Basic Information
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Targeting strategy
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Gene targeting strategy for B-hCXCL13/hCXCR5 mice. The exons 1~4 of mouse Cxcl13 gene that encode the full-length protein were replaced by human CXCL13 exons 1~4 in B-hCXCL13/hCXCR5 mice. The exons 1~2 of mouse Cxcr5 gene that encode the full-length protein were replaced by human CXCR5 exons 1~2 in B-hCXCL13/hCXCR5 mice.
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mRNA expression analysis
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Species-specific CXCL13 and CXCR5 gene expression analysis in wild-type and humanized B-hCXCL13/hCXCR5 mice by RT-PCR. Murine Cxcl13 and Cxcr5 mRNA was detected in splenocytes isolated from wild-type C57BL/6 (+/+) mice, while human CXCL13 and CXCR5 mRNA was detected in homozygous B-hCXCL13/hCXCR5 (H/H) mice.
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Protein expression analysis
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Species-specific CXCL13 protein expression analysis in wild-type and humanized B-hCXCL13/hCXCR5 mice. Serum was isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXCL13/hCXCR5 (H/H) mice, and analyzed by ELISA using species-specific CXCL13 kits. Murine CXCL13 protein was detected in wild-type mice, while human CXCL13 protein was detected in B-hCXCL13/hCXCR5 mice.
Species-specific CXCR5 protein expression analysis in wild-type and humanized B-hCXCL13/hCXCR5 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXCL13/hCXCR5 mice, and analyzed by flow cytometry using species-specific anti-CXCR5 antibodies. Murine CXCR5 protein was detected in wild-type mice, while human CXCR5 protein was detected in B-hCXCL13/hCXCR5 mice.
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Analysis of spleen leukocytes
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Analysis of spleen leukocytes in wild-type and humanized B-hCXCL13/hCXCR5 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXCL13/hCXCR5 (H/H) mice (n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live cells were gated on CD45+ and used for further analysis as indicated. (B) Percent of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes and macrophages in homozygous B-hCXCL13/hCXCR5 mice were similar to those in wild-type mice, demonstrating that introduction of hCXCL13/hCXCR5 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these splenic cell types. Values are expressed as mean ± SEM.
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Analysis of spleen T cells
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Analysis of spleen T cells in wild-type and humanized B-hCXCL13/hCXCR5 mice. Splenocytes were isolated from wild-type C57BL/6 (+/+) and homozygous B-hCXC13/hCXCR5 (H/H) mice (female, n=3, 6-week-old), and analyzed by flow cytometry to assess leukocyte subpopulations. (A) Representative flow cytometry plots. Single live CD45+ cells were gated on CD3+ T cells and used for further analysis as indicated. (B) Percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCXCL13/hCXCR5 mice were similar to those in wild-type mice, demonstrating that introduction of hCXCL13/hCXCR5 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these splenic T cell subtypes. Values are expressed as mean ± SEM.
