• ASPH, whose full name is Aspartate β-hydroxylase, is a member of the α-ketoglutarate-dependent dioxygenase family and belongs to the type II transmembrane proteins. The main enzymatic functional domain of ASPH is located at the C-terminus, and the C-terminal fragment of the ASPH molecule possesses hydroxylase activity. Mice lacking Asph exhibit syndactyly, facial deformities, mild hard palate defects, and reduced fertility in females. ASPH has natural substrates, namely aspartic acid and asparagine residues within the EGF-like repeat sequences of various proteins, particularly the Notch receptors and ligands. The Notch signaling pathway is crucial for cell differentiation, cell fate determination, adhesion, proliferation, migration, invasion, and stemness. ASPH is silent in normal adult tissues but re-emerges and is significantly upregulated during tumorigenesis through growth factor signaling pathways such as IN/IGF1/IRS1/RAS/RAF/MAPK/ERK, IN/IGF1/IRS1/PI3K/AKT, and WNT/β-catenin, which are associated with the development and progression of malignant tumors. ASPH protein is overexpressed in various malignant tumors, including non-small cell lung cancer (NSCLC), pancreatic cancer, cholangiocarcinoma, hepatocellular carcinoma, and colorectal cancer. Upregulation of ASPH enhances cell migration and invasion. In summary, ASPH can serve as a TAA target for cancer therapy.
• Gene targeting strategy for B-hASPH mice. The exons 4-24 of mouse Asph gene that encode extracellular domain and 3’ UTR region are replaced by human counterparts in B-hASPH mice. The genomic region of mouse Asph gene that encodes cytoplasmic portion is retained. The promoter and 5’UTR region of the mouse gene are retained. The chimeric ASPH expression is driven by endogenous mouse Asph promoter, while mouse Asph gene transcription and translation will be disrupted.
• Protein expression analysis: ASPH was detected in liver, lung and colon of both wild-type mice and homozygous B-hASPH mice, but weakly detected in brain, as anti-ASPH antibody was cross-reactive between human and mouse.
• mRNA expression analysis: Mouse Asph mRNA was only detectable in wild-type mice. Human ASPH mRNA was exclusively detectable in homozygous B-hASPH mice but not in wild-type mice..

Gene targeting strategy for B-hASPH mice. The exons 4-24 of mouse Asph gene that encode extracellular domain and 3’ UTR region are replaced by human counterparts in B-hASPH mice. The genomic region of mouse Asph gene that encodes cytoplasmic portion is retained. The promoter and 5’UTR region of the mouse gene are retained. The chimeric ASPH expression is driven by endogenous mouse Asph promoter, while mouse Asph gene transcription and translation will be disrupted.

Western blot analysis of ASPH protein expression in homozygous B-hASPH mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hASPH mice (H/H), and then analyzed by western blot with cross reatived anti-ASPH antibody (Santa, sc-271391). 40 μg total proteins were loaded for western blotting analysis. ASPH was detected in liver, lung and colon of both wild-type mice and homozygous B-hASPH mice, but weakly detected in brain.

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