Murine Ccr4 was replaced by the human CCR4 coding sequence and the luciferase sequence in B-hCCR4-luc EL4 cells. Human CCR4 is highly expressed on the surface of B-hCCR4-luc EL4 cells.
B-hCCR4-luc EL4 cells have the capability to establish tumors in vivo and can be used for efficacy studies.
Gene Targeting Strategy
The exogenous promoter, human CCR4 coding sequence and the luciferase sequence were inserted to replace part of murine exon 2. This insertion disrupts the endogenous murine Ccr4 gene, resulting in a non-functional transcript.
Protein Expression Analysis
Luminescence Signal Intensity Analysis
The luminescence signal intensity of B-hCCR4-luc EL4 cells. The luminescence intensity was detected by Bright-GloTM luciferase Assay System (Promega, Cat E2610). Clones 3-F10 and 3-F11 from B-hCCR4-luc EL4 cells displayed strong luminescence signal.
CCR4 expression analysis in B-hCCR4-luc EL4 cells by flow cytometry. Single cell suspensions from wild-type EL4 and B-hCCR4-luc EL4 cultures were stained with species-specific anti-CCR4 antibody. Human CCR4-luc was detected on the surface of B-hCCR4-luc EL4 cells but not wild-type EL4 cells. The clones 3-F10 and 3-F11 of B-hCCR4-luc EL4 cells were used for in vivo tumor growth assays.
B-hCCR4-luc EL4 cells were subcutaneously implanted into C57BL/6 mice (n=5). Tumor cells were harvested on day 21 post inoculation and assessed for species-specific CCR4 expression by flow cytometry. As shown, human CCR4 was highly expressed on the surface of humanized B-hCCR4-luc EL4 tumor cells, indicating B-hCCR4-luc EL4 cells can be used for in vivo efficacy studies of CCR4 therapeutics.
Tumor Growth Curve
Subcutaneous homograft tumor growth of B-hCCR4-luc EL4 cells. B-hCCR4-luc EL4 cells (2×105) and wild-type EL4 cells (2×105) were subcutaneously implanted into C57BL/6 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-hCCR4-luc EL4 cells were able to form tumors in vivo, which can be used for efficacy studies.