Development of evaluation system for bioactive substances using human artificial chromosome-mediated osteocalcin gene expression.

Bioactive substances in daily food and supplements are expected to prevent various lifestyle-related diseases. Recently, many evaluation systems for bioactive substances were developed with cell lines integrated with green fluorescence protein (GFP) reporter gene. To evaluate osteogensis activity in functional food, we developed a novel cell line that reports osteocalcin gene expression using the human artificial chromosome (HAC) vector. HAC vectors are able to avoid various problems in usual plasmid vector such as difficulty in control of transgene copy number. HAC is transmitted to cells as an independent chromosome from host chromosomes, and expresses transgenes depending on host cell circumstances. We established Chinese hamster ovary cell lines that carried GFP gene regulated by osteocalcin gene promoter on the HAC. Expression of GFP was responded to vitamin D(3) [1alpha,25(OH)(2)D(3)]. Furthermore, we constructed HAC vector bearing tandem repeats of reporter gene unit, to enhance intensity of gene expression. GFP expression in these reporter cells is related to the copy number of reporter gene units. Using the evaluation system for bioactive substances, we could show osteogenic activity in some fish oils.