Basic Information
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Targeting strategy
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Gene targeting strategy for B-hCD3EDG/hCD28 mice.
The exons 2-8 of mouse Cd3e gene were 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. The 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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Strain specific analysis of CD3DG gene expression in wild type (WT) mice and B-hCD3EDG/hCD28 mice by RT-PCR.
Mouse Cd3d and Cd3g mRNA were detectable in thymus of WT mice (+/+). Chimeric CD3D and CD3G mRNA were detectable in homozygous B-hCD3EDG /hCD28 mice (H/H) but not in WT mice (+/+).
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Protein expression analysis
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Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD28 mice by flow cytometry.
Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (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-hCD3EDG/hCD28 mice (H/H) but not in WT mice (+/+).
Strain specific CD28 expression analysis in homozygous B-hCD3EDG/hCD28 mice by flow cytometry.
Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (H/H), and analyzed by flow cytometry with species-specific anti-CD28 antibody. Mouse CD28 was detectable in WT mice (+/+). Human CD28 was exclusively detectable in homozygous B-hCD3EDG/hCD28 mice (H/H) but not in WT mice (+/+).
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Analysis of spleen leukocytes cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of spleen leukocyte subpopulations by FACS.
Splenocytes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 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 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
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Analysis of spleen T cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of spleen T cell subpopulations by FACS.
Splenocytes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 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 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.
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Analysis of lymph node leukocytes cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of lymph node leukocyte subpopulations by FACS.
Leukocytes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (n=3, 7 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
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Analysis of lymph node T cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of lymph node T cell subpopulations by FACS.
Leukocytes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (n=3, 7 week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in lymph node. Values are expressed as mean ± SEM.
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Analysis of blood leukocytes cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of blood leukocyte subpopulations by FACS.
Blood cells were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (n=3, 7 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, granulocytes, monocytes and macrophages in homozygous B-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
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Analysis of blood T cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of blood T cell subpopulations by FACS.
Blood cells were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (n=3, 7 week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in blood. Values are expressed as mean ± SEM.
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Analysis of thymus T cell subpopulations in B-hCD3EDG/hCD28 mice
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Analysis of thymus T cell subpopulations by FACS.
Thymocytes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (n=3, 7 week-old). Flow cytometry analysis of the thymocytes was performed to assess leukocyte subpopulations. 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-hCD3EDG/hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD3EDG/hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in thymus. Values are expressed as mean ± SEM. DN: double negative. DP: double positive.
