Engineered transcription factor-binding arrays for DNA-based gene expression control in mammalian cells

Area of Science:

• Synthetic Biology
• Molecular Biology
• Mammalian Cell Engineering

Background:

• Gene expression control is crucial for cell engineering.
• Existing methods often require additional expression components.
• Novel tools for precise gene regulation in mammalian systems are needed.

Purpose of the Study:

• To demonstrate the use of transcription factor recognition element (RE) arrays as DNA tools for controlling gene expression in mammalian cells.
• To develop a method for assembling custom RE arrays.
• To show the application of these arrays in cell engineering.

Main Methods:

• Assembly of transcription factor recognition element (RE) arrays using the Cloning Troublesome Repeats in Loops (CTRL) technique.
• Demonstration of TetR-based RE arrays for synthetic gene circuit modulation.
• Transfection of custom RE array plasmids into mammalian cells.

Main Results:

• TetR-based RE arrays were shown to alter synthetic gene circuit performance.
• The CTRL technique successfully assembled plasmids with up to 256 RE repeats.
• Custom RE array plasmids modified host cell gene regulation by sequestering transcription factors (TFs).
• Both synthetic and native TFs were sequestered, impacting cell fate.

Conclusions:

• Transcription factor (TF) binding RE arrays are effective DNA tools for controlling gene expression in mammalian cells.
• The CTRL technique facilitates the assembly of complex repetitive DNA arrays.
• This approach expands possibilities for mammalian cell engineering, including gene circuit control and cell fate determination.