Generation of Stable Cell Lines Expressing Exogenous Circular RNA

• Luisa Abadia, Biological Sciences, University of Delaware

• Mona Batish, Medical & Molecular Sciences, University of Delaware

Circular RNAs are a newly appreciated class of regulatory RNAs. The circular RNAs are produced as a result of back splicing and do not have an open 5’ or 3’ end; and thus, are highly stable. In order to study their function, it is important to express these circular RNAs exogenously in a heterologous system. In this project, plasmids expressing all the signature motifs required for circularization were used so that the gene of interest expressed through these plasmids should lead to formation of circular RNA in recipient cells. A mutant version of the plasmid, which had a mutation in the region critical for circularization was used as a control. The mutant plasmid was expected to lead to the formation of a linear RNA. Both wild type and mutant plasmids were linearized with the enzyme PvuI, which cuts in the ampicillin resistance region of the plasmid. The Linearized DNA was then transfected into HeLa cells. Both plasmids contain a neomycin resistance gene which was used as the selective marker for this experiment. The recombinant cells were screened by growing the cells in presence of neomycin. The surviving cells were expanded and verified using single molecule fluorescent in situ hybridization. This method uses a set of small oligonucleotide probes terminally labeled with fluorophores. These probes bind to the length of target RNA in tandem and renders it visible in a florescence microscope as a diffraction limited spot. The circular and linear forms of RNAs were confirmed by treating the cells with exonuclease, Rnase R, which only digests linear RNA. We were able to develop cell lines that stably express the circular wild type, and linear mutant sequences of the gene of interest. These cell lines will be very useful for downstream studies for the function of circular RNAs.