Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hGAS6 mice. The exons 1-15 of mouse Gas6 gene that encode the full-length protein were replaced by human GAS6 exons 1-15 in B-hGAS6 mice.
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mRNA expression analysis
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Species-specific GAS6 gene expression analysis in wild-type and humanized B-hGAS6 mice by RT-PCR. Murine Gas6 mRNA was detected in ovarian tissue isolated from wild-type C57BL/6 (+/+) mice, while human GAS6 mRNA was exclusively detected in homozygous B-hGAS6 (H/H) mice.
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Protein expression analysis
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Species-specific GAS6 protein expression analysis in wild-type and humanized B-hGAS6 mice. Plasma was isolated from wild-type (+/+) and homozygous B-hGAS6 (H/H) mice, and analyzed by ELISA using species-specific GAS6 kits. Murine GAS6 protein was detected in wild-type mice, while human GAS6 protein was exclusively detected in B-hGAS6 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-hGAS6 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/B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hGAS6 mice were similar to those in the C57BL/6 mice, demonstrating that GAS6 humanized 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-hGAS6 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 CD8+ T cells and CD4+ T cells in homozygous B-GAS6 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGAS6 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. Leukocytes were isolated from female C57BL/6 and B-hGAS6 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 CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis Percent of T cells, B cells, NK cells, in homozygous B-hGAS6 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGAS6 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 lymph node
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Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hGAS6 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. 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 CD8+ T cells, CD4+ T cells and Treg cells in homozygous B-hGAS6 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGAS6 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 leukocytes cell subpopulation in blood
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Analysis of blood leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hGAS6 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 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-hGAS6 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGAS6 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 leukocyte subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hGAS6 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. 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+, CD8+, Treg cells in homozygous B-hGAS6 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGAS6 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.
