Phenotypes of genetically altered mice can be validated and enhanced by conducting basic immune function assays. These assays provide information to distinguish whether phenotypic characteristics are associated with secondary changes and ill health, or are the direct or indirect result of genetic manipulation. Basic immune analyses provide further evidence of the validity of these observations. In addition, novel phenotypes can be identified for understanding molecules and pathways involved in immunocyte development and immune function, including immunodeficiency, cancer, autoimmunity, biology of aging, and resistance to infectious diseases. The identification of lineage-specific developmental defects in mutant mice is also possible.
Complete blood counts (CBC) on each mutant line are performed to determine the total white blood cell counts and relative representation of each subpopulation (lymphocytes, neutrophils, bands, eosino-phils, basophils, monocytes). In addition, hematocrits and other red blood cell indices, as well as platelet counts from each mutant line are obtained. Peripheral lymphocytes are further characterized using flow cytometry by staining lymphocytes with fluorochrome-labeled antibodies specific for CD4 helper T cells, CD8 cytotoxic T cells, CD3e (T cell receptor component), B220 (pan-B cell marker), IgM, and IgD (separates immature from mature B cells). B and T lymphocyte function in mutant mice is assessed by stimulating splenocytes with T cell mitogens anti-CD3e plus anti-CD28, and separately, B lymphocytes with anti-IgM or lipopolysaccharide (LPS).
Lymphocyte development in bone marrow and thymus is assessed, because transgenic and gene-targeted mutant mice with moderate to slight defects in hematopoietic development can compensate and show normal peripheral WBC indices. B cell development can be broken down into seven different stages by the expression of various surface markers using flow cytometry and the following combinations of antibodies: B220BIO, IgMFITC, CD43PE combination, which divides the B cell lineage into mature B cells (fraction F-B220hi, IgM,+ CD43-), immature B cells (fraction E-B220to, IgM,+ CD43-), and pre-B cells (B220,+ IgM,- CD43+). If defects in B cell development are detected, further flow cytometry is done utilizing the following combination of antibodies: Allophycocyanin-conjugated B220, PE-conju-gated CD43, FITC-conjugated BP-1, and biotinylated HSA combination, which divides B lineage into large pre-B (fraction C'-B220,+ CD43,+ HSAhi, BP-1+), pro-B (fraction C -B220,+ CD43,+ HSAto, BP-1+), pro-B (fraction B-B220,+ CD43,+ HSA,+ BP1-), and pre-pro-B cells (fraction A-B220,+ CD43,+ HSA,-BP-1-).
A similar approach is used to characterize T cell development by flow cytometry in mutant mice. Thymocytes are harvested and total thymus cellularity measured, as well as the relative representation of CD4-CD8- (double-negative) CD4+CD8+ (double-positive) and CD4+8,- CD4-8+ (single-positive) developmental populations. For this purpose, a combination of anti-CD4/anti-CD8/anti-CD3e (T cell receptor) fluorochrome-labeled antibodies is used. CD69 levels, which are a measure of T cell activation, will also be measured. If any defects in thymocyte development are detected, further testing is warranted utilizing antibodies against other markers such as CD44, CD25, and Qa-2, a marker for positive selection (terminal differentiation of double-positive thymocytes into the single-positive compartment).
Myeloid and erythroid progenitors in total bone marrow are assessed by flow cytometry utilizing a combination of the following fluorochrome-labeled antibodies: Gr-1/Mac1, which identifies myeloid progenitors, and CD61 and Ter119, which identify megakaryocytes and erythroblasts, respectively.
A noninvasive assay to test for T cell function is the contact hypersenstivity assay (CHS). An exaggerated and sustained cutaneous swelling to the hapten dinotrofluoro-benzene (DNFB) is suggestive of a T cell defect. The procedure consists of sensitizing mice with DNFB on the abdominal skin with subsequent challenge with DNFB 4 days later on both ears. Cutaneous swelling is measured over time. The mean increase in ear thickness following DNFB challenge is calculated in units of 10-4 inches ± SEM.
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