Basic Information
Description
The mouse B2m gene was replaced by human B2M and HLA-G coding sequence in B-hB2M/HLA-G MC38. Human HLA-G is highly expressed on the surface of B-hB2M/HLA-G MC38 cells.
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Targeting strategy
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Gene targeting strategy for B-hB2M/HLA-G MC38 cells. The exogenous promoter and human B2M and HLA-G coding sequence was inserted to replace part of murine exon 1 and all of exon 2 and 3. The insertion disrupts the endogenous murine B2m gene, resulting in a non-functional transcript.
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Protein Expression Analysis
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HLA-G expression analysis in B-hB2M/HLA-G MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hB2M/HLA-G MC38 cells cultures were stained with species-specific anti-HLA-G antibody. Human HLA-G was detected on the surface of B-hB2M/HLA-G MC38 cells but not wild-type MC38 cells. The 2-A08 clone of B-hB2M/HLA-G MC38 cells was used for in vivo experiments.
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Tumor growth curve & Body weight changes
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Subcutaneous homograft tumor growth of B-hB2M/HLA-G MC38 cells. B-hB2M/HLA-G MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into B-Tg(hLILRB2/hLILRB3) mice (female, 7-week-old). 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-hB2M/HLA-G MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
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Protein expression analysis of tumor cells
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B-hB2M/HLA-G MC38 cells were subcutaneously transplanted into B-Tg(hLILRB2/hLILRB3) mice (n=3), and on 28 days post inoculation, tumor cells were harvested and assessed for human HLA-G expression by flow cytometry. As shown, human HLA-G was highly expressed on the surface of tumor cells. Therefore, B-hB2M/HLA-G MC38 cells can be used for in vivo efficacy studies of novel HLA-G therapeutics.
