The mouse AMHR2 gene was replaced by human AMHR2 coding sequence in B-hAMHR2 MC38 cells. Human AMHR2 is highly expressed on the surface of B-hAMHR2 MC38 cells.
Gene Targeting Strategy
Gene targeting strategy for B-hAMHR2 MC38 cells. The exogenous promoter and human AMHR2 coding sequence was inserted to replace part of murine exon 2 and all of exon 3. This insertion disrupts the endogenous murine Amhr2 gene, resulting in a non-functional transcript.
Protein Expression Analysis
AMHR2 expression analysis in humanized B-hAMHR2 MC38 cells. Single cell suspensions from wild-type MC38 and B-hAMHR2 MC38 (clone #1-A01) cultures were stained with an anti-human AMHR2 antibody and analyzed by flow cytometry. Human AMHR2 protein was detected on the surface of B-hAMHR2 MC38 cells compared to wild-type MC38 cells. The 1-A01 clone was used for in vivo experiments.
B-hAMHR2 MC38 cells were subcutaneously transplanted into wild-type C57BL/6 mice (n=5). Tumor cells were harvested on day 30 post inoculation and assessed for human AMHR2 expression by flow cytometry using an anti-human AMHR2 antibody. As shown, human AMHR2 protein was highly expressed on the surface of tumor cells, indicating B-hAMHR2 MC38 cells can be used for in vivo efficacy studies of novel AMHR2 therapeutics.
Tumor Growth Curve
Subcutaneous homograft tumor growth of B-hAMHR2 MC38 cells. B-hAMHR2 MC38 cells (5×105) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6N mice (female, 7-week-old, n=5). (A) Average tumor volume ± SEM, and (B) body weight (Mean ± SEM) were measured twice a week. Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hAMHR2 MC38 cells were able to establish tumors in vivo, which can be used for efficacy studies.