Mammalian cells part (2014) Goal and objectives Mammalian cells are routinely being used for production of specific biochemical compounds with therapeutic or diagnostic functions. These cells have distinct properties, such as slow growth and anchorage-dependent behavior, which affect production efficiency. By identifying genes and microRNA responsible for specific properties, it may be possible to change cell behavior and to improve production capability. Summary a. In our early work we were able to convert the anchorage dependent MDCK cells to anchorage independent cells by stable transfection with the human siat7e gene (ST6GalNac V), a type II membrane glycosylating sialyltransferase. The converted cells were able to produce the influenza virus in bioreactors demonstrating their capability to replace the current egg based production process. Another approach to improve cellular properties of mammalian cells was based on identifying microRNAs that affect cells apoptosis. By implementing microarray, bioinformatics and experimental tools we identified the miR-466h as a possible candidate. By stably inhibiting mmu-miR-466h-5p in CHO cells it was possible to increase their 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. The above 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 to improve functional expression of NTSR1 by as much as 48%. In parallel, an HEK293 cell line expressing luciferase was also screened with the same human miRNA mimic library. All five identified microRNA mimics were also found to enhance the luciferase expression which could indicate that these molecules may have a wider role in recombinant protein expression from these cells. c. The research work related to the gene identification was expanded to create multi tissue computational model of type 2 diabetes in mice based on microarray analysis. In this study, a new algorithm for generating tissue-specific metabolic models is presented, along with the resulting multi-confidence level (MCL) multi-tissue model. The effect of T2DM on liver, muscle, and fat in MKR mice was first studied by microarray analysis and subsequently the changes in gene expression of frank T2DM MKR mice versus healthy mice were applied to the multi-tissue model to test the effect. Using the first multi-tissue genome-scale model of all metabolic pathways in T2DM, we found out that branched-chain amino acids degradation and fatty acids oxidation pathway is down-regulated in T2DM MKR mice. Microarray data showed low expression of genes in MKR mice versus healthy mice in the degradation of branched-chain amino acids and fatty-acid oxidation pathways. In addition, the flux balance analysis using the MCL multi-tissue model showed that the degradation pathways of branched-chain amino acid and fatty acid oxidation were significantly down-regulated in MKR mice versus healthy mice. Validation of the model was performed using data derived from the literature regarding T2DM. Microarray data was used in conjunction with the model to predict fluxes of various other metabolic pathways in the T2DM mouse model and alterations in a number of pathways were detected.The Type 2 Diabetes MCL multi-tissue model may explain the high level of branched-chain amino acids and free fatty acids in plasma of Type 2 Diabetic subjects from a metabolic fluxes perspective. 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 β-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.
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