Targeting strategy

Gene targeting strategy for B-hCD3E/hMSLN mice. The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hMSLN mice. The mouse Msln gene sequences that encode the full length of coding region were replaced by human MSLN coding gene sequence in B-hCD3E/hMSLN mice.

Protein expression analysis

Strain specific CD3E expression analysis in homozygous B-hCD3E/hMSLN mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/hMSLN mice (H/H; H/H), and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was detectable in WT mice (+/+). Human CD3E was exclusively detectable in homozygous B-hCD3E/hMSLN mice (H/H; H/H) but not in WT mice (+/+).

Strain specific MSLN expression analysis in homozygous B-hCD3E/hMSLN mice by western blot. Lung was collected from wild type mice (+/+) and homozygous B-hCD3E/hMSLN mice (H/H; H/H), and analyzed by western blot with anti-MSLN antibody. Mouse MSLN was detectable in wild type mice (+/+). Human MSLN was detectable in homozygous B-hCD3E/hMSLN mice (H/H; H/H).

Analysis of spleen leukocytes cell subpopulations in B-hCD3E/hMSLN mice

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD3E/hMSLN mice (n=3, 8 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for 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, neutrophils, monocytes and macrophages in homozygous B-hCD3E/hMSLN mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and MSLN humanized do not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocytes cell subpopulations in B-hCD3E/hMSLN mice

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3E/hMSLN mice (n=3, 8 week-old). Flow cytometry analysis of the blood leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for 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, neutrophils, monocytes and macrophages in homozygous B-hCD3E/hMSLN mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and MSLN humanized do not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Blood routine test in B-hCD3E/hMSLN mice

Complete blood count (CBC). Blood from C57BL/6 and B-hCD3E/hMSLN mice (n=8, 8 week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hCD3E/hMSLN mice, indicating that CD3E and MSLN humanized do not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood biochemistry of B-hCD3E/hMSLN mice

Blood biochemistry tests of B-hCD3E/hMSLN mice. Serum from the C57BL/6 and B-hCD3E/hMSLN mice (n=8, 8 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hCD3E/hMSLN mice, indicating CD3E and MSLN humanized do not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.