Basic Information
Description
The mouse Epcam gene was replaced by human EPCAM coding sequence in B-hEPCAM B16-F10 cells. Human EPCAM is highly expressed on the surface of B-hEPCAM B16-F10 cells.
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Targeting strategy
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Gene targeting strategy for B-hEPCAM B16-F10 cells. The exogenous promoter and human EPCAM coding sequence was inserted to replace part of murine exon 4 and all of exons 5~7. The insertion disrupts the endogenous murine Epcam gene, resulting in a non-functional transcript.
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Protein Expression Analysis
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EPCAM expression analysis in B-hEPCAM B16-F10 cells by flow cytometry. Single cell suspensions from wild-type B16-F10 and B-hEPCAM B16-F10 cultures were stained with species-specific anti-EPCAM antibody. Human EPCAM was detected on the surface of B-hEPCAM B16-F10 cells but not wild-type B16-F10 cells. The 2-H11 clone of B-hEPCAM B16-F10 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-hEPCAM B16-F10 cells. B-hEPCAM B16-F10 cells (2×105) and wild-type B16-F10 cells (2×105) were subcutaneously implanted into C57BL/6 mice (female, 9-week-old, n=8). Tumor volume and body weight were measured thrice 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-hEPCAM B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies.
Animals with tumor volume over 3000mm3 in the control group were euthanized.
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Protein expression analysis of tumor cells
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B-hEPCAM B16-F10 cells were subcutaneously transplanted into C57BL/6 mice (n=8). At the end of the experiment, tumor cells were harvested and assessed for human EPCAM expression by flow cytometry. As shown, human EPCAM was highly expressed on the surface of tumor cells. Therefore, B-hEPCAM B16-F10 cells can be used for in vivo efficacy studies of novel EPCAM therapeutics.
