• CMTM6 represents the CMTM family member proteins, a family consisting of 8 members. The CMTM6 gene is located on chromosome 3p22, and all the CMTM proteins belong to a chemokine-like factor gene superfamily, similar to the chemokine and transmembrane 4 superfamilies. CMTM6 interacts and colocalizes with PD-L1 in plasma membrane. Again, it prevents the lysosome mediated degradation of PD-L1, therefore, it stabilizes PD-L1 expression.

Gene targeting strategy for B-hCMTM6 mice.
The exons 1-4 of mouse Cmtm6 gene that encode extracellular domain, transmembrane domain, cytoplasmic region and 3’UTR region are replaced by human counterparts in B-hCMTM6 mice. The promoter and 5’UTR region of the mouse gene are retained. The human CMTM6 expression is driven by endogenous mouse Cmtm6 promoter, while mouse Cmtm6 gene transcription and translation will be disrupted.

Strain specific analysis of CMTM6 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hCMTM6 mice by RT-PCR. Liver RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCMTM6 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CMTM6 primers. Mouse Cmtm6 mRNA was detectable in wild-type mice. Human CMTM6 mRNA was detectable only in homozygous B-hCMTM6 mice (H/H) but not in wild-type mice.

Western blot analysis of CMTM6 protein expression in homozygous B-hCMTM6 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCMTM6 mice (H/H), and then analyzed by western blot with anti-CMTM6 antibody (Abcam, ab264067). 40 μg total proteins were loaded for western blotting analysis. CMTM6 was detected in liver, lung and spleen in wild-type mice and homozygous B-hCMTM6 mice, as the antibody is cross-recognized between human and mouse CMTM6.