Basic Information
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Targeting strategy
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Gene targeting strategy for B-hCRTAM mice. The exons 1~8 of mouse Crtam gene that encode the extracellular domain was replaced by human CRTAM exons 1~8 in B-hCRTAM mice.
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Protein expression analysis in CD8+ T cells of spleen
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Strain specific CRTAM expression analysis in homozygous B-hCRTAM mice by flow cytometry. CD8+ T cells were sorted from the splenocytes in wild-type C57BL/6 mice (+/+) and homozygous B-hCRTAM mice (H/H) and stimulated with anti-mCD3e in vitro (0.5 μg/mL, stimulation for 72 hours) and anti-mCD28 in vitro (1 μg/mL, stimulation for 72 hours). Protein expression was analyzed with anti-mouse CRTAM antibody (Biolegend, 142007) and anti-human CRTAM antibody (Biolegend, 339106) by flow cytometry. Mouse CRTAM was exclusively detectable in wild-type mice. Human CRTAM was exclusively detectable in homozygous B-hCRTAM mice but not in wild-type mice.
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mRNA expression analysis
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Strain specific analysis of CRTAM mRNA expression in wild-type C57BL/6 mice and B-hCRTAM mice by RT-PCR. Spleen RNA were isolated from wildtype C57BL/6 mice (+/+) and homozygous B-hCRTAM mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CRTAM primers. Mouse Crtam mRNA were detectable only in wild-type C57BL/6 mice. Human CRTAM mRNA was detectable only in homozygous B-hCRTAM mice but not in wild-type mice.
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Functional analysis
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Induction of mouse IFN-γ was induced with human NECL2 in wild-type C57BL/6 mice and homozygous B-hCRTAM mice. CD8+ T cells were isolated and induced with anti-mCD3e (0.25 μg/mL)(BioXcell, BE0001-1), anti-mCD28 (0.5 μg/mL) (BioXcell, BE0015-1) with or without hNECL2 (5 μg/mL) (ACROB, CA1-H5225) for 72h. Concentration of mouse IFN-γ secreted from CD8+ T cells was assayed with anti-mouse IFN-γ antibody (Biolegend, 430804) by ELISA. Mouse IFN-γ was successfully induced with human hENCL2 in homozygous B-hCRTAM mice. These results indicate that humanized CRTAM can recognize human NECL2 ligands to activate CD8+ T in homozygous B-hCRTAM mice. Values are expressed as mean ± SEM. Significance was determined by Multiple t tests. *P < 0.05, **P < 0.01, ***P < 0.001.
