This project’s goal was to discover and relate poorly-annotated genes to respiratory function and inflammatory lung disease as part of a high-throughput mouse production and phenotyping pipeline. Sixty strains of knockout mice were produced, expanded, and screened using 16 mice (8 females; F, 8 males; M) per strain. Twelve mice (6F, 6M) were homozygous knockouts and 4 mice were co-raised wild type controls (2F, 2M). All mice were sensitized with an inert allergen (ovalbumin; OVA) over 21 days. On day 22 mice were challenged with a non-allergen airway agonist (Methacholine; MCh) followed by measurement of enhanced pause (Penh), respiratory rate, tidal volume, peak expiratory flow (PEF), and expiratory time (Te) using unrestrained whole-body plethysmography (WBP) to assess respiratory function, lung development, and airway hyperreactivity. Measurements were taken at baseline and at concentrations of 0, 12.5, 25, and 50 mg/ml MCh. In addition serum immunoglobulin (OVA-specific IgG1 and IgE) was measured by ELISA and standardized semi-quantitative histopathology scoring was used to assess allergic airway disease and acquired immune response/TI hypersensitivity. Scoring evaluates airway wall thickening, inflammatory cell infiltration, fibrosis, and Clara cell hyperplasia characterized by hyper secretion, mucus metaplasia, and airway mucus plugging.
The 60 strains of knockout mice had cumulative histopathology scores between 1.25 and 2.94 compared to the wild-type average of 1.93 (n=231). The 5 genes which caused the highest scores (2.31 – 2.94) were: Svs2, Slc47a2, Adipoq, Fbxl22, and Arap1. These 5 strains also had either significantly increased Penh (p≤0.05), or decreased PEF (p≤0.05), or increased Te (p≤0.05), or a combination of increased Penh and decreased PEF compared to wild type mice. The 5 knockout strains with the lowest histopathology scores had no abnormal WBP measurements.
We report a standardized and validated method to screen presumptive null mutant mouse strains with poorly annotated genes for abnormal phenotypes associated with respiratory function and inflammatory lung disease. Strains with high histopathology scores correlated with abnormal lung function results. One strain with decreased tidal volume and Penh (airway hyperreactivity) after sensitization may be particularly interesting as a potential drug target. All of the mouse strains used in this screen are freely available to the research community and may provide new models for hypothesis-driven mechanistic studies or purpose-driven drug target research.