Core C: The Murine Genetics and Phenotyping Core
|Flow cytometry strategy to isolate inflammatory cell populations in homogenized murine lungs. We developed a minimal and complete panel of antibodies that recognize surface markers on lung inflammatory cells that can accurately distinguish the inflammatory cell populations in the lung (minimal as shown) and their activation status. We have validated this panel in the normal and injured lung. (Misharin et al. AJRCMB 2013).|
The purpose of this PPG is to determine how the alveolar epithelium alone and in coordination with inflammatory cells in the lung contributes to the development of acute lung injury. In the first cycles of this PPG, the project investigators made important discoveries, which have enhanced our understanding of the pathophysiology of acute lung injury. All of these discoveries were supported by murine models provided by this Core. Dr. Sznajder (Project 1) used mice genetically deficient in HOIL specifically in the alveolar epithelium to identify a critical role for LUBAC in the development of lung injury. Dr. Ridge (Project 2) used mice globally deficient in vimentin to show that this intermediate filament protein serves as a scaffold required for activation of the inflammasome. Dr. Chandel (Project 3) has used mice harboring cell specific knockouts of key metabolic proteins to demonstrate that alterations in metabolism play a causal role in the pathogenesis of ALI. In this renewal application, all of the project investigators have designed careful gain and loss-of-function experiments that use murine genetics to target putative genes/proteins/pathways involved in the development of acute lung injury in specific cells and tissues. To examine the effects of these interventions, the project investigators have identified a need for accurate breeding and genotyping of these murine strains and the performance of reliable, reproducible and complementary measurements to determine the severity of the resulting lung injury. To accomplish these goals, the murine phenotyping core will pursue three specific aims.
- Aim 1. To perform breeding and genotyping of all of the murine strains, including the generation of tissue and cell type-specific knockout animals required by the project investigators.
- Aim 2. To provide investigators with several well-characterized models of acute lung injury.
- Aim 3. To provide quantitative, complementary measurements of lung inflammation, lung injury and lung regeneration following the induction of lung injury.
Support from the experienced personnel in the Core will provide resources that are unlikely to be achieved without support from the PPG mechanism. Careful attention to murine breeding techniques and rapid genotyping will accelerate the generation of tissue specific strains and reduce breeding costs. The highly reproducible murine model of influenza A pneumonia will provide a platform for the study of acute lung injury in each of the three projects. Findings in this model can be further examined in the other models of lung injury and fibrosis provided by the Core. Standardized, accurate, controlled and blinded measures of outcomes in the influenza A pneumonia and other models will allow investigators to compare the effects of their respective genes/pathways of interest in the same animal model opening new avenues for synergy.
1. Misharin AV, Morales-Nebreda L, Mutlu GM, Budinger GRS, Perlman H. Flow Cytometric Analysis of the Macrophages and Dendritic Cell Subsets in the Mouse Lung. American Journal of Respiratory Cell and Molecular Biology 2013.
Faculty Associated With Core C:
GR Scott Budinger, Core Leader
Gökhan Mutlu, Co-Leader