Description

• Human A33, also known as GPA33, is a 43 kDa type I transmembrane glycoprotein that belongs to the CTX (cortical thymocyte marker in Xenopus) family of cell adhesion molecules within the immunoglobulin superfamily.
• The exons 2-6 of mouse Gpa33 gene that encode extracellular domain and part of transmembrane domain were replaced by human counterparts in B-hGPA33 mice. The genomic region of mouse Gpa33 gene that encode signal peptide, cytoplasmic portion and part of transmembrane domain were retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric GPA33 expression was driven by endogenous mouse Gpa33 promoter, while mouse Gpa33 gene transcription and translation will be disrupted.
• Mouse Gpa33 mRNA was only detected in wild-type C57BL/6JNifdc mice. Human GPA33 mRNA was only detected in B-hGPA33 mice.
• hGPA33 was detected in small intestine from B-hGPA33 mice but not in wild-type mice.
• Humanized mice can be used for the efficacy validation and safety evaluation.

Targeting strategy

Gene targeting strategy for B-hGPA33 mice. The exons 2-6 of mouse Gpa33 gene that encode extracellular domain and part of transmembrane domain were replaced by human counterparts in B-hGPA33 mice. The genomic region of mouse Gpa33 gene that encode signal peptide, cytoplasmic portion and part of transmembrane domain were retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric GPA33 expression was driven by endogenous mouse Gpa33 promoter, while mouse Gpa33 gene transcription and translation will be disrupted.

mRNA expression analysis

Strain specific analysis of GPA33 mRNA expression in wild-type C57BL/6JNifdc mice and B-hGPA33 mice by RT-PCR. Small intestinal RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hGPA33 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GPA33 primers. Mouse Gpa33 mRNA was only detectable in wild-type mice. Human GPA33 mRNA was exclusively detectable in homozygous B-hGPA33 mice but not in wild-type mice.

Protein expression analysis in small intestinal cells

Strain specific GPA33 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hGPA33 mice by flow cytometry. Small intestinal cells were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hGPA33 mice (H/H) (male, 12-week-old, n=1). Protein expression was analyzed with anti-human GPA33 antibody (R&D, FAB3080P) by flow cytometry. Human GPA33 was exclusively detectable in homozygous B-hGPA33 mice, but not in wild-type C57BL/6JNifdc mice.