Generation of a B-1 lymphocyte-specific conditional knockout mouse model Bhlhe41tdTomato-Cre

Abstract B-1 lymphocytes play indispensable roles in defensing a wide range of pathogens prior to adaptive immune response arising. Although several transgenic mouse models targeting different stages of B lymphocytes have been established e.g. Cd19-Cre and Mb1-Cre, a B-1 lymphocyte specific conditional knockout mouse model for illuminating the role of a gene of interest in B-1 lymphocytes is still missing. In this study, we aimed to generate a B-1 lymphocyte-specific knockout mouse model by expression of Cre recombinase under the control of the promoter of Bhlhe41, which is preferentially expressed in B-1 lymphocyte and microglia in immune system. A tdTomato-2A-Cre-polyA cassette was inserted to the locus of Bhlhe41 by CRISPR-CAS9 technology. Bhlhe41tdTomato-Cre/+Rosa26EYFP/+ mice were generated for determining Cre-mediated recombination efficiency. The accuracy of insertion of tdTomato-2A-Cre-polyA cassette to Bhlhe41 locus was confirmed by significantly reduced peritoneal B-1a lymphocytes in Bhlhe41tdTomato-Cre/tdTomato-Cre mice. Flow cytometric analysis of tdTomato in immune cells from Bhlhe41tdTomato-Cre/+confirmed the specific expression of BHLHE41 in B-1 lymphocytes rather than other B subsets. Further analysis of EYFP in immune cells from Bhlhe41tdTomato-Cre/+Rosa26EYFP/+ mice revealed larger than 90% recombination in splenic and peritoneal B-1 lymphocytes while less than 10% recombination in other B subsets. Hence, we generated a B-1 lymphocyte-specific conditional knockout mouse model Bhlhe41tdTomato-Cre, which can be used for studying the function of a specific gene in B-1 lymphocyte-mediated immune responses.