Gene targeting strategy for B-hCD3E/hEGFR 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/hEGFR mice. The exons 2-17 of mouse Egfr gene that encode the extracellular domain were replaced by human EGFR exons 2-17 in B-hCD3E/hEGFR mice. 

Strain specific analysis of CD3E gene expression in wild type (WT) mice and B-hCD3E/hEGFR mice by RT-PCR. Mouse Cd3e mRNA was detectable only in spleen of WT mice (+/+). Human CD3E mRNA was detectable only in homozygous B-hCD3E/hEGFR mice (H/H; H/H) but not in WT mice (+/+). 

Strain specific CD3E expression analysis in homozygous B-hCD3E/hEGFR mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3E/hEGFR 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/hEGFR mice (H/H; H/H) but not in WT mice (+/+).

Immunohistochemical (IHC) analysis of EGFR expression in homozygous B-hCD3E/hEGFR mice. The liver and kidney were collected from WT and homozygous B-hCD3E/hEGFR mice (H/H) and analyzed by IHC with anti-EGFR antibody. Mouse EGFR was detectable in WT mice, and human EGFR was detectable in homozygous B-hCD3E/hEGFR mice. The arrow indicates tissue cells with positive EGFR staining (brown). 

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 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/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 8 week-old). Flow cytometry analysis of the splenocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3E/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these T cell subtypes  in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 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/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 8 week-old). Flow cytometry analysis of the leukocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3E/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these T cell subtypes  in blood. Values are expressed as mean ± SEM.

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 8 week-old). Flow cytometry analysis of the 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 and NK cells in homozygous B-hCD3E/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD3E/hEGFR mice (n=4, 8 week-old). Flow cytometry analysis of the leukocytes was performed to assess T cell subsets. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hCD3E/hEGFR mice were similar to those in the C57BL/6 mice, demonstrating that CD3E and EGFR humanized do not change the overall development, differentiation or distribution of these T cell subtypes  in lymph node. Values are expressed as mean ± SEM.

Complete blood count (CBC).  Blood from female C57BL/6 and B-hCD3E/hEGFR 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/hEGFR mice, indicating that CD3E and EGFR humanized do not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood biochemistry tests of B-hCD3E/hEGFR mice. Serum from the C57BL/6 and B-hCD3E/hEGFR 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/hEGFR mice, indicating that CD3E and EGFR humanized do not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.