Inducible gene expression using an autoregulatory, tetracycline-controlled system.

Area of Science:

• Molecular Biology
• Cell Biology
• Gene Expression Systems

Background:

• Stable transfection is crucial for long-term gene expression studies.
• Inducible systems allow controlled gene activation, essential for dissecting gene function.
• Fibroblast cell lines are commonly used models in molecular biology research.

Purpose of the Study:

• To describe robust protocols for the stable transfection of adherent cells.
• To establish methods for generating cell lines with inducible transactivator and target gene expression.
• To provide supporting protocols for testing inducible gene expression and detecting transfected genes.

Main Methods:

• Cotransfection of fibroblast cells with plasmids encoding a transactivator (pTet-tTAk) and a selectable marker.
• Sequential stable cotransfection with plasmids for target genes and a second selectable marker.
• Testing of transfected cell lines for inducible gene expression and gene detection.

Main Results:

• Successful derivation of stable fibroblast cell lines with inducible transactivator expression.
• Establishment of cell lines with inducible expression of target genes.
• Validated methods for assessing transfection efficiency and inducible gene product levels.

Conclusions:

• The described protocols enable the generation of stable, inducible mammalian cell lines for gene expression studies.
• These methods facilitate the investigation of gene function through controlled expression.
• The protocols are adaptable for various gene targets and selectable markers.