IPTG-inducible episomal expression system for exogenous genes in primate cells

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• MCF7 cells are a widely used model for human breast carcinoma research.
• Regulated expression of exogenous genes is crucial for studying gene function and developing therapies.
• Existing expression systems may lack efficiency or reversibility in primate cells.

Purpose of the Study:

• To establish a novel, inducible episomal expression system for the human breast carcinoma cell line MCF7.
• To enable tightly regulated and reversible expression of exogenous genes in primate cells.
• To assess the system's efficiency and applicability for various gene types.

Main Methods:

• Development of a two-component system: pEpiLac episomal vector and MCF7/LAP5 cell line expressing LAP267 transactivator.
• Utilized isopropyl beta-D-thiogalactopyranoside (IPTG) for inducible gene expression.
• Validated system performance using reporter genes (luciferase) and functional genes (p27KIP1, Nur77, Cyr61).

Main Results:

• Achieved efficient and IPTG-inducible expression of exogenous genes in MCF7/LAP5 cells.
• Demonstrated up to 300-fold induction with luciferase as a reporter.
• Confirmed prompt cessation of gene expression upon IPTG removal.
• Successfully expressed cell cycle inhibitor p27KIP1, nuclear receptor Nur77, and angiogenic inducer Cyr61.
• Showcased stable maintenance and easy recovery of the episomal vector.

Conclusions:

• The developed IPTG-inducible episomal expression system provides a robust tool for regulated gene expression in primate cells.
• This system is particularly suitable for studying the effects of growth inhibitory or cytotoxic genes.
• The system offers efficient, reversible, and stable gene expression for research and potential therapeutic applications.