Basic Information
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Gene targeting strategy
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The exons 2-7 of mouse Cd20 gene that encode the full-length protein were replaced by human CD20 exons 2-7 in B-hCD20 mice.
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mRNA expression analysis
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Strain specific analysis of CD20 gene expression in wild type (WT) mice and B-hCD20 mice by RT-PCR.
Mouse CD20 mRNA was detectable only in splenocytes of WT mice (+/+). Human CD20 mRNA was detectable only in homozygous B-hCD20 mice (H/H) but not in WT mice (+/+).
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Protein expression analysis in B cells
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Strain specific CD20 expression analysis in homozygous B-hCD20 mice by flow cytometry.
Splenocytes were collected from WT and homozygous B-hCD20 (H/H) mice, and analyzed by flow cytometry with species-specific anti-CD20 antibody. Mouse CD20 was detectable in WT mice. Human CD20 was exclusively detectable in homozygous B-hCD20 but not WT mice.
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Analysis of spleen leukocytes cell subpopulations in B-hCD20 mice
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Analysis of spleen leukocyte subpopulations by FACS.
Splenocytes were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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-hCD20 mice
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Analysis of spleen T cell subpopulations by FACS.
Splenocytes were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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-hCD20 mice
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Analysis of lymph node leukocyte subpopulations by FACS.
Leukocytes were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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-hCD20 mice
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Analysis of lymph node T cell subpopulations by FACS.Leukocytes were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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-hCD20 mice
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Analysis of blood leukocyte subpopulations by FACS.
Blood cells were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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-hCD20 mice
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Analysis of blood T cell subpopulations by FACS.
Blood cells were isolated from female C57BL/6 and B-hCD20 mice (n=3, 9 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-hCD20 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD20 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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Blood routine test in B-hCD20 mice
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Complete blood count (CBC).
Blood from C57BL/6 and B-hCD20 mice (n=8, 8 week-old) was collected and analyzed for CBC. There was no differences among any measurement between C57BL/6 and B-hCD20 mice, indicating that introduction of hCD20 in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.
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Blood biochemistry of B-hCD20 mice
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Blood biochemistry tests of B-hCD20 mice.
Serum from the C57BL/6 and B-hCD20 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-hCD20 mice, indicating that introduction of hCD20 in place of its mouse counterpart does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.
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Summary
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mRNA expression analysis:
Mouse CD20 mRNA was detectable in splenocytes of WT mice (+/+). Human CD20 mRNA was exclusively detectable in homozygous B-hCD20 mice (H/H) but not in WT mice (+/+).
Protein expression analysis:
Human CD20 was exclusively detectable on B cells of homozygous B-hCD20 mice (H/H) but not in WT mice (+/+), and mouse CD20 was detectable only in WT mice (+/+).
Leukocytes cell subpopulation analysis：
CD20 humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, lymph node and blood.
Blood routine test and Blood biochemical test
CD20 humanized does not change the blood cell composition and morphology, ALT and AST levels or health of liver.
