The mouse Ccr9 gene was replaced by human CCR9 coding sequence in B-hCCR9 MC38 cells. Human CCR9 is highly expressed on the surface of B-hCCR9 MC38 cells.

Gene targeting strategy for B-hCCR9 MC38 cells. The exogenous promoter and human CCR9 coding sequence were inserted to replace part of murine exon 4. The insertion disrupts the endogenous murine Ccr9 gene, resulting in a non-functional transcript.

CCR9 expression analysis in B-hCCR9 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCCR9 MC38cultures were stained with species-specific anti-CCR9 antibody. Human CCR9 was detected on the surface of B-hCCR9 MC38 cells, but not onthe surface of wild-type MC38 cells. The 1-G09 clone of B-hCCR9 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCCR9 MC38 cells. B-hCCR9 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into heterozygous B-hCCR9 mice (female, 8-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-hCCR9 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.Tip:B-hCCR9 MC38 can not establish tumors in C57BL/6 mice, even in high dose (5E6 cells per mice).