• ADAM28 encodes a desintegrin metalloproteinase, a type I transmembrane protein with two isomers: membrane anchoring (ADAM28m) and secreting (ADAM28s). In human tissues, ADAM28 is also expressed in epithelial cells of various organs such as the epididymis, bronchus and stomach. Knockout mice are viable, and proteolysis is mediated by increased metastatic lung colonization after injection of tumor cells due to reduced CD8+T invasion in homozygous KO lungs. ADAM28 is a multifunctional protein that regulates cell-cell and cell-matrix interactions. ADAM28 is overexpressed in cancer cells of various cancer types (including breast cancer, renal cell carcinoma, non-small cell lung cancer, leukemia, prostate cancer, chondrosarcoma, bladder cancer, pancreatic cancer and head and neck cancer), and is involved in the proliferation, invasion and metastasis of various tumors in vivo.
• mRNA expression analysis: Mouse Adam28 mRNA was only detectable in lung of wild-type C57BL/6JNifdc mice. Human ADAM28 mRNA was only detectable in lung of homozygous B-hADAM28 mice but not in wild-type mice.
• Protein expression analysis: ADAM28 was detected in both wild-type mice and homozygous B-hADMA28 mice, as anti-ADAM28 antibody was cross-reactive between human and mouse. The pro-ADAM28 was detected in heart, liver, spleen, lung and stomach, while the active ADAM28 was detected in heart and liver of both wild-type mice and homozygous mice.

Gene targeting strategy for B-hADAM28 mice. The exons 3-18 of mouse Adam28 gene that encode extracellular domain is replaced by human counterparts in B-hADAM28 mice. The genomic region of mouse Adam28 gene that encodes signal peptide, cytoplasmic portion and transmembrane domain is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric ADAM28 expression is driven by endogenous mouse Adam28 promoter, while mouse Adam28 gene transcription and translation will be disrupted.

Strain specific analysis of ADAM28 mRNA expression in wild-type C57BL/6JNifdc mice and B-hADAM28 mice by RT-PCR. Lung RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hADAM28 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human ADAM28 primers. Mouse Adam28 mRNA was detectable in wild-type mice and homozygous mice. Human ADAM28 mRNA was detectable only in homozygous B-hADAM28 mice but not in wild-type mice.

Western blot analysis of ADAM28 protein expression in homozygous B-hADAM28 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hADAM28 mice (H/H), and then analyzed by western blot with cross reactive anti-ADAM28 antibody (Invitrogen, PA5-97223). 40 μg total proteins were loaded for western blotting analysis. The red box represents the pro-ADAM28, and the red arrow represents the active form of ADAM28. The pro-ADAM28 was detected in heart, liver, spleen, lung and stomach, while the active ADAM28 was detected in heart and liver of both wild-type mice and homozygous mice.