Work

A Dual-Selection System for Enhanced Efficiency and Fidelity of Circular RNA Overexpression

Year: 2025

Type: article

Abstract: • Dual fluorescent-antibiotic selection system enhances specificity of circRNA overexpression. • Flow cytometry sorting eliminates false positives in stable circRNA-expressing cell lines. • Novel lentiviral design enables controlled and enriched inducible circRNA expression. Circular RNAs (circRNAs) are essential regulators of cellular processes, but are challenging to study using traditional methods. Overexpression approaches, such as the use of linearized plasmids and viral vectors, often result in high rates of false-positive clones, where cells retain selection markers without expressing the target circRNA. This study addresses this limitation by developing a dual-selection circRNA system designed to enhance the accuracy and reliability of circRNA overexpression. Our system integrates a fluorescent reporter gene upstream of the circRNA expression cassette, under a shared promoter, and a downstream antibiotic resistance marker, allowing for both antibiotic selection and flow cytometric cell-sorting to identify and enrich cells with genuine circRNA expression. We successfully incorporated this system into an inducible lentiviral vector for controlled overexpression in various cell types. The dual-selection circRNA system offers a significant advance for circRNA research and studies of other RNA species where accurate and reliable overexpression is essential.

Source: Journal of Molecular Biology

DOI: 10.1016/j.jmb.2025.169064