The mouse B7h4 gene was replaced by human B7-H4 coding sequence in B-hB7-H4 MC38 cells. Human B7-H4 is highly expressed on the surface of B-hB7-H4 MC38 cells.
The exogenous promoter and human B7-H4 coding sequence was inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine B7h4 gene, resulting in a non-functional transcript.
Protein expression analysis
B7-H4 expression analysis in B-hB7-H4 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hB7-H4 MC38 cultures were stained with species-specific anti-B7-H4 antibody. Human B7-H4 was detected on the surface of B-hB7-H4 MC38 cells but not wild-type MC38 cells. The 3-F09 clone of B-hB7-H4 MC38 cells was used for in vivo experiments.
Tumor growth curve
Subcutaneous homograft tumor growth of B-hB7-H4 MC38 cells. B-hB7-H4 MC38 cells (1×106) were subcutaneously implanted into B-h4-1BB mice (female, n=8). 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-hB7-H4 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Recommendations for inoculation :
1.Cell inoculation amount is recommended to be tried between 5×105-1×107;
2.Inoculated cells can be tried to be suspended with DMEM stock solution;
3.The use of humanized mice for cell inoculated is recommended.