The human GAS6 CDS was inserted into mouse Gas6 gene, so that the mouse Gas6 was not expressed anymore, human GAS6 was expressed in B-GAS6 MC38 cells.

Gene targeting strategy for B-hGAS6 MC38 cells. The exogenous promoter and human GAS6 coding sequence were inserted to replace part of murine exon 1 and all of exon 2. The insertion disrupts the endogenous murine Gas6 gene, resulting in a non-functional transcript.

GAS6 expression analysis in B-hGAS6 MC38 cells by ELISA. Supernatant from wild-type MC38 and B-hGAS6 MC38 cultures were stained with species-specific anti-GAS6 antibody. Human GAS6 was detected in the supernatant of B-hGAS6 MC38 cells, but not in the supernatant of wild-type MC38 cells. The 8-H12 clone of B-hGAS6 MC38 cells was used for in vivo experiments. ND: Not detected.

Subcutaneous homograft tumor growth of B-hGAS6 MC38 cells. B-hGAS6 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 6-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-hGAS6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGAS6 MC38 tumor growth of individual mice. B-hGAS6 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 6-week-old, n=6). As shown in panel, B-hGAS6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.