A scalable platform for the development of cell-type-specific viral drivers
- Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
Experimental strategy and GRE selection.
(a) Paralleled Enhancer Single Cell Assay (PESCA). Comparative ATAC-Seq is used to identify candidate GREs. A library of gene regulatory elements (GREs) is inserted upstream of a minimal …
Figure 1—figure supplement 1
Hierarchical clustering of the Mo et al. (2015) and our own ATAC-seq datasets.
Any ATAC-seq peak identified in any of the PV, SST, or VIP ATAC-seq datasets of this manuscript was given a score of 0 or one depending on whether any reads fell into that peak for a given sample. …
Figure 1—figure supplement 2
Identification of conserved GREs.
Left: For each of the 323,369 reference genomic regions that were identified by ATAC-Seq, a region of the same size was chosen exactly 100,000 bases away from the GRE. The mean sequence conservation …
Figure 2 with 6 supplements
PESCA screen identifies GREs highly enriched for SST+ interneurons.
(a) PESCA library plasmid map. ITR, inverted terminal repeats; GRE, gene regulatory element; pr, HBB minimal promoter; int, intron; GFP, green fluorescent protein; WPRE, Woodchuck Hepatitis Virus …
Figure 2—figure supplement 1
Schema for PESCA library construction.
PCR is used to amplify GREs from the genomic DNA and to introduce appropriate restriction enzyme sites and, subsequently, a 10 bp barcode sequence. Each GRE is amplified three times using three …
Figure 2—figure supplement 2
UMI, gene and GRE detection metrics.
(a) Dot plot of the number of unique molecular identifiers (UMIs) and the number of genes for each nucleus that was analyzed. (b) Plot showing the density distribution of number of UMIs and genes …
Figure 2—figure supplement 3
t-SNE plots of 32,335 nuclei from V1 cortex of two analyzed animals.
Color gradient denotes number of unique transcripts per nucleus of the indicated cellular marker gene.
Figure 2—figure supplement 4
Pairwise comparison between SST fold-enrichment values.
Dot plot of pairwise comparison between SST fold-enrichment values across three pairs of barcodes associated with the same GRE (left) and across randomly shuffled barcodes (right). The values on …
Figure 2—figure supplement 5
GRE specificity metrics.
(a) t-SNE plot of 32,335 nuclei from V1 cortex of two analyzed animals showing the mean viral expression across all GREs. Plot is pseudocolored based on the mean expression in each cell type. (b) …
Figure 2—figure supplement 6
Analysis of computationally subsampled data.
Data from each of the five most cell-type-specific GRE hits was computationally subsampled to decrease the number of viral transcripts by 2, 4, 8, or 16 fold (x-axis) (Materials and methods). Each …
Figure 3 with 5 supplements
In situ characterization of rAAV-GRE reporter expression.
(a–d) Fluorescent images from adult Sst-Cre; Ai14 mouse visual cortex twelve days following injection with rAAV-GRE-GFP as indicated. Scale bars 100 µm. (e) Identification of rAAV-GRE-GFP+ cells …
Figure 3—figure supplement 1
Fluorescent images from adult Sst-Cre; Ai14 mouse visual cortex twelve days following injection with rAAV-GRE-GFP as indicated.
https://doi.org/10.7554/eLife.48089.013
Figure 3—figure supplement 2
Analysis of mDlx5/6-GFP+ cells.
(a) Fluorescent images from adult Sst-Cre; Ai14 mouse visual cortex immunostained for PVALB twelve days following injection with rAAV-mDlx5/6-GFP as indicated. Scale bar 100 µm. (b) Quantification …
Figure 3—figure supplement 3
Quantification of the number of GFP+ SST+ cells normalized for area of infection.
Each dot represents one animal. Box plot represents mean ± standard error of the mean (s.e.m). Values are 73.0 ± 17.2, 146.9 ± 19.7, 144.8 ± 38.6 and 125.6 ± 26.4 cells/mm2 for AAV-[ΔGRE, GRE12, …
Figure 3—figure supplement 4
Fluorescent images from adult Vip-Cre; Ai14 mouse visual cortex immunostained for PVALB twelve days following injection with rAAV-GRE-GFP as indicated.
https://doi.org/10.7554/eLife.48089.016
Figure 3—figure supplement 5
Quantification of the fraction of GFP+ cells that are present it each cortical layer.
