185 CELLULAR VIABILITY AND EXPRESSION OF GREEN FLUORESCENT PROTEIN IN BOVINE FETAL FIBROBLASTS FOLLOWING TRANSFECTION OF SYNTHETIC mRNA INCLUDING MODIFIED BASES

Abstract

Synthetic RNA transfection has been an invaluable tool in understanding the mammalian genome because of its ability to deliver exogenous protein without mutagenic effects caused by double-stranded DNA. A common problem associated with the introduction of exogenous mRNA into mammalian cells is the stimulated interferon response. This innate immune response can be avoided with the addition of modified bases during the in vitro transcription process of synthetically derived mRNA. The bases cytidine triphosphate (CTP) and uridine triphosphate (UTP) are replaced with 5-methylcytidine-5′-triphosphate (5-Methyl-CTP) and pseudouridine-5′-triphosphate (Pseudo-UTP) during in vitro transcription. Cellular reprogramming is achieved by the delivery of this mRNA into the cytoplasm. Previous cellular reprogramming experiments lacking modified bases resulted in increased toxicity and a decrease in cellular viability, which lead to the incorporation of modified bases. In the first experiment, bovine fetal fibroblasts were transfected with modified synthetic mRNA encoding green fluorescent protein (GFP) to evaluate the effects on cellular viability and fluorescence. The cellular viability was measured by counting a final number of cells after seeding a constant number of cells in all treatment groups. The control group consisted of bovine fetal fibroblasts cultured in normal growth medium. A no-RNA (NR) group was held under the same conditions with the addition of the transfection reagent, Lipofectamine (Invitrogen, Carlsbad, CA, USA), to account for toxicity resulting from the transfection reagent alone. The cells were transfected every other day for 12 days and were evaluated on days 3, 6, 9, and 12 for viability and fluorescence by flow cytometry. There was no difference in viability of all cells treated with synthetic mRNA encoding GFP when compared to controls (P = 0.9). There was a significant difference in fluorescence on all time points when compared to controls (Day 3, P = 0.004; Day 6, P = 0.004; Day 9, P = 0.007; Day 12, P = 0.04). The second experiment consisted of bovine fetal fibroblasts transfected with modified synthetic mRNA encoding pluripotency factors. The controls were identical to the previous experiment, but treatment groups were transfected with modified synthetic mRNA encoding either three factors (3F: OCT4, SOX2, KLF4) or four factors (4F: OCT4, SOX2, KLF4, c-MYC). The treated cells were transfected every other day and evaluated on Day 24 for cellular viability. There was no difference in cellular viability in all treatment groups when compared to controls (P = 0.2). The introduction of synthetic mRNA containing modified bases maintains cellular viability when compared to controls. The decreased immune response by the inclusion of modified bases may be advantageous in a variety of applications from the introduction of transcription activator-like effector nuclease (TALEN) or zinc finger nucleases for genomic editing to increased efficiency of the development of induced pluripotent stem cells.