Genetically Encoded Approaches to Target Specific Neural Subtypes in Vivo
Abstract
Neural cell-types are incredibly heterogenous. How the diversity of neural cell subtypes comes to be during development is a topic of intense study. New tools and techniques are essential for examining cell-type diversity and its implications for healthy brain functions. Differences in development likely contribute to the varied responses of cell subpopulations during neuroinflammation. This collection of works presents the design and generation of three tools to label and manipulate subtypes of cells. In Chapter 1, we introduce the Expression by Boolean Exclusion (ExBoX) AAV-based tool. ExBoX encodes expression of a gene of interest (GOI) that is turned on by one recombinase (Cre or Flp) and turned off by the other. Its simple design allows for insertion of larger cargo than similar tools. ExBoX can be used to express a GOI in a subset of neural cells, or to regulate GOI expression for a defined critical period. We show proof of concept utilization of ExBoX in the tight regulation of labeling both in vitro and in vivo. In chapter 2 we describe the design, generation, and characterization of a knock-in mouse line (Lcn2CreERT2) to specifically target subsets of astrocytes that become reactive in disease. Lcn2CreERT2 labels a subset of reactive astrocytes (RAs) across a wide range of neuroinflammatory insults. Using the Lcn2CreERT2 line we show RAs labeled during systemic inflammation remain in the brain following the resolution of inflammation and largely revert to a pre-inflammatory state. Next, we provide an extended protocol for induction and labeling of RAs in the Lcn2CreERT2 mouse. Finally, we designed and generated the C3-FlpOERT2 mouse line. The C3-FlpOERT2 line can label a subset of RAs under some neuroinflammatory conditions. We show this subset of RAs is labeled only in certain models of neuroinflammation caused by direct insult to the brain, and that C3-FlpOERT2 expressing RAs are not proliferative. Additionally, we show this tool can be used in combinatorial ways to drive robust and specific reporter expression in RAs. Overall, we show the design, generation, and utility of these newly generated mouse lines and viral tools for use in investigating cellular diversity during development and disease.