Description
The mouse Car12 gene was replaced by human CAXII (CA12) coding sequence in B-hCAXII MC38 cells. Human CAXII is highly expressed on the surface of B-hCAXII MC38 cells.
Targeting strategy
Gene targeting strategy for B-hCAXII MC38 cells. The exogenous promoter and human CAXII coding sequence was inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine Car12 gene, resulting in a non-functional transcript.
Protein expression analysis
CAXII expression analysis in B-hCAXII MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCAXII MC38 cultures were stained with species-specific anti-CAXII antibody. Human CAXII was detected on the surface of B-hCAXII MC38 cells but not wild-type MC38 cells. The 2-D05 clone of B-hCAXII MC38 cells was used for in vivo experiments.
Protein expression analysis of tumor cells
B-hCAXII MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6), and on 35 days post inoculation, tumor cells were harvested and assessed for human CAXII expression by flow cytometry. As shown, human CAXII was highly expressed on the surface of tumor cells. Therefore, B-hCAXII MC38 cells can be used for in vivo efficacy studies of novel CAXII therapeutics.
Tumor growth curve & Body weight changes
Subcutaneous homograft tumor growth of B-hCAXII MC38 cells. B-hCAXII MC38 cells (5x106) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 11-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hCAXII MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Tumor growth curve of individual mice
B-hCAXII MC38 tumor cells growth of individual mice. B-hCAXII MC38 cells (5x106) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 11-week-old, n=6). As shown in panel, B-hCAXII MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
