Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hIL7R mice. The exons 1~6 of mouse Il7r gene that encode the extracellular region were replaced by human IL7R exons 1~6 in B-hIL7R mice.
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mRNA expression analysis
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Species-specific IL7R gene expression analysis in wild-type and humanized B-hIL7R mice by RT-PCR. Murine Il7r mRNA was detected in splenocytes isolated from wild-type (+/+) mice, while human IL7R mRNA was detected in homozygous B-hIL7R (H/H) mice.
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Protein expression analysis
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Species-specific IL7R protein expression analysis in wild-type and humanized B-hIL7R mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hIL7R (H/H) mice, and analyzed by flow cytometry using species-specific anti-IL7R antibodies. Murine IL7R protein was detected in wild-type mice, while human IL7R protein was detected in B-hIL7R mice.
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T cell proliferation analysis
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Assessment of T cell proliferation (Ki-67) in humanized B-hIL7R mice. Human IL7 was intraperitoneally injected into B-hIL7R mice. Blood was collected before IL7 injection and on days 2, 5, 8 and 13 after injection. (A) Percentages of CD4+Ki67+ T cells and CD8+Ki67+ T cells were analyzed by flow cytometry. The change fold was shown in (B). Proliferation of CD4 + T cells and CD8 + T cells was significantly evident after stimulation with human IL7. Results demonstrate that introduction of hIL7R in place of its mouse counterpart does not change the proliferation function of CD4+ T cells and CD8+ T cells in blood.
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Induction of STAT5 phosphorylation analysis
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Mouse pSTAT5 was induced with mouse IL7 and human IL7 in homozygous B-hIL7R mice analyzed by flow cytometry. Splenocytes were collected from wild type C57BL/6 mice (+/+) and homozygous B-hIL7R mice (H/H), and stimulated with culture medium, mIL7 or hIL7. The induction of STAT5 phosphorylation on CD4+ T cells with the indicated stimulators was assayed by flow cytometry. STAT5 phosphorylation was successfully induced with mouse and human IL7 in wild type C57BL/6 mice and homozygous B-hIL7R mice. Results demonstrated that IL7 and IL7R were cross-reactive in mouse and human.
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Immune Cell Profile
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Analysis of leukocytes cell subpopulation in spleen
Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that IL7R humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.
Analysis of T cell subpopulation in spleen
Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL7R 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.
Analysis of leukocytes cell subpopulation in lymph node
Analysis of lymph node leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that IL7R humanized does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.
Analysis of T cell subpopulation in lymph node
Analysis of lymph node T cell subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL7R 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.
Analysis of leukocytes cell subpopulation in blood
Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that IL7R humanized does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.
Analysis of T cell subpopulation in blood
Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and B-hIL7R mice (n=3, 10-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-hIL7R mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL7R 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.
