The mouse Siglec15 gene was replaced by human SIGLEC15 coding sequence in B-hSIGLEC15 MC38 cells. Human SIGLEC15 is highly expressed on the surface of B-hSIGLEC15 MC38 cells.
The exogenous promoter and human SIGLEC15 coding sequence was inserted to replace part of murine exon 3 ~ 3’UTR. The insertion disrupts the endogenous murine Siglec15 gene, resulting in a non-functional transcript.
Protein expression analysis
SIGLEC15 expression analysis in B-hSIGLEC15 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hSIGLEC15 MC38 cultures were stained with species-specific anti-SIGLEC15 antibody. Human SIGLEC15 was detected on the surface of B-hSIGLEC15 MC38 cells but not wild-type MC38 cells. The 3-D10 clone of B-hSIGLEC15 MC38 cells was used for in vivo experiments.
Subcutaneous homograft tumor growth of B-hSIGLEC15 MC38 cells. B-hSIGLEC15 MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6N mice (female, 5-8-week-old, n=5). 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-hSIGLEC15 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
Recommendations for inoculation in other mouse strains:
1.Cell inoculation amount is recommended to be tried between 5×105-1×107;
2.Inoculated cells can be tried to be suspended with DMEM stock solution.