A low-background Tet-On system based on post-transcriptional regulation using Csy4

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• Biotechnology

Background:

• Tetracycline-inducible systems are widely used for controlled gene expression in eukaryotes.
• Leaky expression from these systems can be problematic, especially when expressing toxic proteins.
• The Tet-On 3G system exhibited significant leaky expression in sf9 insect cells, leading to cell death.

Purpose of the Study:

• To address the issue of leaky expression in the Tet-On 3G system in sf9 cells.
• To develop a novel, low-background inducible expression system for insect cells.
• To improve the safety and efficacy of inducible gene expression systems.

Main Methods:

• Evaluated leaky expression of the Tet-On 3G system using the kid toxin gene.
• Tested the efficacy of tetracycline-controlled transcriptional silencer (tTS) in reducing background expression.
• Investigated the use of Csy4, a CRISPR-associated protein, for reducing leaky expression.
• Engineered a modified Tet-On 3G system incorporating Csy4 for post-transcriptional regulation.

Main Results:

• The Tet-On 3G system showed significant leaky expression, causing toxicity in sf9 cells.
• tTS was ineffective in reducing background expression in this system.
• Csy4 effectively reduced background expression but also lowered maximum induced expression.
• The modified system with anti-sense and sense Csy4 significantly reduced leaky expression and enhanced induction levels.

Conclusions:

• A novel, low-background inducible expression system was developed using Csy4-mediated post-transcriptional regulation.
• This optimized system effectively minimizes cell death from toxic protein background expression.
• The developed system holds potential for applications in insect cells and other organisms, including mammals.