Inducible expression based on regulated recombination: a single vector strategy for stable expression in cultured...

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• Site-specific recombinases (SSRs) fused to ligand binding domains (LBDs) gain ligand-dependent activity.
• Existing inducible expression systems often rely on regulated transcription.

Purpose of the Study:

• To develop a single-step inducible expression system using SSR-LBD fusion proteins.
• To demonstrate ligand-inducible gene expression regulated by recombination, not transcription.
• To show the system's capability for expressing toxic proteins.

Main Methods:

• Constructing a single transgene with a bi-directional promoter driving SSR-LBD and a selectable marker/inducible gene cassette.
• Utilizing puromycin acetyltransferase (pac) gene for selection and recombination.
• Inducing recombination via ligand activation of SSR-LBD to excise the pac gene and enable inducible gene expression.

Main Results:

• The system allows for ligand-dependent activation of gene expression through recombination.
• Successfully demonstrated inducible expression of diphtheria toxin, a highly toxic protein.
• Showed that graded selection pressure can select for clones with varying induction properties.

Conclusions:

• This system offers a powerful tool for ligand-inducible gene expression, distinct from transcriptional control.
• The single-transgene approach simplifies cellular introduction and regulatory circuit integration.
• The system is versatile and applicable for expressing toxic proteins and fine-tuning induction levels.