• Claudin family is a kind of tight junction proteins discovered in recent years. The changes of claudins directly affect the structure and function of tight junctions. Claudins are the main skeleton protein of the important membrane junction complex between tightly connected cells.
• Clostridium perfringens enterotoxin (CPE) is a natural ligand of claudin-3 (CLDN3) and claudin-4 (CLDN4). CLDN3 is highly expressed in human esophageal cancer, epithelial ovarian cancer and other cancer tissues. CPE can effectively inhibit malignant tumors expressed by CLDN3, which provides the possibility for CLDN3 to be used as a targeted therapy for malignant tumors. CLDN3 is not only expressed in various human gland cells such as prostate, pancreas and liver cells, but also in mammary epithelial cell, endometrium, esophageal mucosa, alveolar epithelium and biliary epithelial cell.
• In ovarian cancer, overexpression of CLDN4 induces epithelial-mesenchymal transition through the PI3K/Akt and Twist1 signal pathway. In pancreatic cancer, TGF-β can negatively regulate the expression of CLDN4, and the high expression of CLDN4 can inhibit tumor cell invasion. In breast cancer, CLDN4 promotes tumor cell migration and invasion through the PAK4-CEBPB-CLDN4 axis.
• Mouse Cldn3 and Cldn4 mRNA were detectable in colon of wild-type C57BL/6 mice. Human CLDN3 and CLDN4 mRNA were detectable in colon of B-hCLDN3/hCLDN4 mice.
• CLDN3 was detected in lung and colon of both wild-type C57BL/6 mice and homozygous B-hCLDN3/hCLDN4 mice, as anti-CLDN3 antibody was cross-reactive between human and mouse. Human CLDN4 was exclusively detectable in lung and colon of homozygous B-hCLDN3/hCLDN4 mice.
• Application: This product is used for pharmacodynamics and safety evaluation of tumor and other autoimmune diseases.

Gene targeting strategy for B-hCLDN3/hCLDN4 mice.
Mouse Cldn3 and Cldn4 gene were replaced by human CLDN3 and CLDN4 gene in B-hCLDN3/hCLDN4 mice.

Strain specific analysis of CLDN3 and CLDN4 mRNA expression in wild-type C57BL/6 mice and B-hCLDN3/hCLDN4 mice by RT-PCR. Colon RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCLDN3/hCLDN4 mice (H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CLDN3 or CLDN4 primers. Mouse Cldn3 and Cldn4 mRNA were detectable only in wild-type C57BL/6 mice. Human CLDN3 and CLDN4 mRNA were detectable only in homozygous B-hCLDN3/hCLDN4 mice but not in wild-type mice.

Western blot analysis of CLDN3 protein expression in homozygous B-hCLDN3/hCLDN4 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (male，5-week-old) and homozygous B-hCLDN3/CLDN4 mice (male，5-week-old), and then analyzed by western blot with the human-mouse cross reactivity anti-CLDN3 antibody (abcam, ab317319). 40 μg total proteins were loaded for western blotting analysis. CLDN3 expression was detected in the liver, lung, colon and pancreas from homozygous B-hCLDN3/CLDN4 mice, but not in the spleen.

Immunohistochemical analysis of organs in B-hCLDN3/hCLDN4 mice. Major organs were collected from wild-type C57BL/6JNifdc mice (male=1, 5-week-old) and homozygous B-hCLDN3/hCLDN4 mice (female=1, male=1, 5-week-old), and analyzed by IHC using the human-mouse cross reactivity anti-CLDN3 antibody (abcam, ab317319). The arrow indicates tissue cells with positive CLDN3 staining (brown). “+” indicates that the tissue is positive, and “-” indicates that the tissue is negative.

Western blot analysis of CLDN4 protein expression in homozygous B-hCLDN3/hCLDN4 mice. Various tissue lysates were collected from wild-typeC57BL/6 mice (male, 13-week-old) and homozygous B-hCLDN3/CLDN4 mice (male, 13-week-old) , and then analyzed by western blot with the human-specific anti-CLDN4 antibody (Invitrogen, 32-9400). 40 μg total proteins were loaded for western blotting analysis. CLDN4 expression was detected in thelung and colon from homozygous B-hCLDN3/CLDN4 mice, but not in the heart, liver, spleen, kidney, brain and stomach.