Background: In previous work we have been able to improve growth properties of mammalian cells based on work in which we identified microRNAs that affect cells apoptosis in CHO cells. By using microarray, bioinformatics and experimental tools we identified miR-466h -5p as an apoptosis enhancer agent. By stably inhibiting this microRNA in CHO cells it was possible to increase the cells ability to resist apoptosis and to increase their production properties. These results demonstrated the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression. This work was followed by conducting high throughput screening to explore the possibility of enhancing expression of membrane proteins by using microRNA. A stable T-REx-293 cell line expressing the neurotensin receptor 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR), was constructed. The cell line was subjected to high-throughput human miRNA mimic library screening. Five microRNA mimics: ( hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429 and hsa-miR-2110 were identified) these microRNAs improved functional expression of NTSR1 by as much as 48%. In parallel, HEK293 cell lines expressing luciferase, serotonin receptor and a secreted protein were also screened with the same human miRNA mimic library. The five identified microRNA mimics were found to enhance the expression of these proteins which is an indication that these molecules may have a wider role in recombinant protein expression from these cells. The next step for identifying genes that can affect recombinant protein production was to conduct an unbiased, high-throughput whole-genome RNA interference screen using HEK 293 cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1- the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Creating HEK cell line in which the OAZ1 gene is not functioning was the next step in this project. By using the crisper technology an HEK cell line lacking OAZ1 was created. The cells were found to have three times increase expression of luciferase, at the same time the cells growth properties and metabolic activities were not affected In addition we demonstrated a novel approach for identification of the target genes of microRNA by integrating the differential gene expression analysis with information obtained from our previously-conducted high-throughput siRNA screening. After identifying the down-regulated genes and cross-referencing them with the predicted targets of the miR-22-3p, they were further cross-referenced with our previously conducted siRNA screen. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. When repressed, a phosphorylating protein was found to increase luciferase and GPC3 expression by 3.3-fold and 2.2-fold respectively.
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