The goal of this project is to build and distribute a community library of more than 100 mouse intersectional genetic alleles to facilitate the developmental, anatomical, molecular, and functional characterization of neural circuit organization in behavior and physiology. Even within narrowly defined cell types, significant diversity is found at multiple levels including genetic and molecular signatures, activity patterns, and synaptic connectivity. Current challenges now center on tools to identify, access, and study cell populations with increasing precision. Intersectional genetics offers exceptionally high resolution to consistently delineate distinct cell types in the embryo and adult mouse for functional, molecular and anatomical studies. Intersection genetics utilizes a ubiquitously expressed conditional allele that is activated by Cre and Flp site specific recombinases. Upon activation, the intersectional genetic alleles may express any number of Genetically Encodable Effector Molecules (GEEMS), such as channelrhodopsins for neural activity perturbations or an L10-GFP ribotrap fusion to affinity purify translating mRNAs. The intersectional allele is activated by overlapping expression of both Flp and Cre recombinases in the same cell. Traditionally, these recombinases have been deployed as gene knock-in or transgenic alleles that are designed to express in a cell or gene specific fashion. The use of two selectors (Cre and Flp) to define a cellular population enables high specificity and modularity in combining any set of Cre, Flp and intersectional alleles to fit an experiment. The number of Cre and Flp recombinase mouse lines are constantly growing, giving greater access to increasing numbers of cell types. Additionally, these recombinases are the focus of multiple efforts to deploy them in ways that select cells based on other unique properties such as neuronal activity and synaptic connectivity. Despite the modularity and advantages offered by intersectional genetic mouse tools, their use remains limited due to the small number of publically available intersectional responsive alleles that express unique GEEMS and the difficulty in producing new intersectional mouse lines. Toward increasing the number of intersectional lines available to the mouse community, we propose a production pipeline in the following three aims create a suite of resources for anyone to easily make their own intersectional mouse line, to produce over 100 targeted ES cell lines that can be developed into mouse lines, as well as 10-15 high demand mouse lines.
Aim 1) Assemble community input and generate a facile pipeline for the rapid production of intersectional targeting vectors. Our lab has engineered several different intersectional genetic targeting cassettes to quickly build new alleles based on public input and rapidly integrate new effector molecules.
Aim 2) Use multiplex gene targeting for cost-efficient production of a large library of intersectional ES cell lines. Our lab has re-designed the frequently used Rosa26 targeting vector to be stable and amenable to Cas9 mediated homologous recombination while increasing the rate of targeting to facilitate multiplexed ES cell electroporation.
Aim 3) Distribute targeted alleles either as ES cells or, for select alleles, generate mouse lines for deposit in a public repository. Based on public input, a select number of ES lines will be prioritized for mouse line generation. All resources derived here will be placed in public repositories.
It is now clear that cells and tissues once thought to be homogeneous are diverse in their molecular makeup and specific functions in physiology, behavior, and disease and thus tools that enable the targeting and access of highly defined cellular populations for in vivo studies are needed. Based on community input, our work will create a vast resource of recombinant DNA vectors, targeted ES cell lines, and intersectional genetic mouse lines to delineate, access, and study highly defined cell populations for multifaceted studies at the anatomical, molecular, and functional levels. All plasmids, ES cell lines, and mouse lines produced under this proposal will be freely available to the research community through publically managed repositories.