It takes a village! While you usually hear this phrase when discussing raising children, it can also be applied to research. Sharing knowledge and tools is the best way to help propel research forward and have the biggest impact. We should know — Addgene has built its foundation on this idea! And we believe in the power of partnerships with like-minded individuals and organizations, like the Allen Institute, to achieve greater outcomes together.
The Allen Institute is a research organization that is committed to understanding some of the basic principles of life and health and works to advance foundational research. There are many focus areas of the Allen Institute, one being the Allen Institute for Brain Science (AIBS). Scientists at AIBS have allocated tremendous time and resources to generate highly specific tools to target different cell populations and regions of the brain. This has culminated in a collection of AAV enhancer plasmids with high specificity and brightness, now available at Addgene!
Using enhancers to target cell populations
Enhancers are non-coding sequences of DNA that regulate gene expression by interacting with proteins such as transcription factors and chromatin modifiers. These proteins recognize and bind motifs within the enhancer sequence, then interact with mediator complexes and the RNA polymerase at the promoter region of a gene to initiate transcription (Figure 1). Although all cell types share the same DNA sequence, not all enhancers are functional in every cell. Differential expression of transcription factors, which recognize and bind to different DNA motifs, results in a given DNA sequence serving as a functional enhancer in one cell type but not in others. The same mechanism that allows enhancers to regulate gene expression in a cell type-specific manner also makes them useful tools for targeting those specific cell types to express exogenous genes — a feature the AIBS has capitalized on.
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| Figure 1: Schematic of transcriptional regulation by an enhancer. Created with BioRender.com. |
Developing AAV enhancer tools for brain visualization
Putative enhancer sequences for specific cell types can be identified based on the wear-and-tear footprint that they leave on the DNA only in target cells. This pattern is termed “differential DNA accessibility,” and AIBS uses it to find potential enhancer candidates.
To use these sequences for making functional genetic tools, enhancers were nominated from the genome based on their differential chromatin accessibility across different cell types. Most enhancers were derived from the human, mouse, and macaque genomes, and are designated by a letter following the enhancer ID (h, m, or q, respectively). These sequences were then cloned into an AAV backbone containing a minimal promoter to drive expression of a fluorescent gene and packaged into viral vectors that can be administered systemically (retro-orbitally, RO; or intracerebroventricularly, ICV) or locally (stereotaxically, STX) to the brain.
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| Figure 2: Selection, prep, and testing of AAV enhancers in mice. Adapted from the Allen Institute's Genetic Tools Atlas. |
To evaluate the resulting expression of the fluorescent reporter, the brain tissue was extracted, sectioned, and imaged, with all image sets available at the Allen Institute’s Genetic Tools Atlas (GTA). The GTA has a wealth of information and includes annotated images of the brain sections describing the labeled regions, as well as the strength and density of labeling in each. This allows users to quickly find the tools that best fit their specific experimental requirements.
Best-in-class collection at Addgene
The GTA contains data on thousands of AAV enhancer plasmids, which are all available at Addgene. This allows scientists to choose enhancers that may have value in certain situations or target a very specific cell type. However, Addgene has partnered with the AIBS to showcase the “best-in-class” enhancers. The Allen Institute for Brain Science AAV Enhancer Collection is a quick guide to a smaller set of tried-and-true winners, containing enhancers with the highest specificity and brightness. You will find enhancers to target many different cell populations and brain regions and the genetic tools that target them the best.
More from the Allen Institute
The Allen Institute has many other projects outside of the enhancers and their neuroscience research. This includes other data, tools, and resources. Luckily for us, they like sharing! You can find data, analysis tools, and other scientific and educational resources on their website. You can find more neuroscience-focused tools in their Brain Map portal, and cell science-focused tools at the Allen Institute for Cell Science. There are also more resources available at Addgene, including the Allen Institute for Cell Science Plasmid Collection and the BRAIN Armamentarium Collection, a collaborative effort that the Allen Institute contributes to.
