Basic Information
Description
The mouse Pdl1 gene and Ceacam5 gene were replaced by human PD-L1 and CEACAM5 coding sequence in B-hPD-L1 plus/hCEA MC38 cells. Human PD-L1 and CEACAM5 are highly expressed on the surface of B-hPD-L1 plus/hCEA MC38 cells.
-
Target Strategy
-
Gene targeting strategy for B-hPD-L1 plus/hCEA MC38 cells.
The exogenous promoter and human PD-L1 coding sequence was inserted to replace part of murine exon 3. The insertion disrupts the endogenous murine Pdl1 gene, resulting in a non-functional transcript.
The exogenous promoter and human CEACAM5 coding sequence was inserted into the ROSA26 locus in murine.
-
Protein expression analysis
-
PD-L1 and CEACAM5 expression analysis in B-hPD-L1 plus/hCEA MC38 cells by flow cytometry. Single cell suspensions from MC38 and B-hPD-L1 plus/hCEA MC38 cultures were stained with species-specific anti-PD-L1 and anti-CEACAM5 antibodies. Human PD-L1 was detected on the surface of B-hPD-L1 plus/hCEA MC38 cells. Human CEACAM5 was detected on the surface of B-hPD-L1 plus/hCEA MC38 cells. The 2-D05 clone of B-hPD-L1 plus/hCEA MC38 cells was used for in vivo experiments.
-
Tumor growth curve & Body weight changes
-
Subcutaneous homograft tumor growth of B-hPD-L1 plus/hCEA MC38 cells. B-hPD-L1 plus/hCEA MC38 cells (5×105) and B-hPD-L1 MC38 plus cells (5×105) were subcutaneously implanted into C57BL/6 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-hPD-L1 plus/hCEA MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.
