Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells.

Area of Science:

• Synthetic biology
• Molecular and cell biology
• Genetic engineering

Background:

• Mammalian cell engineering relies on precise control of gene expression.
• Transcription factors (TFs) are key regulators of gene expression.
• Existing methods for TF manipulation have limitations.

Purpose of the Study:

• To explore transcription factor recognition element (RE) arrays as DNA tools for modulating TF levels and gene expression in mammalian cells.
• To develop a method for assembling long repetitive RE arrays for TF sequestration.
• To demonstrate the application of RE arrays in controlling synthetic and native TF activity for cell engineering.

Main Methods:

• Proof-of-concept using Tet TF-binding RE arrays of varying lengths to tune gene expression.
• Development of the Cloning Troublesome Repeats in Loops (CTRL) method to assemble plasmids with up to 256 RE repeats.
• Transfection of RE array plasmids into mammalian cells to assess TF sequestration and gene regulation.
• Demonstration of RE arrays targeting both synthetic and native mammalian TFs.

Main Results:

• Tet RE arrays demonstrated predictable tuning of gene expression and gene circuit performance.
• The CTRL method successfully assembled plasmids with extensive RE repeats.
• Longer RE array sizes showed potential for modifying host cell gene regulation via TF sequestration.
• RE array plasmids effectively modulated genetic circuits and influenced cell fate by targeting TFs.

Conclusions:

• TF-binding RE arrays represent a novel DNA tool for manipulating mammalian gene expression.
• The CTRL method facilitates the construction of complex repetitive DNA arrays for biological applications.
• This approach expands the toolkit for mammalian cell engineering, enabling precise control over gene regulation and cell fate determination.