Gene targeting strategy for B-hCD3E/hGPRC5D mice. The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hGPRC5D mice. The exons 2-4 of mouse Gprc5d gene that encode the full-length protein were replaced by human GPRC5D exons 1-3 in B-hCD3E/hGPRC5D mice. 

Strain specific CD3E expression analysis in homozygous B-hCD3E/hGPRC5D mice by flow cytometry. Splenocytes were collected from 4 week old wild type mice (+/+) and homozygous B-hCD3E/hGPRC5D mice (H/H; H/H). Protein expression was analyzed with anti-mouse CD3e antibody (Biolegend, 100312) and anti-human CD3e antibody (BD Horizon™, 562426) by flow cytometry. Mouse CD3E was detectable in wild type mice. Human CD3E was exclusively detectable in homozygous B-hCD3E/hGPRC5D mice but not in wild type mice.

GPRC5D expression analysis in homozygous B-hCD3E/hGPRC5D mice by western blot. Spleen, liver and testis were collected from 4 week old wild type mice (+/+) and homozygous B-hCD3E/hGPRC5D mice (H/H; H/H) analyzed by western blot with anti-GPRC5D antibody (abcam, ab55044). GPRC5D was detectable in wild type mice and homozygous B-hCD3E/hGPRC5D mice due to the cross-reactivity of antibody.

Strain specific analysis of GPRC5D mRNA expression in wild-type C57BL/6 mice and B-hCD3E/hGPRC5D mice by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3E/hGPRC5D mice (H/H; H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GPRC5D primers. Mouse Gprc5d mRNA was detectable in wild-type C57BL/6 mice. Human GPRC5D mRNA was detectable only in homozygous B-hCD3E/hGPRC5D mice but not in wild-type mice. 

Complete blood count (CBC). Blood from C57BL/6 and B-hCD3E/hGPRC5D mice (n=8, 8 week old) was collected and analyzed for CBC. There were no differences among any measurement between C57BL/6 and B-hCD3E/hGPRC5D mice, indicating that CD3E and GPRC5D humanized do not change blood cell composition and morphology. Values are expressed as mean ± SEM.

Blood biochemistry tests of B-hCD3E/hGPRC5D mice. Serum from the C57BL/6 and B-hCD3E/hGPRC5D mice (n=8, 8 week old) was collected and analyzed for levels of ALT and AST. There were no differences on either measurement between C57BL/6 and B-hCD3E/hGPRC5D mice, indicating that CD3E and GPRC5D humanized do not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.