Basic Information
Description
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.
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Application
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B-hCCR4-luc EL4 cells have the capability to establish tumors in vivo and can be used for efficacy studies.
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Gene Targeting Strategy
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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.
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Protein Expression Analysis
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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.
Pre-inoculation
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.
Post-inoculation
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.
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Tumor Growth Curve
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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.
