FUNCTIONAL CHARACTERIZATION OF RECOMBINANT GENE CIRCUITS IN REGULATING THERAPEUTIC GENE EXPRESSION IN MAMMALIAN CELLS

Abstract

This paper will design and functionally characterize recombinant gene circuits that enable the full and accurate regulation of therapeutic gene expression in mammalian cells. Optimized techniques of transfection were used to introduce synthetic gene circuits which included inducible, feedback-controlled and logic-gated architectures into mammalian cell lines including HEK293 and CHO. The monitored expression of genes was done by reporter assays which incorporated GFP and luciferase systems and quantitative assessment done by RT-qPCR and Western blot analysis. Dose-response studies and time-course expression profiling were done to verify the dynamic behavior, stability, and tunability of the engineered circuits, which was referred to as functional validation. The findings reveal that the recombinant gene circuits allow expression of genes with a high degree of precision, with high tunability, and stability which is highly improved in comparison with the traditional systems of expressing genes. On the whole, these results indicate that synthetic gene circuits have the potential to be effective and programmable therapeutic systems of genetics in mammalian cells.