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.

Gene targeting strategy for 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.

B-hB7-H4 MC38 cells (1x10⁶ and 3x10⁶) were subcutaneously implanted into B-hB7-H4 mice (n=5), and on 24 days post inoculation, tumor cells were harvested and assessed for human B7-H4 expression by flow cytometry. As shown, human B7-H4 was highly expressed on the surface of tumor cells. Therefore, B-hB7-H4 MC38 cells can be used for in vivo efficacy studies of novel B7-H4 therapeutics.

Subcutaneous homograft tumor growth of B-hB7-H4 MC38 cells. B-hB7-H4 MC38 cells (1x10⁶) 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 5x10⁵-1x10⁷; 

2.Inoculated cells can be tried to be suspended with DMEM stock solution;

3.The use of humanized mice for cell inoculated is recommended.