Generation of novel cell type specific mouse genetic tools

Ngai, John

Abstract

Understanding how neural circuits process sensory information, generate movement and give rise to other complex behaviors requires an inventory of the cell types comprising these circuits and a means of studying their intrinsic properties and functional connectivity. Recent years have witnessed a revolution in our ability to identify and characterize cell types from highly complex organs based on their transcriptomes. Such transcriptomic analysis ? enabled by remarkable advances in single-cell RNA-sequencing technologies ? not only allows a definitive classification of cell types but also genetic information that can be used to access these cells experimentally. In parallel, significant advances have been made over the last decade in interrogating neuronal anatomy and function by driving expression of fluorescent protein reporters and optogenetic and chemogenetic effectors in specific cell types using cell type-specific transcriptional promoters. However, many if not most cell types in the brain cannot be defined by a single gene marker alone but rather require combinations of markers to allow their unambiguous identification. Thus, comprehensive access to cell types in the mammalian brain will require a strategy to create ? at scale ? mouse lines in which cell types are labeled intersectionally based on co-expression of multiple marker genes. In this Research Segment, we will deploy an efficient mouse transgenic pipeline based on CRISPR/Cas9-mediated gene editing technologies to generate intersectional mouse driver/reporter lines that label cell types discovered by transcriptomics in Research Segment 1 and from other groups around the world. Our mouse genome engineering pipeline is scalable and designed to allow the rapid generation and screening of mouse reporter lines for downstream anatomical and functional analysis.

Public Health Relevance

Unraveling the principles underlying the coding of information and generation of complex behaviors by the brain requires an understanding of the cell types comprising the brain?s underlying neural circuits and a means to interrogate these cells experimentally. Recent advances in single cell transcriptome profiling have provided an approach to identify and classify the myriad cell types in the mammalian brain. In this research segment, we will deploy an efficient and scalable genome engineering pipeline for creating mouse reporter lines that will enable broad experimental access to neuronal cell types discovered by single cell transcriptomics.