Gene targeting strategy for B-CAG-hROR1 MC38. The human full-length ROR1 CDS region was inserted in the Ror1 exon3~4 of wild-type MC38.
Protein expression analysis
The ROR1 expression analysis in B-CAG-hROR1 MC38 cells by flow cytometry. Single cells were collected from wild-type MC38 and B-CAG-hROR1 MC38 cells, and analyzed by flow cytometry with species-specific anti-ROR1 antibody. Human ROR1 was detectable in B-CAG-hROR1 MC38 cells but not wild-type MC38 cells.
Tumor Growth Curve & Body Weight Changes
Subcutaneous xenograft tumor growth of B-CAG-hROR1 MC38 cells. B-CAG-hROR1 MC38 cells(5E5) and wild-type MC38 cells(5E5) were subcutaneously implanted into the C57BL/6N mice (female, 8 week-old, n=8).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-hROR1 MC38 were able to establish tumor in vivo and can be used for efficacy study.
Protein expression analysis in tumor cells
B-CAG-hROR1 MC38 cells were transplanted into C57BL/6 mice(n=8), and on 34 days post inoculation, certain volumes of tumors as was taken to detect the expression level of the cells. As Shown,human ROR1 was highly expressed on the surface of tumor cells. B-CAG-hROR1 MC38 can be used for in vivo efficacy validation.