Enhancing science
How research is conceived, developed, funded, and shared has no doubt changed drastically over the past few decades. But no matter how much it changes, it will still take a community to advance our knowledge. We need to continue to develop improved, reliable tools to help carry out that research. Organizations like the Allen Institute are committed to just that, and their painstaking efforts towards screening thousands of enhancer sequences has culminated in this best-in-class collection. Hopefully, this collection of highly specific AAV enhancers can help scientists around the world better understand the brain, how it functions, and how we can help keep it healthy.
Thank you to Dr. Yoav Ben-Simon for reviewing this post! Dr. Ben-Simon is an assistant investigator at the Allen Institute in the department of Molecular Genetics.
References and Resources
References
Ben-Simon, Y., Hooper, M., Narayan, S., Daigle, T. L., Dwivedi, D., Way, S. W., Oster, A., Stafford, D. A., Mich, J. K., Taormina, M. J., Martinez, R. A., Opitz-Araya, X., Roth, J. R., Alexander, J. R., Allen, S., Amster, A., Arbuckle, J., Ayala, A., Baker, P. M., . . . Tasic, B. (2025). A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types. Cell. https://doi.org/10.1016/j.cell.2025.05.002
Hunker, A. C., Wirthlin, M. E., Gill, G., Johansen, N. J., Hooper, M., Omstead, V., Vargas, S., Lerma, M. N., Taskin, N., Weed, N., Laird, W. D., Bishaw, Y. M., Bendrick, J. L., Gore, B. B., Ben-Simon, Y., Opitz-Araya, X., Martinez, R. A., Way, S. W., Thyagarajan, B., Otto, S., … Ting, J. T. (2025). Enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits. Neuron https://doi.org/10.1016/j.neuron.2025.04.035
Graybuck, L. T., Daigle, T. L., Sedeño-Cortés, A. E., Walker, M., Kalmbach, B., Lenz, G. H., Morin, E., Nguyen, T. N., Garren, E., Bendrick, J. L., Kim, T. K., Zhou, T., Mortrud, M., Yao, S., Siverts, L. A., Larsen, R., Gore, B. B., Szelenyi, E. R., Trader, C., . . . Tasic, B. (2021). Enhancer viruses for combinatorial cell-subclass-specific labeling. Neuron, 109(9), 1449-1464.e13. https://doi.org/10.1016/j.neuron.2021.03.011
Mich, J. K., Graybuck, L. T., Hess, E. E., Mahoney, J. T., Kojima, Y., Ding, Y., Somasundaram, S., Miller, J. A., Kalmbach, B. E., Radaelli, C., Gore, B. B., Weed, N., Omstead, V., Bishaw, Y., Shapovalova, N. V., Martinez, R. A., Fong, O., Yao, S., Mortrud, M., . . . Levi, B. P. (2021). Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. Cell Reports, 34(13), 108754. https://doi.org/10.1016/j.celrep.2021.108754
Panigrahi, A., & O'Malley, B. W. (2021). Mechanisms of enhancer action: the known and the unknown. Genome Biology, 22(1). https://doi.org/10.1186/s13059-021-02322-1
Pennacchio, L. A., Bickmore, W., Dean, A., Nobrega, M. A., & Bejerano, G. (2013). Enhancers: five essential questions. Nature Reviews Genetics, 14(4), 288–295. https://doi.org/10.1038/nrg3458
Ray-Jones, H., & Spivakov, M. (2021). Transcriptional enhancers and their communication with gene promoters. Cellular and Molecular Life Sciences, 78(19–20), 6453–6485. https://doi.org/10.1007/s00018-021-03903-w
Thomas, H. F., & Buecker, C. (2023). What is an enhancer? BioEssays, 45(10). https://doi.org/10.1002/bies.202300044
Resources on the Addgene blog
- Plasmids 101: Gene, enhancer, and promoter trapping
- Plasmids 101: The Promoter Region – Let's Go!
- Plasmids for Endogenous Gene Tagging in Human Cells
Resources on addgene.org
Topics: Open Science, Other Plasmid Tools, AAV, Material Sharing
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