Basic Information
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Targeting strategy
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Gene targeting strategy for B-hTLR8/hTLR7 mice. The exon 3 of mouse Tlr8 gene that encodes the extracellular domain was replaced by human TLR8 exon 3 B-hTLR8/hTLR7 mice. The exon 3 of mouse Tlr7 gene that encodes the extracellular domain was replaced by human TLR7 exon 3 in B-hTLR8/hTLR7 mice.
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mRNA expression analysis
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Strain specific analysis of TLR8 and TLR7 gene expression in wild-type C57BL/6 mice and homozygous B-hTLR8/hTLR7 mice by RT-PCR. Mouse Tlr8 and Tlr7 mRNA was detectable in wild-type C57BL/6 mice (+/+). Human TLR8 and TLR7 mRNA was detectable only in homozygous B-hTLR8/hTLR7 mice but not in wild-type mice.
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Protein expression analysis in monocytes
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Strain specific TLR8 expression analysis in homozygous B-hTLR8/hTLR7 mice by flow cytometry. Splenocytes were collected from wild-type mice (+/+) and homozygous B-hTLR8/hTLR7 mice (H/Y;H/Y), and analyzed by flow cytometry with species-specific anti-TLR8 antibody. Mouse TLR8 was detectable in wild-type mice and homozygous B-hTLR8/hTLR7 mice due to the anti-mouse TLR8 antibody cross-reacts with human TLR8. Human TLR8 was exclusively detectable in homozygous B-hTLR8/hTLR7 mice but not WT mice.
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Protein expression analysis in macrophages
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Strain specific TLR7 expression analysis in homozygous B-hTLR8/hTLR7 mice by flow cytometry. Splenocytes were collected from wild-type mice (+/+) and homozygous B-hTLR8/hTLR7 mice (H/Y;H/Y), and analyzed by flow cytometry with species-specific anti-TLR7 antibody. Mouse TLR7 was exclusively detectable in WT mice but not in homozygous B-hTLR8/hTLR7 mice. Human TLR7 was exclusively detectable in homozygous B-hTLR8/hTLR7 mice but not WT mice.
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Protein expression analysis in DCs
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Strain specific TLR7 expression analysis in homozygous B-hTLR8/hTLR7 mice by flow cytometry. Splenocytes were collected from wild-type mice (+/+) and homozygous B-hTLR8/hTLR7 mice (H/Y;H/Y), and analyzed by flow cytometry with species-specific anti-TLR7 antibody. Mouse TLR7 was exclusively detectable in WT mice but not in homozygous B-hTLR8/hTLR7 mice. Human TLR7 was exclusively detectable in homozygous B-hTLR8/hTLR7 mice but not WT 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-hTLR8/hTLR7 mice (n=3, 8-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-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that TLR8 humanized does not change the overall development, differentiation or distribution of these cell types in spleen.
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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-hTLR8/hTLR7 mice (n=3, 8-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 , CD4+ T cells and Tregs in homozygous B-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that TLR8 humanized does not change the overall development, differentiation or distribution of these cell types in spleen.
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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-hTLR8/hTLR7 mice (n=3, 8-week-old). Flow cytometry analysis of the blood 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, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that TLR8 humanized does not change the overall development, differentiation or distribution of these cell types in blood.
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Analysis of T cell subpopulation in blood
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Analysis of blood T cell subpopulations by FACS. Blood were isolated from female C57BL/6 and B-hTLR8/hTLR7 mice (n=3, 8-week-old). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. 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. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hTLR8 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in blood.
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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 node cells were isolated from female C57BL/6 and B-hTLR8/hTLR7 mice (n=3, 8-week-old). Flow cytometry analysis of the 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，CD4+ T cells，CD8+ T cells in homozygous B-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that TLR8 humanized does not change the overall development, differentiation or distribution of these cell types in lymph node.
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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. lymph node cells were isolated from female C57BL/6 and B-hTLR8/hTLR7 mice (n=3, 8-week-old). Flow cytometry analysis of the 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 Tregs in homozygous B-hTLR8/hTLR7 mice were similar to those in the C57BL/6 mice, demonstrating that TLR8 humanized does not change the overall development, differentiation or distribution of these cell types in lymph node.
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Analysis of mTNF-α and mIFN-γ protein expression by ELISA
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Strain specific mTNF-α and mIFN-γ expression analysis in homozygous B-hTLR8/hTLR7 mice by ELISA. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTLR8/hTLR7(H/Y, H/Y) and stimulated with TLR8/TLR7 agonist in vitro for 24 h, then cell supernatants were collected and analyzed by ELISA with species-specific mTNF-α (A) and mIFN-γ (B) ELISA kit.
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Summary
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mRNA expression analysis:
Mouse Tlr8 and Tlr7 mRNA was detectable in wild-type C57BL/6 mice (+/+). Human TLR8 andTLR7 mRNA was detectable only in homozygous B-hTLR8/hTLR7 mice but not in wild-type mice.
Protein expression analysis:
- Mouse TLR8 was detectable in WT mice and homozygous B-hTLR8/hTLR7 mice due to the anti-mouse TLR8 antibody cross-reacts with human TLR8. Human TLR8 was exclusively detectable in homozygous B-hTLR8/hTLR7 mice but not WT mice.
- Mouse TLR7 was exclusively detectable in WT mice but not in homozygous B-hTLR8/hTLR7 mice. Human TLR7 was exclusively detectable in homozygous B-hTLR8/hTLR7 mice but not WT mice.
Leukocytes cell subpopulation analysis：
TLR8 and TLR7 humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, blood and lymph node in B-hTLR8/hTLR7 mice.
Functional analysis:
TLR8 agonists GS-9688 or TL8-506 and TLR7 agonist CL264 can activate mTNF-α and mIFN-γ in both wild-type C57BL/6 mice (+/+) and homozygous B-hTLR8/hTLR7 mice.
