Case Study 1

1. Case Study 1 • Dilated cardiomyopathy in RAG-1-/- mice • ? Viral ? Genetic • Sporadic occurrence in up to 20% of mice • Increased incidence after extensive backcrossing to B10.BR - almost universal on histopathology, and clinically apparent particularly in female breeding stock • Unable to segregate RAG-1-/- and cardiomyopathy, no way to genotype for susceptibility to cardiomyopathy • Fortunately, RAG-1-/- subline sent to ARC was free of cardiomyopathy. Entire backcross to B10.BR was repeated starting with breeding nucleus from ARC

2. Case Study 2 • Colitis in RAG-1-/- colony • Originally suspected due to Citrobacter but microbiological tests negative • Genetic analysis of every case in past 12 months revealed that colitis occurred only in mice with a particular genotype - T cell receptor transgenic with T cells produced by the thymus • Solution 1: breed T cell receptor transgenic colony on a background that does not produce T cells in the thymus, and cross onto T-producing background only when necessary to produce experimental mice. Use mice in experiments before onset of colitis • Solution 2: Infection with Helicobacter identified as a necessary co-factor for development of colitis. C-section colony onto Helicobacter-negative foster mothers from ARC

3. Case Study 3 • Early death in homozygous CTLA-4-/- mice due to abnormal immune activation • Occurrence of pathology and need for technically difficult genetic screening in breeding colonies led to problems identifying sick animals quickly enough, maintaining lines and generating sufficient homozygous mice for experiments • Solution: Breed two homozygous colonies of CTLA-4-/- mice on RAG-1-/- and RAG-2-/- backgrounds, respectively. The lack of immune system prevents immune pathology due to abnormal immune activation. When mice needed for experiments, intercross the lines on RAG-1-/- and RAG-2-/- backgrounds. This generates RAG-1+/- and RAG-2+/- mice with an immune system. All mice will be homozygous CTLA-4-/-, and experiments can be planned knowing how many mice are available. • Note - total number of mice bred while establishing CTLA-4-/- lines on RAG-1-/- and RAG-2-/- backgrounds was significantly larger than required to maintain the previous strategy