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

Area of Science:

• Biotechnology
• Cell Biology
• Nutritional Science

Background:

• Bioactive compounds in food and supplements are crucial for preventing lifestyle diseases.
• Existing reporter gene systems face challenges in controlling transgene copy number.
• Evaluating osteogenesis activity is vital for functional food development.

Purpose of the Study:

• To develop a novel cell line for assessing osteogenesis activity in functional foods.
• To utilize a human artificial chromosome (HAC) vector for stable transgene expression.
• To establish a reliable system for identifying bioactive substances with osteogenic potential.

Main Methods:

• Engineered Chinese hamster ovary (CHO) cells with a green fluorescence protein (GFP) reporter gene on a HAC vector.
• The GFP gene was regulated by the osteocalcin gene promoter.
• Constructed HAC vectors with tandem repeats of reporter gene units to enhance expression intensity.
• Validated the system's response to vitamin D3 and assessed fish oil samples.

Main Results:

• Established CHO cell lines exhibiting GFP expression controlled by the osteocalcin promoter.
• Demonstrated that GFP expression correlated with vitamin D3 stimulation.
• Confirmed that GFP expression intensity relates to the copy number of reporter gene units on the HAC.
• Identified osteogenic activity in tested fish oil samples using the developed system.

Conclusions:

• The novel HAC-based cell line effectively reports osteocalcin gene expression, serving as a robust tool for evaluating osteogenesis.
• This system overcomes limitations of traditional plasmid vectors, offering stable and controllable transgene expression.
• The developed evaluation system successfully identified bioactive compounds with osteogenic effects, particularly in fish oils.