a. In earlier work we have been able to improve growth properties of mammalian cells as a result of identifying microRNAs that affect cells apoptosis in CHO cells. By using microarray, bioinformatics and experimental tools we identified the 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 engineered cells reached higher maximum viable cell density compared with parental CHO, and the expression of secreted alkaline phosphatase (SEAP) from these cells was 43% higher compared with the stable pools of negative control CHO cells. These results demonstrated the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression. b. We followed this work with implementing high throughput method 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 then 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 microRNA were able to improve functional expression of NTSR1 by as much as 48%. In parallel, an HEK293 cell lines expressing luciferase, serotonin receptor and a secreted protein were also screened with the same human miRNA mimic library. All 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. c. Another approach for identifying genes that can affect recombinant protein production was to conduct an unbiased, high-throughput whole-genome RNA interference screen using human embryonic kidney 293 (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. d. Generation of Immortalized MEF cell lines to study O-GlcNAc Metabolism and Neurodegeneration: O-GlcNAcylation is an abundant post-translational modification in which the monosaccharide -Nacetyl-D-glucosamine (O-GlcNAc) is added to and removed from Ser/Thr residues by O GlcNAc transferase (OGT) and O GlcNAcase (OGA), respectively. Signalling and metabolism are affected in null alleles of OGT and OGA in C. elegans and D. melanogaster, hence suggesting that O-GlcNAc metabolism serves an essential function. The implications of O-GlcNAc metabolism are difficult to study in vertebrates as conditional knockout mutants of the enzymes of O GlcNAc cycling result in low viability of embryos or embryonic lethality. OGA and OGT knockout mutants have been generated in mice, and primary MEFs have been used to study O-GlcNAc cycling. But since the use of primary MEFs is time consuming and results can be difficult to replicate immortalized wild type, OGA null allele, and OGT floxed allele MEF cell lines were generated to study O-GlcNAc metabolism. We evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for >40 passages with a median doubling rate of 4555hrs (n=8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5uM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 259 hours (n=9) and viability greater than 90%.
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