Gene targeting strategy for B-CAG-hB7-H3 EL4.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 EL4.
Protein expression analysis
The B7-H3 expression analysis in B-CAG-hB7-H3 EL4 cells by flow cytometry. Single cells were collected from wild-type EL4 and B-CAG-hB7-H3 EL4 cells, and analyzed by flow cytometry with species-specific anti-B7-H3 antibody. Mouse B7-H3 was not detectable in wild-type EL4 cells. Human B7-H3 was exclusively detectable in B-CAG-hB7-H3 EL4 cells but not wild-type EL4 cells.
Tumor Growth Curve & Body Weight Changes
Subcutaneous xenograft tumor growth of B-CAG-hB7-H3 EL4 cells. B-CAG-hB7-H3 EL4 cells and wild-type EL4 cells (2×105) were subcutaneously implanted into the C57BL/6N mice (female, 6-9 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 EL4 were able to establish tumor in vivo and can be used for efficacy study.
Protein expression analysis in tumor cells
FMO Control 1: B-CAG-hB7-H3 EL4 tumor cells only stained hB7-H3 PE antibody;
FMO Control 2: B-CAG-hB7-H3 EL4 tumor cells only stained mB7-H3 APC antibody
B-CAG-hB7-H3 EL4 tumor cells were transplanted into C57BL/6N mice(n=5), and on 28 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 EL4 can be used for in vivo efficacy validation.