Gene targeting strategy for B-CAG-hB7-H3 MC38.The exons 3-4 of mouse B7-H3 gene that encode the extracellular domain were replaced by human B7-H3 CDS region in B-CAG-hB7-H3 MC38.
Protein expression analysis
The B7-H3 expression analysis in B-CAG-hB7-H3 MC38 cells by flow cytometry. Single cells were collected from wild-type MC38 and B-CAG-hB7-H3 MC38 cells, and analyzed by flow cytometry with species-specific anti-B7-H3 antibody. Human B7-H3 was exclusively detectable in B-CAG-hB7-H3 MC38 cells but not wild-type MC38 cells.
Tumor Growth Curve & Body Weight Changes
Subcutaneous xenograft tumor growth of B-CAG-hB7-H3 MC38 cells. B-CAG-hB7-H3 MC38 cells and wild-type MC38 cells (5×105) were subcutaneously implanted into the C57BL/6N mice (female, 5-8 week-old, n=5).Tumor size and mice body weight were measured twice a week. (A) Tumor average volume ± SEM, (B) Mice body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5a X b2, where a and b were the long and short diameters of the tumor, respectively. As shown in panel A, B-CAG-hB7-H3 MC38 were able to establish tumor in vivo and can be used for efficacy study.
Protein expression analysis in tumor cells
B-CAG-hB7-H3 MC38 tumor cells were transplanted into C57BL/6N mice(n=5), and on 31 days post inoculation, certain volumes of tumors as was taken to detect the expression level of the cells. As Shown,human B7-H3 was highly expressed on the surface of tumor cells. B-CAG-hB7-H3 MC38 can be used for in vivo efficacy validation.