (minBG, minRho, minCMV) or an endogenous promoter in an AAV plasmid backbone, driving the expression of yellow fluorescent protein SYFP.

Figure 2. Enhancer AAV vector information on Addgene

(https://www.addgene.org/browse/article/28248370/)

The plasmids were packaged into PHP.eB AAV vectors, capable of crossing the blood-brain barrier, and delivered to C57BL/6J mice via retro-orbital (RO) injection. About four weeks post-injection, brain slice fluorescence images were first screened visually, followed by serial two-photon tomography (STPT) for whole-brain labeling assessment, and single-cell RNA sequencing (scRNA-seq) for determining cell-type specificity. Experiments focused on the mouse visual cortex to ensure data consistency.

Through core deletion and tandem optimization, SYFP2 mRNA expression levels from enhancer AAVs increased by an average of 839%, while maintaining stable specificity. Recombinase versions (e.g., Cre, FlpO) were also developed, with modifications such as Cre(R297T) mutation and adjustments to WPRE sequences to reduce off-target expression.

Figure 3. Screening enhancer AAVs in the mouse brain

The most valuable tools—those enabling genetic access to specific sub-classes or clusters—were designated with a “Hall of Fame” (HoF) label. This naming, manually assigned by Allen Institute scientists, considers factors such as specificity determined from scRNA-seq data and signal intensity of enhancer AAVs, helping researchers identify the best tools for targeting defined cell populations.

Figure 4. Hall of Fame (HoF) enhancer AAVs

Establishment of Transgenic Driver and Reporter Mouse Lines

Finally, researchers established and evaluated 15 transgenic driver mouse lines and 2 reporter lines, which can be used either independently or in combination with enhancer AAVs to achieve genetic access to even the most finely classified cortical subpopulations.
The two reporter lines (Ai193 and Ai224) contain independent transcription units that report the expression of Cre and Flp recombinases by expressing different fluorescent proteins:
Ai193: TICL-EGFP-WPRE-ICF-tdT-WPRE-hyg
Ai224: TICL-NLS-EGFP-ICF-NLS-dT-hyg

In these lines: GFP is expressed when Cre is present, tdTomato is expressed when Flp is present, Both fluorescent proteins are expressed simultaneously when Cre and Flp are co-expressed.
Based on the transcriptomic taxonomy of mouse cortical cell types, marker genes capable of labeling specific cell types (subclasses, supertypes, or clusters) were selected. Fifteen genes were chosen to generate Cre or Flp driver lines.

Glutamatergic driver lines: 11 marker genes were used, including Batf3, Parm1, Rxfp1, Chrna6, Slco2a1, Cplx3, Cpne4, Ctxn3, Gpr139, Npnt, and Slc17a7.
·Batf3-IRES2-FlpO-WPRE-neo primarily targeted the L5_IT_VISp_Batf3 cluster within the L5_IT subclass, but also labeled some L2/3 IT, other L5_IT, L6_CT cells, and microglia.
·Chrna6-IRES2-FlpO specifically labeled the L5_ET_Chrna6 cell type.

GABAergic driver lines: genes such as Lamp5, Sncg, and Chodl were used to construct driver lines.
·Lamp5-P2A-FlpO labeled Lamp5 GABAergic neurons but also labeled glutamatergic cells in layers 2–3, 5, and 6.
·Sncg-IRES-FlpO;Ai65F labeled the Sncg neuronal population as well as endothelial cells.

Figure 5. Comparison of transgenic reporter and driver mouse lines with selected enhancer AAVs.

Significance of the Study

This article reports the creation and public release of over 1,000 enhancer AAVs and 17 transgenic mouse lines as genetic tools. Their specificity was validated through single-cell sequencing and imaging technologies, and further optimized to improve performance. By establishing a cross-species cortical cell taxonomy, the study provides a standardized toolkit and data resource for cortical cell-type research, advancing our understanding of brain function and disease mechanisms. Moreover, the large-scale development workflow sets a methodological foundation for future innovations in neuroscience tool development.

Original article: https://doi.org/10.1016/j.cell.2025.05.002

The following table was compiled by the Brain Case Team, with information sourced from the supplementary materials of DOI: 10.1016/j.cell.2025.05.002. If you are interested in the related vectors, please feel free to contact us. bd@ebraincase.com

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[Research Tool Highlight] Featured in Cell! Brain Case launches new product — Enhancer AAV Vectors, a breakthrough for research Tool !

Enhancer AAV Vector Screening and Optimization

Establishment of Transgenic Driver and Reporter Mouse Lines

Significance of the Study

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[Research Tool Highlight] Featured in Cell! Brain Case launches new product — Enhancer AAV Vectors, a breakthrough for research Tool !

Enhancer AAV Vector Screening and Optimization

Establishment of Transgenic Driver and Reporter Mouse Lines

Significance of the Study

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