Genetic Tools Atlas

ENHANCER AAVs  

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. 

Mouse transgenes

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.

Epifluorescence imaging (EPI) 

EPI datasets consist of five 30 µm-thick sagittal sections distributed along the medio-lateral axis of the mouse brain, imaged with an epifluorescence microscope. In most experiments, enhancers are used to drive expression of SYFP2 (green), with DAPI (blue) and PI (red) providing nuclear/cytoplasmic counterstain. In experiments where the enhancer drives a recombinase, the signal will depend on the recombinase reporter and the interfering counterstain will be omitted (e.g. if a tdTomato-expressing recombinase reporter was used, PI stain was omitted).

Serial Two-Photon Tomography (STPT) 

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. 

Single Cell/Single Nucleus RNA-sequencing 

Single cell or single nucleus RNA-sequencing (sc/snRNA-seq) is used to determine the transcriptomic identity of labeled cells. In this approach, following virus administration, a region of interest (ROI) is dissected, and the tissue is dissociated for isolation of individual labeled cells/nuclei with FACS. After RNA-sequencing with Smart-seq v4 (SSv4) and next generation sequencing, the transcriptome of each cell is mapped to the transcriptomic cell type taxonomy most relevant to the dissected ROI. The proportions of cells/nuclei mapped to each of the region’s cell types are determined. This approach provides a detailed transcriptomic characterization of the labeled cells, but it can be prone to biases, which stem from the relatively small number of sequenced cells, as well as differential sensitivity of cell types to the dissociation and sorting process.