An auto-inducible vector conferring high glucocorticoid inducibility upon stable transformant cells

Area of Science:

• Molecular Biology
• Gene Regulation
• Mammalian Cell Culture

Background:

• Developing controllable gene expression systems is crucial for molecular biology research.
• Inducible systems offer precise control over gene activity, minimizing off-target effects.
• The glucocorticoid receptor (GR) is a well-characterized transcription factor responsive to synthetic hormones.

Purpose of the Study:

• To engineer a novel inducible gene expression system in mammalian cells.
• To utilize the glucocorticoid receptor (GR) as a positive feedback factor for enhanced gene expression.
• To validate the system's efficacy in driving high-level expression of reporter and target genes.

Main Methods:

• Transfection of mouse Ltk- cells with plasmids encoding GR and lacZ reporter gene.
• Fusion of GR and lacZ genes with the mouse mammary tumor virus (MTV) glucocorticoid-inducible enhancer/promoter.
• Induction of gene expression using dexamethasone, a synthetic glucocorticoid hormone.

Main Results:

• Stable transformants showed a 60-140-fold increase in beta-galactosidase activity upon dexamethasone induction.
• The human alpha-interferon gene fused to the MTV promoter exhibited over 12,000-fold induction.
• The system achieved high gene expression levels without significantly increasing basal expression.

Conclusions:

• A novel, highly efficient inducible gene expression system utilizing GR-mediated positive feedback has been established.
• This system enables significant amplification of gene expression in mammalian cells.
• The developed system is a valuable tool for investigating gene functions, especially for cytotoxic or growth-inhibiting genes.