Description

Background:

• ITIH4 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 4) belongs to the Intermediate trypsin inhibitor family. Inter-alpha-trypsin inhibitors (ITIs) are a group of structurally related plasma serine protease inhibitors involved in extracellular matrix stabilization and the prevention of tumor metastasis. ITIH4 is a 120 kDa acute-phase glycoprotein primarily produced in the liver and secreted into the bloodstream, where it is cleaved by plasma kallikrein into two smaller forms. ITIH4 not only plays a role in liver development and extracellular matrix (ECM) stabilization but also exhibits altered expression in liver diseases. Additionally, ITIH4 expression may serve as a biomarker for breast cancer, liver cancer, and fatty liver disease.
• Research suggests that ITIH4 can inhibit tumor migration and progression, with higher expression of this protein correlating with better prognosis in cancer patients. Therefore, agonists targeting ITIH4 may hold potential as tumor-suppressing therapeutics. Meanwhile, the long non-coding RNA ITIH4-AS1, located within the ITIH4 gene, promotes colorectal cancer proliferation and metastasis by activating the JAK/STAT3 signaling pathway. Consequently, nucleotide-based inhibitors targeting this lncRNA could also be developed as a therapeutic strategy.

Targeting strategy:

• The exon 2~23 of mouse Itih4 gene that encodes the full-length protein except signal peptide was replaced by human ITIH4 exon 2~24 in B-hITIH4 mice.

Verification: 

• Human ITIH4 protein was only detected in homozygous B-hITIH4 mice, but not in C57BL/6JNifdc mice.
• Mouse Itih4 mRNA was only detected in wild-type mice, human ITIH4 mRNA was only detected in homozygous B-hITIH4 mice.

Application:

• This product can be used to evaluate the efficacy and toxicity of drugs for breast cancer, liver cancer and other tumors with high expression of this target.

Targeting strategy

Gene targeting strategy for B-hITIH4 mice. The exons 2~23 of mouse Itih4 gene that encodes the full-length protein except signal peptide, including 3’UTR were replaced by human ITIH4 exons 2~24 in B-hITIH4 mice. The promoter and 5’UTR region of the mouse gene are retained. The human ITIH4 expression is driven by endogenous mouse Itih4 promoter, while mouse Itih4 gene transcription and translation will be disrupted.  

Protein expression analysis

Strain specific ITIH4 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hITIH4 mice by ELISA. Plasma were collected from wild-type C57BL/6JNifdc mice (+/+) (n=3, 7-week-old) and homozygous B-hITIH4 mice (H/H) (n=3, 7-week-old). Protein expression level of ITIH4 was analyzed by ELISA with anti-human ITIH4 kit (R&D, DY8157-05). Human ITIH4 was exclusively detectable in homozygous B-hITIH4 mice but not in wild-type mice.

mRNA expression analysis

Strain specific analysis of ITIH4 mRNA expression in wild-type C57BL/6JNifdc mice and B-hITIH4 mice by RT-PCR. Liver RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hITIH4 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human ITIH4 primers. Mouse Itih4 mRNA was only detectable in wild-type mice, human ITIH4 was detectable in homozygous B-hITIH4 mice.