Background

Precise control of gene expression is critical for biological research and biotechnology. For example, the function of a protein can depend on its abundance in a cell. Thus, it is often essential to ensure consistent protein expression between each cell in a cell line. In an ideal compensation circuit, the per- plasmid expression rate is inversely proportional to the copy number; the total protein expression thus remains constant (Fig 1). Compensation circuits promise to combine the versatility and convenience of plasmids with the lower cell-to-cell variability of chromosomal expression.

Transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity and varying concentrations of the protein between cells. Existing mammalian circuits buffer gene dosage variation across a limited range of plasmid copy numbers. Moreover, their ability to reduce cell-to-cell expression variability within a transfected population has not been demonstrated or has been incompletely quantified.

Technology Overview

Baylor College researchers have developed a suite of new gene expression circuits that provide uniform protein expression thereby reducing intercellular variability. “The Equalizer” is a plasmid-based synthetic circuit that buffers copy-number variation at the single-cell level. Further, these circuits also enable the generation of extrachromosomal cell lines that are much faster and easier to produce than typical cell lines made by chromosomal integration yet produce the same low cell-to-cell variability. 

Stage of Development

Data indicates that Equalizers outperform other gene dosage compensation topologies and perform as well as chromosomally integrated genes. Further, episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

Further Details

Yang, J., Lee, J., Land, M.A. et al. A synthetic circuit for buffering gene dosage variation between individual mammalian cells. Nat Commun 12, 4132 (2021).

Engineered Circuit for Gene Expression Could Be Great Equalizer (genengnews.com)

Benefits

• Equalizers function in multiple cell types
• Cell lines ready for use 1 – 3 days post transfection vs several weeks for chromosomalintegration
• Equalizers outperform other compensation circuits at the single-cell level
• Equalizers enable long-term gene expression with cell-to-cell variation comparable tochromosomal expression