Gene targeting strategy for B-hCD93 mice. The exon 1 of mouse Cd93 gene that encode the extracellular domain was replaced by human CD93 exon 1 in B-hCD93 mice.

Strain specific CD93 expression analysis in heterozygous B-hCD93 mice by flow cytometry. Bone marrow were collected from wild type and heterozygous B-hCD93 mice (H/+) , and analyzed by flow cytometry with species-specific CD93 antibody. Mouse CD93 was detectable in wild type mice and heterozygous B-hCD93 mice. Human CD93 was exclusively detectable in heterozygous B-hCD93 mice but not in wild type mice.

Strain specific CD93 expression analysis in homozygous B-hCD93 mice by flow cytometry. Bone marrow were collected from C57BL/6 wild type and homozygous B-hCD93 mice (H/H) , and analyzed by flow cytometry with species-specific CD93 antibody. Mouse CD93 was detectable in wild type mice. Human CD93 was exclusively detectable in homozygous B-hCD93 mice but not in wild type mice.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD93 mice (n=3, 7-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD93 mice were similar to those in the C57BL/6 mice, demonstrating that CD93 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. The same results were observed in lymph node and blood, data were not shown. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD93 mice (n=3, 7-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hCD93 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD93 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. The same results were observed in lymph node and blood, data were not shown. Values are expressed as mean ± SEM.