The mouse H2-T23 gene was replaced by human HLA-E coding sequence in B-hHLA-E MC38 plus cells. Human HLA-E is highly expressed on the surface of B-hHLA-E MC38 plus cells.
The exogenous promoter and human HLA-E coding sequence was inserted to replace part of murine exon 3 and all of exons 4~7. The insertion disrupts the endogenous murine H2-T23 gene, resulting in a non-functional transcript.
Protein expression analysis
HLA-E expression analysis in B-hHLA-E MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hHLA-E MC38 plus cultures were stained with species-specific anti-HLA-E antibody. Human HLA-E was detected on the surface of B-hHLA-E MC38 plus cells but not wild-type MC38 cells. The 1-A07 clone of B-hHLA-E MC38 plus cells was used for in vivo experiments.
Tumor growth curve
Subcutaneous homograft tumor growth of B-hHLA-E MC38 plus cells. B-hHLA-E MC38 plus cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6N mice (female, 7-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-hHLA-E MC38 plus 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.