Foundational Porcine Stem Cell and Genetic Tools to Enable Rigorous, Reproducible Generation of Large-Animal Models

About This Grant

Project Summary The discovery of embryonic stem cells (ESCs) and the development of genetic tools for cell type-specific manipulation, lineage tracing, and functional analysis have elevated the mouse to a foundational role in biomedical research. However, limitations inherent to mice prevent them from serving as “universal” models. Large animal “bridge” models, such as pigs that share a close similarity in size, anatomy, and physiology with humans, combined with the feasibility of genetic engineering (GE), offer a transformative alternative. These attributes make pigs uniquely suited for xenotransplantation research and for modeling chronic diseases that account for over 90% of the $4.1 trillion annual U.S. healthcare costs. Despite this potential, the utility and broad adoption of pigs have been limited by the lack of authentic ESCs that enable iterative, complex genetic modifications required for generating Cre-driver and Cre-responder lines, developing sophisticated models for disease research, and for xenotransplantation. This R24 resource proposal aims to close this critical gap through three Specific Aims: • Aim 1: Validate a site-specific serine recombinase-based porcine embryonic stem cell (pESC) platform. A landing pad compatible with Bxb1, PA01, and KP03 recombinases has been integrated into the pROSA26 safe harbor locus, enabling the scalable and seamless insertion of large transgenes. As a proof-of-concept, we will generate three pESC-derived pig models of increasing complexity: (i) a dual fluorescent/PET reporter (~5 kb) for lineage tracing, non-invasive imaging, and conditional ablation; (ii) an ~20 kb transgene designed to overcome innate immune barriers to xenotransplantation; and (iii) a human cholesteryl ester transfer protein (CETP) BAC (~50 kb) to model human lipid metabolism. • Aim 2: Generate and characterize lineage-specific, tamoxifen-inducible CreERT2 driver lines targeting alveolar cells, cardiomyocytes, endothelial cells, enterocytes, hepatocytes, and pancreatic β-cells, chosen for their relevance to vascular biology, metabolism, and xenotransplantation. Additional drivers will be developed in response to community needs. • Aim 3: Implement transparent prioritization, access, and resource sharing via the National Swine Resource and Research Center (NSRRC), supported by standardized MTAs, cost-recovery mechanisms, and community outreach. In summary, this project will deliver: (i) the first standardized pESC-based GE platform for pigs, (ii) a suite of lineage-specific, temporally inducible Cre-driver lines, and (iii) a transparent distribution framework to ensure broad access. By filling critical infrastructure gaps and ensuring rigorous validation and dissemination, this proposal aims to transform the utility of pigs as a genetically tractable model for biomedical research.