Basic Information
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Targeting strategy
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Gene targeting strategy for B-hTLR9 mice. The exon 2 of mouse Tlr9 gene that encode the extracellular domain was replaced by human TLR9 exon 2 in B-hTLR9 mice.
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mRNA expression analysis
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Strain specific analysis of TLR9 gene expression in WT and hTLR9 mice by RT-PCR. Mouse Tlr9 mRNA was detectable only in splenocytes of wild-type C57BL/6 mice (+/+). Human TLR9 mRNA was detectable only in homozygous B-hTLR9 mice (H/H), but not in wild-type mice. -
Expression analysis in B cells
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Strain specific TLR9 expression analysis in homozygous B-hTLR9 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hTLR9 (H/H) mice, and analyzed by flow cytometry with species-specific anti-TLR9 antibody. Mouse TLR9 was detectable in WT mice (middle) and homozygous B-hTLR9 mice (right) compared to isotype (left) due to the cross-reactivity of the anti-mTLR9 antibody between mice and humans. Human TLR9 was exclusively detectable in homozygous B-hTLR9 mice (right) but not WT mice (middle).
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Expression analysis in NK cells
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Strain specific TLR9 expression analysis in homozygous B-hTLR9 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hTLR9 (H/H) mice, and analyzed by flow cytometry with species-specific anti-TLR9 antibody. Mouse TLR9 was detectable in WT mice and homozygous B-hTLR9 due to the anti-mTLR9 antibody is cross-reactive between mice and humans. Human TLR9 was exclusively detectable in homozygous B-hTLR9 but not WT mice.Protein expression analysis in NK cells
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Expression analysis in macrophages
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Protein expression analysis in macrophages
Strain specific TLR9 expression analysis in homozygous B-hTLR9 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hTLR9 (H/H) mice, and analyzed by flow cytometry with species-specific anti-TLR9 antibody. Mouse TLR9 was detectable in WT mice and homozygous B-hTLR9 due to the anti-mTLR9 antibody is cross-reactive between mice and humans. Human TLR9 was exclusively detectable in homozygous B-hTLR9 but not WT mice.Protein expression analysis in macrophages
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Cytokine secretion
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Functional analysis
Methods:
B-hTLR9 mice and wild-type C57BL/6 mice (n=3, female, 4~6 weeks) were treated with TLR9 agonists at different doses, and blood were collected at 2 h, 6 h and 24 h post s.c. injection of TLR9 agonist in saline. Cytokines in serum of individual mouse were determined in duplicate using multiplex assay.Results:
1. TLR9 agonists induced a number of cytokines in vivo as expected. In general, the cytokine response was dose and time dependent for most cytokines in both B-hTLR9 and wild-type mice. However, the cytokine response was attenuated in B-hTLR9 mice compared with wild-type mice. No adverse clinical symptoms and mortality were observed during the experiment, indicating that the test compounds were well tolerated by the animals.
2. The mouse TLR9 selective oligo only activated the signaling in the wild-type C57BL/6 mice but not in B-hTLR9 mice, and increased the expression of IL-6, TNF-α, IFN-γ.
3.The human/mouse cross-reactive oligo both increased the expression of IL-6, TNF-α, IFN-γ in the homozygous B-hTLR9 mice and C57BL/6 mice.
4.B-hTLR9 mice provide a powerful preclinical model for in vivo evaluation of antibodies conjugated TLR9 oligo. -
References
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1.Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene. 2008 Jan 7;27(2):161-7.
2.Mifsud EJ, Tan AC, Jackson DC. TLR Agonists as Modulators of the Innate Immune Response and Their Potential as Agents Against Infectious Disease. Front Immunol. 2014 Mar 3;5:79.
3.Krieg M. Development of TLR9 agonists for cancer therapy. J Clin Invest. 2007 May;117(5):1184-94.