Each dot represents one animal. Box plot represents mean ± standard error of the mean (s.e.m). Gray represents all SST+ cells, colored plots represent GFP+ SST+ cells respectively, for AAV-[GRE12, …
Figure 4
Electrophysiology of neurons expressing an rAAV-GRE-driven reporter and modulation of neuronal activity with rAAV-GREs.
(a) Representative current-clamp recordings from SST neurons in the visual cortex of Sst-Cre; Ai14 mice injected with rAAV-GRE44-GFP. Top: Representative traces from a cortical SST neuron with …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Sst | NCBI Gene ID: 20604 | ||
| Genetic reagent (M. musculus) | Sst-IRES-Cre | Jackson Laboratory Stock # 013044 | IMSR Cat# JAX:013044, RRID:IMSR_JAX:013044 | |
| Genetic reagent (M. musculus) | Vip-IRES-Cre | The Jackson Laboratory Stock # 010908 | IMSR Cat# JAX:010908, RRID:IMSR_JAX:010908 | |
| Genetic reagent (M. musculus) | Pv-Cre | The Jackson Laboratory Stock # 017320 | IMSR Cat# JAX:017320, RRID:IMSR_JAX:017320 | |
| Genetic reagent (M. musculus) | SUN1-2xsfGFP-6xMYC | The Jackson Laboratory Stock # 021039 | IMSR Cat# JAX:021039, RRID:IMSR_JAX:021039 | |
| Genetic reagent (M. musculus) | Ai14 | The Jackson Laboratory Stock # 007914 | IMSR Cat# JAX:007914, RRID:IMSR_JAX:007914 | |
| Strain, strain background (Escherichia coli) | High Efficiency NEB 5-alpha | New England Biolabs | C2987H | Competent cells |
| Antibody | anti-GFP (Rabbit monoclonal) | Thermo Fisher | Cat# G10362; RRID:AB_2536526 | 0.012 ug/ul |
| Antibody | anti-Parvalbumin (Mouse monoclonal) | EMD Millipore | Cat# MAB1572; RRID:AB_2174013 | IF(1:2000) |
| Recombinant DNA reagent | pAAV-mDlx-GFP-Fishell-1 (plasmid) | PMID: 27798629 | Addgene # 83900; RRID:Addgene_83900 | |
| Recombinant DNA reagent | pAAV-ΔGRE -GFP- (plasmid) | This paper | ||
| Recombinant DNA reagent | pAAV-GRE12-GFP- (plasmid) | This paper | ||
| Recombinant DNA reagent | pAAV-GRE22-GFP- (plasmid) | This paper | ||
| Recombinant DNA reagent | pAAV-GRE44-GFP- (plasmid) | This paper | ||
| Commercial assay or kit | Nextera DNA Library Prep Kit | Illumina | FC-121–1030 | |
| Commercial assay or kit | In-Fusion HD cloning kit | Takara Bio | 639645 | |
| Commercial assay or kit | Agencourt AMPure XP | Beckman Coulter | # A63881 | |
| Commercial assay or kit | Hot Start High-Fidelity Q5 polymerase | New England Biolabs | M0494L |
Additional files
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Supplementary file 1
Genomic coordinates and conservation scores for the 323,369 reference putative GRE regions.
- https://doi.org/10.7554/eLife.48089.019
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Supplementary file 2
Genomic coordinates, distance to nearest TSS, nearest gene, ATAC-seq-specificity and PESCA-specificity of the 287 GREs that were included in the library.
- https://doi.org/10.7554/eLife.48089.020
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Supplementary file 3
Vector sequences for the in situ evaluated vectors rAAV-ΔGRE/GRE12/GRE22/GRE44-GFP.
Gray highlighting indicates the ITRs, green indicates GFP and yellow indicates GRE.
- https://doi.org/10.7554/eLife.48089.021
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Supplementary file 4
Electrophysiological Parameters of GRE44- and GRE44+ SST Neurons in Visual Cortex.
- https://doi.org/10.7554/eLife.48089.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.48089.023
