298 STIMULATION OF ENDOGENOUS OCT4 IN BOVINE FETAL FIBROBLASTS WITH SYNTHETIC mRNA TRANSFECTION

Abstract

Induced pluripotent cells have been produced with viral vectors and other transgenic approaches that modify the genome of the host cell. Transfection with mRNA encoding transcription factors eliminates genomic modification and has been successful with human cells. The objective of this study was to deliver synthetic mRNA to bovine fetal fibroblasts in a manner consistent with induced pluripotency. Plasmids encoding green fluorescent protein (GFP) and human OCT4 were used as templates for in vitro transcription of mRNA (ivtRNA) and used to transfect bovine fetal fibroblasts. Expression of GFP was measured by flow cytometry and cell viability by seeding a constant cell number and determining final cell count. Differences between treatments were tested by ANOVA at P ≤ 0.05. In the first experiment, ivtRNA concentrations of 140, 280, and 560 ng/well of a 24-well plate were used and cell number and fluorescence determined after 24 h. Transfected treatments had significantly fewer cells and greater fluorescence than controls. Cells transfected with 280 ng/well ivtRNA had the highest fluorescence. Cells were then transfected with 280 ng/well of ivtRNA and total cells and fluorescence determined after 12, 24, 36, 48, and 60 h. Cell number was significantly lower than controls for all time points but similar between time points. Fluorescence was greater than controls for all time points and peaked at 24 to 36 h. There was no difference in fluorescence between 24 and 36 h, but these time points were significantly greater than all other time points. From these data, we determined that sustained expression could be maintained with transfection every other day. Cells were transfected every other day with ivtRNA encoding GFP and fluorescence measured every 3 days. Fluorescence was greater than controls for all time points but did not increase with repeated transfection. After 6 transfections, there were no viable cells left. In the final experiment, cells were transfected every other day for 12 days with 140, 280, or 560 ng/well ivtRNA encoding human Oct4. After 12 days, RNA was isolated and bovine OCT4 transcripts measured by RT-PCR. Primers and PCR conditions were designed so that bovine OCT4 transcripts were amplified but human OCT4 ivtRNA was not. The bovine poly-adenylate polymerase (PAP) transcript was also amplified. After gel electrophoresis of RT-PCR products, band intensities were determined and a ratio of OCT4/PAP calculated. This ratio was 0.7 for controls and 1.2 for the 140 ng/well ivtRNA treatment. No bovine OCT4 expression was detected in the higher ivtRNA treatments because of poor cell viability. From these experiments, we conclude that ivtRNA can be transfected into and expressed by bovine fetal fibroblasts. Repeated transfections with ivtRNA encoding human OCT4 induced expression of the endogenous bovine OCT4 but cell viability was reduced. Further experiments utilising ivtRNA with modified bases are planned to reduce the cell toxicity of ivtRNA.