Gene targeting strategy for B-hPD-1/hPD-L1/hIL1B mice. The exon 2 of mouse PD-1 gene that encodes the IgV domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/hIL1B mice. The exon 3 of mouse pdl1 gene that encodes the IgV domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/hIL1B mice. The exons 2-7 of mouse Il1b gene that encode the full-length protein were replaced by human IL1B exons 2-7 in B-hPD-1/hPD-L1/hIL1B mice.
Protein expression analysis
Strain specific PD-1 and PD-L1 expression analysis in wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hIL1B mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hIL1B mice (H/H) after stimulated with or without anti-CD3ε in vivo. Mouse PD-1 and PD-L1 were detectable in wild-type C57BL/6 mice. Human PD-1 and PD-L1 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hIL1B mice but not in wild-type mice.
Strain specific IL1B expression analysis in homozygous B-hPD-1/hPD-L1/hIL1B mice by ELISA. Bone marrow cells were collected from C57BL/6 (+/+) and homozygous B-hPD-1/hPD-L1/hIL1B mice (H/H) and stimulated with LPS for 24 hours in vitro. The supernatant was analyzed with species-specific IL1B ELISA kit. Mouse IL1B was detectable in C57BL/6 mice. Human IL1B was exclusively detectable in homozygous B-hPD-1/hPD-L1/hIL1B mice but not C57BL/6 mice. ND, not detectable.