This project will credential two novel types of broadly applicable mouse models, which we will demonstrate in the lung. First, we will express genes that drive disease susceptibility on one side of the lungs only, a model we call a Unilateral Mosaic, creating disease-prone and disease resistant (internal control) lung tissue in a single animal. The lungs serve as a proof-of-concept organ, but this Unilateral Mosaic model applies to other asymmetric or paired organ systems such as the liver, and also to the limbs. The second type of engineered mouse will harbor an allele that installs permanent genetic marks within niche or neighbor cells, for in vivo cellular contact tracing in mice. Using a secreted and cell-permeant Tat-Cre protein, cells will be targeted for recombination in vitro using co-culture and media transfer experiments, and in vivo using transplantation of engineered Tat-Cre expressing cells into loxP-activated fluorescent reporter mice. There is an urgent need for mouse models that reduce animal to animal variability, and increase the in vivo resolving power for tracking cellular behavior. The models we present begin to address these challenges, and will appeal to a wide variety of investigators that employ mice to study complex human diseases. The proposed genetic tools are designed to address numerous categories of biological problems in living animals, but are not intrinsically confined to a specific disease state or organ. These studies employ the well-established Cre-LoxP system for targeted in vivo gene expression, applicable to the numerous floxed alleles in existence. This work will broadly impact the available applications for tissue- or lineage-specific expression of genes in mice, enabling investigators to track cells or induce gene expression in neighboring or transacting cells that contact driver cells of a given lineage.

Specific Aim 1. Generate a Unilateral Mosaic model of disease susceptibility. A. Test whether human Ace2 excision can restrict SARS-CoV2 infection to one side of the lung. B. Determine whether excision of Cox2/Ptgs2 unilaterally in the lungs can compartmentalize metastatic disease after intravenous injection of metastatic mouse mammary cancer cells.

Specific Aim 2. Test and deploy a Tat-Cre allele for in vivo lineage contact tracing. We anticipate this project will yield rapid results, as we already have the ASE-Cre Unilateral Mosaic driver mice in house and have exciting preliminary data to reveal the target organs for its asymmetric expression. We have performed many SARS-CoV-2 viral challenge studies in human Ace2 knockin/knockout mice with a currently approved and active animal BSL-3 program in the lab. In addition to our experience in mouse mammary tumor models and lung developmental and cancer biology in mice, we have established collaborations to ensure the success of syngeneic mammary tumor transplant studies. For Aim 2, we already have cloned and present preliminary data to demonstrate the utility of the new Tat-Cre construct. In summary, we have the required experience and existing preliminary data and materials to bring the proposed studies to a successful completion.