The chimera CDS composed of mouse signal peptide, human extracellular region, moue transmembrane region and mouse intracellular region of LAIR1 was inserted to replace part of mouse exon 3 and intron 3. Human LAIR1 is highly expressed on the surface of B-hLAIR1-luc EL4 cells.
Gene Targeting Strategy
Gene targeting strategy for B-hLAIR1-luc EL4 cells. The chimera CDS composed of mouse signal peptide, human extracellular region, moue transmembrane region and mouse intracellular region of LAIR1 was inserted to replace part of mouse exon 3 and intron 3. The insertion disrupts the endogenous murine Lair1 gene, resulting in a non-functional transcript.
Protein Expression Analysis
Human LAIR1 expression analysis in B-hLAIR1-luc EL4 cells. Single cell suspensions from wild-type EL4 and B-hLAIR1-luc EL4 cultures were stained with an anti-human LAIR1 antibody. Human LAIR1 protein was detected on the surface of B-hLAIR1-luc EL4 cells compared to wild-type EL4 cells. The 3-E02 clone of B-hLAIR1-luc EL4 cells was used for in vivo experiments.
Human LAIR1 expression analysis. B-hLAIR1-luc EL4 cells were subcutaneously transplanted into C57BL/6 mice (n=5). Tumor cells were harvested on day 18 post inoculation and assessed for human LAIR1 expression by flow cytometry. As shown, human LAIR1 was highly expressed on the surface of tumor cells, indicating B-hLAIR1-luc EL4 cells can be used for in vivo efficacy studies of novel LAIR1 therapeutics.
Tumor Growth Curve
Subcutaneous homograft tumor growth of B-hLAIR1-luc EL4 cells. B-hLAIR1-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, and (B) body weight 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-hLAIR1-luc EL4 cells were able to establish tumors in vivo, which can be used for efficacy studies. Values are expressed as mean ± SEM.