• PD-1 (Programmed Cell Death-1) is mainly expressed on the surface of T cells and primary B cells. PD-L1 (Programmed Cell Death Ligand-1), also known as CD274, is mainly expressed on antigen-presenting cells (APCs) and activated T cells, and is one of the two ligands of PD-1. The interaction between PD-1 and PD-L1 plays an important role in the negative regulation of immune responses. The interaction between PD-1 and PD-L1 can reduce T cell activation and promote tumor immune escape.
• The CDS that encodes human PD-1 signal peptide, extracellular domain, transmembrane and cytoplasmic domain was inserted right after mouse PD-1 ATG to replace the mouse PD-1 gene in B-hPD-1 ad/hPD-L1 plus mice. The exons 1-7 of mouse Pdl1 gene that encode the whole molecule were replaced by human counterparts in B-hPD-1 ad/hPD-L1 plus mice, and the promoter, 5’UTR and 3’UTR region of the mouse gene were replaced by human counterparts.
• Human PD-1 and PD-L1 were only detectable in homozygous B-hPD-1 ad/hPD-L1 plus mice, but not in wild-type C57BL/6JNifdc mice.
• This product can be used as a mouse model to validate the pharmacodynamics and safety of drugs for treating tumors or autoimmune diseases.

Gene targeting strategy for B-hPD-1 ad/hPD-L1 plus mice

The CDS that encode human PD-1 signal peptide, extracellular domain, transmembrane and cytoplasmic domain was inserted right after mouse PD-1 ATG to replace the mouse PD-1 gene. The human PD-1 protein expression was driven by endogenous mouse PD-1 promoter, while mouse PD-1 gene transcription and translation will be disrupted.

The exons 2-7 of the mouse Pdl1 gene were replaced with human PDL1 exons 1-7. The human PD-L1 expression was driven by the endogenous human PDL1 promoter, with both the 5’UTR and 3’UTR sequences being of human origin.

Strain specific PD-1 expression analysis in homozygous B-hPD-1 ad/hPD-L1 plus mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 ad/hPD-L1 plus mice (H/H;H/H) stimulated with or without anti-mouse CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 hrs (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 329904) and anti-human PD-1 antibody (Biolegend, 109104) by flow cytometry. Mouse PD-1 was detectable in wild-type C57BL/6JNifdc mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1 ad/hPD-L1 plus mice but not in wild-type mice.

Strain specific PD-L1 expression analysis in homozygous B-hPD-1 ad/hPD-L1 plus mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 ad/hPD-L1 plus mice (H/H;H/H) stimulated with or without anti-mouse CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 hrs (female, 8-week-old, n=1). Protein expression was analyzed with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry. Mouse PD-L1 was detectable in wild-type C57BL/6JNifdc mice. Human PD-L1 was exclusively detectable in homozygous B-hPD-1 ad/hPD-L1 plus mice but not in wild-type mice.