hPD-1(3)

Fig.1 Detection of PDCD1 expression in spleen by RT-PCR. Spleen RNA was extracted from 7-week-old female wild-type C57BL/6 (WT)(n=2) and homozygous hPD-1(3) knockin mice (HO) (n=2), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mRNA primers. Mouse Pdcd1 mRNA (187 bp) was detectable only in wild-type C57BL/6 mice. Human PDCD1 mRNA (137 bp) was detectable only in homozygous hPD-1(3) knockin mice but not in wild-type mice. 

Fig.2 Detection of PDCD1 expression in spleen by qPCR. H/M Pdcd1 mRNA was detectable both in wild-type mice and homozygous hPD-1(3) knockin mice. Spleen RNA was extracted from 7-week-old female wild-type C57BL/6 (WT)(n=2) and homozygous hPD-1(3) knockin mice (HO) (n=2), then cDNA libraries were synthesized by reverse transcription, followed by qPCR with h/m Pdcd1 and mouse Gapdh primers. Relative expression represents the h/m Pdcd1 mRNA level relative to its average expression in homozygous hPD-1(3) knockin mice. 

Fig.3 Human and mouse PD-1 expression on T cells from WT C57BL/6 and HO hPD-1 homozygous mice. Splenocytes were gated on CD3⁺ or CD4⁺ T cells and analyzed by flow cytometry with anti-human PD-1and anti-mouse PD-1. Flow cytometric analysis shows that human PD‑1 is expressed on CD3⁺ and CD4⁺ T cells in HOhPD-1 mice , while WT C57BL/6 mice lack human PD‑1 expression, confirming successful humanization of the Pdcd1 locus.