The mouse Mpl gene was replaced by human c-MPL coding sequence in B-hc-MPL MC38 cells. Human c-MPL is highly expressed on the surface of B-hc-MPL MC38 cells.

Gene targeting strategy for B-hc-MPL MC38 cells. The exogenous promoter and human c-MPL coding sequence was inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine Mpl gene, resulting in a non-functional transcript.

c-MPL expression analysis in B-hc-MPL MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hc-MPL MC38 cultures were stained with species-specific anti-c-MPL antibody. Human c-MPL was detected on the surface of B-hc-MPL MC38 cells but not wild-type MC38 cells. The 1-H01 clone of B-hc-MPL MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hc-MPL MC38 cells. B-hc-MPL 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× long diameter × short diameter². As shown in panel A, B-hc-MPL MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hc-MPL MC38 tumor cells growth of individual mice. B-hc-MPL 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-hc-MPL MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hc-MPL MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for human c-MPL expression by flow cytometry. As shown, human c-MPL was highly expressed on the surface of tumor cells. Therefore, B-hc-MPL MC38 cells can be used for in vivo efficacy studies of novel c-MPL therapeutics.