Germ Cell Mutations In Lymphocytes The Model Function Tests, Analysis And Application

The fast accumulation of mutant mouse strains in recent years has provided an invaluable resource for phenotype-based genetic screens. However, study of lymphoid phenotypes can be obscured or impractical if homozygous mutations cause early embryonic defects. Here we developed two mitotic recombination systems to aid phenotype screening of germ line mutations in the lymphoid tissues.One method is to induce loss of heterozygosity (LOH) in developing lymphocytes through chromosome deletion. Chromosome deletion was triggered by Cre/loxP-mediated inverse sister chromatid recombination in the G2/M phase of the cell cycle, leading to the generation of daughter cells missing part of or the entire recombinant chromosome. We show that the resulting cells were viable and capable of additional rounds of cell division, thus providing raw materials for subsequent phenotypic assessment. We used the recombination system to induce LOH at the E2A locus in developing B cells. A significant loss of pro-B and pre-B cells was observed when the wild-type allele was removed by chromosome deletion from the E2A heterozygous mice, a result consistent with the required role for E2A in B cell development. We also demonstrated the effectiveness of Cre-mediated chromosome deletion in the LOH assay for HEB function in T cell development. Thus, the Cre-mediated chromosome deletion provides a new and effective method for genome-wide assessment of germ line mutations in the lymphoid system.The other model we developed is to introduce mosaicism in lymphoid tissues by triggering mitotic homologous recombination on Chromosome 1 during mitosis. With lckCre activated in T cell development, we showed homozygous clones arose from heterozygous genetic background through homologous recombination between sister chromatids at pre-defined loxP sites. We further applied this system to investigate the function of DHX9 in T cell development. A severe loss of DHX9 homozygous mutant clones was observed in both thymus and periphery, suggesting DHX9 may affect the survival of developing T cells.