Defining Genetic Variation in Widely Used Congenic and Backcrossed Mouse Models Reveals Varied Regulation of Genes Important for Immune Responses.

Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.

Comparative sensitivity analyses of quantitative polymerase chain reaction and flow cytometry in detecting cellular microchimerism in murine tissues.

Cellular microchimerism is defined as the presence of small populations of cells from one individual in another genetically distinct individual. The pivotal role of cellular microchimerism in a variety of immune settings is increasingly recognized, e.g. in context of pregnancy, transplantation and cancer. However, the detection of chimeric cells is overshadowed by technical limitations. This study aimed to overcome these limitations by testing the sensitivity and detection limit of a molecular biology approach (quantitative polymerase chain reaction, qPCR) and a cellular approach (flow cytometry) in order to identify experimentally induced cellular microchimerism in mice. Leukocytes isolated from lymph nodes or spleens of transgenic enhanced green fluorescent protein (eGFP) and CD45.1 mice respectively were used as targets to be detected as microchimeric cells among wild type (wt) or haploidentical cells. The detection limit of microchimeric cells by flow cytometry was 0.05% or lower for the respective eGFP(+) or CD45.1(+) cell subsets, which equals 48 cells or fewer per 1×10(5) wt cells. The detection limit of CD45.1(+) and CD45.2(+) cells among haploidentical CD45.1(+)2(+) cells by flow cytometry was 48 cells (0.05%) and 198 cells (0.2%), respectively. Using qPCR, a detection limit of 198 eGFP(+) cells per 1×10(5) wt cells, respective 0.2%, could be achieved. We here introduce two technical approaches to reliably detect low number of chimeric cells at a low detection limit and high sensitivity in transgenic mouse systems.