A cell competition system with one gene expression from a single-copy gene in one cell

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Achieving precise control over multi-gene expression in individual cells is a persistent challenge in genetic engineering.
• Existing plasmid and viral vector systems often struggle with variable gene copy numbers and expression levels across transfected cell populations.

Purpose of the Study:

• To develop and validate a novel transgene competition system for precise single-cell, multi-gene expression.
• To assess the impact of expressing 48 different nuclear receptors on HEK293 cell proliferation rates.

Main Methods:

• Utilized a dual recombinase-mediated cassette exchange system combined with Kazusa cDNA clones.
• Established HEK293 cell lines expressing individual nuclear receptors under CMV or EF1 promoter control.
• Monitored and compared cell proliferation rates over 8 weeks in response to nuclear receptor expression.

Main Results:

• Successfully achieved simultaneous expression of all 48 nuclear receptors at consistent levels in individual HEK293 cells.
• Demonstrated significant differences in cell proliferation rates between cell lines expressing different nuclear receptors after 8 weeks.
• Identified specific nuclear receptors (EF1-NR1I2, EF1-NR4A1) that markedly increased or decreased cell proliferation compared to controls.

Conclusions:

• The developed transgene competition system provides a simple, accurate, and wide-ranging method for cell competition assays.
• This system enables robust comparative analysis of gene function, specifically highlighting the differential effects of nuclear receptors on cell proliferation.
• The findings underscore the utility of this system for high-throughput screening and functional genomics studies.