The exogenous promoter and mouse Gpa33 coding sequence were inserted into the mouse genome randomly. Mouse Gpa33 can be secreted by B-Tg(mGpa33) MC38 cells.
 

Gene targeting strategy for B-Tg(mGpa33) MC38 cells. The exogenous promoter and mouse Gpa33 coding sequence were inserted into the mouse genome randomly.

Gpa33 expression analysis in B-Tg(mGpa33) MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-Tg(mGpa33) MC38 cultures were stained with species-specific anti-Gpa33 antibody. Mouse Gpa33 was detected on the surface of B-Tg(mGpa33) MC38 cells but not wild-type MC38 cells. The 1-E08 clone of B-Tg(mGpa33) MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-Tg(mGpa33) MC38 cells. B-Tg(mGpa33) MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 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-Tg(mGpa33) MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-Tg(mGpa33) MC38 tumor growth of individual mice. B-Tg(mGpa33) MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, n=6). As shown in panel, B-Tg(mGpa33) MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-Tg(mGpa33) MC38 cells were subcutaneously transplanted into C57BL/6N mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for mouse Gpa33 expression by flow cytometry. As shown, mouse Gpa33 was highly expressed on the surface of tumor cells. Therefore, B-Tg(mGpa33) MC38 cells can be used for in vivo efficacy studies of novel Gpa33 therapeutics.