Genetic Tools Atlas

Enhancer AAVs contain short genomic regulatory sequences (enhancers) that, in combination with a minimal promoter, drive expression of a chosen genetic cargo. In our collection, most enhancers were selected from the genome based on their differential chromatin accessibility across different cell types, which is indicative of a possible role in cell type-specific regulation of gene expression. Most of our enhancers were derived from the mouse, human, and macaque genomes and were evaluated following delivery to the mouse brain. We used the retro-orbital (RO) delivery to young adult mice, intracerebroventricular (ICV) to newborns, or stereotaxic (STX) injection to adult mice.

Each Allen institute Enhancer (AiE) is given a unique 4-digit identifier, followed by an indication for the species of origin (mouse (m), macaque (q), or human (h). Some enhancers were bashed into fragments, or “Cores”, and a new sequence was constructed by concatenating each Core three times. These sequences contain a suffix denoting the number of the Core (1, 2 or 3) and the number of times the Core was repeated. Plasmids for enhancer AAV production that have been deposited to Addgene have Addgene IDs. Please reach out to Addgene technical support for availability timelines.

Transgenic mouse lines are created by inserting an exogenous DNA sequence (e.g. fluorophore, recombinase, or transcription factor) into the mouse genome. Knock-in transgenic mice are usually created by inserting a single copy of exogenous DNA at a specific position, frequently within a marker gene in the genome. This approach takes advantage of the endogenous regulatory elements in the genome to enable selective expression of various transgenes for cell labeling, monitoring, and/or perturbation. The knock-in transgenes frequently produce expected cell-type labeling, which corresponds to the expression pattern of the marker gene, but their efficiency, specificity, and strength, even in the same locus, can vary depending on the exact components inserted. This contrasts with randomly integrated transgenes, where the endogenous elements have been taken out of context and inserted randomly in the genome. Randomly integrated transgenes produce various expression patterns depending on their size, the regulatory elements included, the copy number inserted, as well as the insertion site, which is frequently unknown.

In most cases, transgenes can be classified as drivers or reporters based on the cargo they express. Driver transgenes express exogenous recombinases or transcription factors. Reporter transgenes express easily detectable ‘readout’ (e.g., fluorescent protein) when crossed to a driver. We characterized driver lines by crosses to appropriate reporters (see table below) and imaging with Serial Two-Photon Tomography (STPT). The imaging pipeline generates high-quality images in three channels, often requiring adjustments to visualize signals in the expected channel.

Datasets are produced by axially-swept light sheet microscopy of whole mouse brains using the SmartSPIM (LifeCanvas) platform. Optical sections are formed by a thin plane of excitation light coincident with the focal plane of the detection objective and imaged onto a sCMOS camera. The resulting dataset samples the fluorescence intensity of the entire brain nearly isotropically at voxel size of 1.8 x 1.8 x 2.0 µm^3. These datasets are used for qualitative evaluation of labeling pattern and brightness of enhancer driven fluorescent transgenes, as well as quantitation of targeted cell count and location throughout the brain.

Cell detection and anatomical placement are performed on the full-resolution three-dimensional data. In addition, we generate a series of maximum intensity projections, representing 30 µm of depth at 100 µm spacing, that can be quickly and consistently visually evaluated for overall expression strength and distribution.

STPT datasets are produced by serial sectioning of an intact brain at 100 µm-intervals along the coronal plane, followed by two-photon imaging of the exposed surface. The resulting dataset is a series of coronal images at high resolution that enable evaluation of the brain regions and cell populations labeled across the entire mouse brain.