Basic Information
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Gene Targeting Strategy
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Exons 2-8 of the mouse Cd3e gene was replaced by human CD3E exons 2-9 in B-hCD3EDG/hCD28 mice. The exons 1-5 of mouse Cd3d and the exons 1-6 of Cd3g gene were replaced by human CD3D exons 1-5 and CD3G exons 1-7 in B-hCD3EDG/hCD28 mice, respectively. Exons 2-3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2-3 in B-hCD3EDG/hCD28 mice.
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mRNA Expression Analysis
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Species-specific CD3DG gene expression analysis in double humanized B-hCD3EDG/hCD28 mice. Murine Cd3d and Cd3g mRNA was detected in the thymus from wild-type C57BL/6 (+/+) mice, while chimeric CD3D and CD3G mRNA was detected in homozygous B-hCD3EDG/hCD28 (H/H) mice.
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Protein Expression Analysis
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Species–specific CD3E and CD28 expression analysis in double humanized B-hCD3EDG/hCD28 mice. Splenocytes were collected from wild-type C57BL/6 (+/+) and homozygous B-hCD3EDG/hCD28 (H/H) mice and analyzed by flow cytometry using species-specific anti-CD3E and anti-CD28 antibodies. Human CD3E and human CD28 protein was exclusively detected in B-hCD3EDG/hCD28 mice compared to wild-type mice. -
Analysis of spleen immune cells
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Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated here. (B) Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these spleen cell subtypes. Values are expressed as mean ± SEM.
Analysis of spleen T cell subpopulations by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 and humanized B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on CD3+ T cells and used for further analysis as indicated. (B) Percent of CD8+ T cells, CD4+ T cells and Tregs humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these spleen T cell subtypes. Values are expressed as mean ± SEM.
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Analysis of lymph node immune cells
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Analysis of lymph node leukocyte subpopulations by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 and humanized B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated. (B) Percent of T cells, B cells and NK cells in homozygous B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these lymph node cell subtypes. Values are expressed as mean ± SEM.
Analysis of lymph node T cell subpopulations by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 and humanized B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on CD3+ T cells and used for further analysis as indicated. (B) Percent of CD8+ T cells, CD4+ T cells and Tregs in humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these lymph node T cell subtypes. Values are expressed as mean ± SEM.
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Analysis of blood immune cells
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Analysis of blood leukocyte subpopulations by flow cytometry. Blood cells were isolated from wild-type C57BL/6 and humanized B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these blood cell subtypes. Values are expressed as mean ± SEM.
Analysis of blood T cell subpopulations by flow cytometry. Blood cells were isolated from wild-type C57BL/6 and humanized B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on CD3+ T cells and used for further analysis as indicated. (B) Percent of CD8+ T cells, CD4+ T cells and Tregs humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these blood T cell subtypes. Values are expressed as mean ± SEM.
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Analysis of thymic T cells
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Analysis of thymic T cell subpopulations by flow cytometry. Thymocytes were isolated from wild-type C57BL/6 and B-hCD3EDG/hCD28 mice (female, n=3, 7 week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on CD3+ T cells and used for further analysis as indicated. (B) Percent of CD8+ T cells, CD4+ T cells and Tregs in humanized B-hCD3EDG/hCD28 mice were similar to those in wild-type mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these thymic T cell subtypes. Values are expressed as mean ± SEM.
