Tomas Lagunas Jr., Stephen P. Plassmeyer, Anthony D. Fischer, Ryan Z. Friedman, Michael A. Rieger et al.
The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.
Tomas Lagunas Jr., Stephen P. Plassmeyer, Anthony D. Fischer, Ryan Z. Friedman, Michael A. Rieger, Din Selmanovic, Simona Sarafinovska, Yvette K. Sol, Michael J. Kasper, Stuart B. Fass, Alessandra F. Aguilar Lucero, Joon-Yong An, Stephan J. Sanders, Barak A. Cohen, Joseph D. Dougherty. A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo. 2023-11-13. https://doi.org/10.1038/s42003-023-05483-w
A Cre-dependent plasmid 3′UTR MPRA library containing 120-bp reference, variant, GC-matched shuffled, and synthetic control elements was transfected into mouse Neuro-2a cells. Six biological replicates were profiled from whole-cell Input RNA and eGFP-RPL10a TRAP RNA, with plasmid DNA used for abundance normalization.
The same 3′UTR library was packaged in AAV9 and injected into perinatal Vglut1-IRES2-Cre-D mice; cortex was dissected at P21 for reporter RNA and viral DNA barcode sequencing. The published summary contains 11 RNA biological replicates and two viral-DNA library replicates.
The 3′UTR library was packaged in AAV9 and injected bilaterally into the striatum of perinatal Slc32a1/Vgat-Cre mice; striatum was dissected at P21 for reporter RNA and viral DNA barcode sequencing. Six animal RNA replicates were used for the reproducible medium-spiny-neuron screen.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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