Basic Information
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Gene targeting strategy for B-hC5/hC5AR1 mice
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Gene targeting strategy for B-hC5/hC5AR1 mice. CDS of human C5 gene was inserted into exon 2 of mouse Hc gene in B-hC5/hC5AR1 mice. The exon 2 of mouse C5ar1 gene that encode the full-length protein was replaced by human C5AR1 exon 2 in B-hC5AR1 mice.
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mRNA expression analysis
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Strain specific analysis of C5 and C5AR1 gene expression in wild-type C57BL/6 mice and B-hC5/hC5AR1 mice by RT-PCR. Mouse Hc and C5ar1 mRNA were detectable only in lung cells of wild-type C57BL/6 mice (+/+). Human C5 and C5AR1 mRNA were detectable only in homozygous B-hC5/hC5AR1 mice (H/H), but not in wild-type mice.
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Protein expression analysis
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Strain specific C5 and C5a expression analysis in homozygous B-hC5/hC5AR1 mice by ELISA. Serum was collected from wild-type C57BL/6 mice (+/+; +/+) or homozygous B-hC5/hC5AR1 mice (H/H; H/H) and analyzed by ELISA with anti-C5 and anti-C5a antibodies. A. Mouse C5 and C5a were detectable in wild-type mice. Human C5 and C5a were exclusively detectable in homozygous B-hC5/hC5AR1 mice but not in wild-type mice. B. The concentration of C5 in male mice is higher than that in female mice.
Strain specific C5AR1 expression analysis in homozygous B-hC5/hC5AR1 mice by FACS. Bone marrow was collected from wild-type C57BL/6 mice (+/+; +/+) and homozygous B-hC5/hC5AR1 mice (H/H; H/H), and analyzed by FACS with anti-C5AR1 antibody. Mouse C5AR1 was only detectable in wild-type mice. Human C5AR1 was exclusively detectable in homozygous B-hC5/hC5AR1 mice but not in wild-type mice.
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Analysis of leukocytes cell subpopulation in spleen
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Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the 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-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 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 T cell subpopulation in spleen
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Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-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 TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.
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Analysis of leukocytes cell subpopulation in lymph node
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Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the 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-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 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 T cell subpopulation in lymph node
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Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-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 TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in lymph node. Values are expressed as mean ± SEM.
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Analysis of leukocytes cell subpopulation in blood
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Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the 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-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 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 T cell subpopulation in blood
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Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hC5/hC5AR1 mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hC5/hC5AR1 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hC5/hC5AR1 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.
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Hemolytic activity of serum complement
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Complement hemolytic activity on sheep erythrocytes. Serum was collected from wild type C57BL/6 mice and homozygous B-hC5/hC5AR1 mice. Hemolysis of sheep erythrocytes was measured after exposure to increasing concentrations of mouse serum. Results are expressed as percentage of hemolysis. Results showed that the complement hemolytic activity of B-hC5/hC5AR1 mice can be detected. But it is lower than that in wild type C57BL/6 mice.
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Summary
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mRNA expression analysis:
Mouse Hc and C5ar1 mRNA were detectable only in lung cells of wild-type C57BL/6 mice. Human C5 and C5AR1 mRNA were detectable only in homozygous B-hC5/hC5AR1 mice.
Protein expression analysis:
- Mouse C5 and C5a were detectable in wild-type mice. Human C5 and C5a were exclusively detectable in homozygous B-hC5/hC5AR1 mice.
- Mouse C5AR1 was only detectable in wild-type mice. Human C5AR1 was exclusively detectable in homozygous B-hC5/hC5AR1 mice.
- The concentration of C5 in male mice is higher than that in female mice.
Leukocytes cell subpopulation analysis：
C5/C5AR1 humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, lymph node and blood.
Complement hemolytic activity：
The complement hemolytic activity of B-hC5/hC5AR1 mice can be detected. But it is lower than that in wild type C57BL/6 mice.
