To identify additional genes functionally related to specific cellular properties, cell lines with different phenotypes were grown in bioreactors and sampled for microarray analysis. A combination of data filtering and clustering algorithms was applied to normalize microarray data. Based on the level of differential expression between samples, clustering techniques, and proposed functionality, several genes were identified. The expression of theses genes was verified using RT-PCR. Gene expression levels were altered using RNAi (gene blocking) and plasmids (gene enhancement). Adhesion was quantified using both a cell counter and a shear flow chamber. The genes siat7e and lama4 were found to impact the adhesion and the morphology of HeLa cells. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells resulted in greater aggregation and morphological changes. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4, which encodes a secreted glycoprotein, was enhanced. In relation to growth rate, two different genes, one encoding a mitochondrial assembly protein and the other encoding a protein with sequence homology to both cyclin-dependent kinases and mitogen-activated protein kinases, were identified as possible enhancers of cellular growth in CHO, HEK-293, and HeLa cells. A provisional patent has been filed for three genes and may be expanded to include other genes. Based on the above work we decided to concentrate on MDCK cells. MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human sia7te gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7x105 cells/ml while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 106 cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 liter of siat7e-expressing cells at a concentration of 106 cells/ml would be equivalent to the amount of HA obtained from 10,000 embryonated eggs. This work was expended further for evaluating the production of other viruses and for the developing pilot scale production method from the anchorage independent MDCK cells. To examine the broad susceptibility of this novel cell line, the scalability of the production process, and the antigenic stability of cell-derived progeny viruses, infection experiments using four current influenza vaccine strains (A/California/07/2009 X-179A H1N1, A/Brisbane/59/2007 IVR-148 H1N1, A/Uruguay/716/2007 X-175C H3N2, and B/Brisbane/60/2008) were performed. In small-scale experiments, this cell line was found to support high-titer replication of all four virus strains. Subsequently, production in a bench-scale bioreactor and the antigenic characteristics of progeny viruses were assessed. High titers of hemagglutinin (at least 1:512) were produced in a 2-L bench-scale bioreactor with all four strains. Immunoblot results comfirmed higher yields than those obtained in chicken embryonated eggs with three of the four tested strains. Progeny viruses collected after serial passages in this cell line exhibited minimal mutations in the HA-encoding gene. Hemagglutination inhibition (HAI) assays using ferret antiserum confirmed antigenic stability. As a proof-of-concept this work demonstrates that by using a proper strategy, high yields of biologically active hemagglutinin can be produced from scalable cultures of suspension MDCK-siat7e cells. Different attempt to improve cellular properties of mammalian cells was done through identification of miRNA that affect cells apopotosis. This study determined the changes in microRNA expression in Chinese hamster ovary (CHO) cells undergoing apoptosis induced by exposing the cells to nutrient-depleted media. The apoptosis onset was confirmed by reduced cell viability and caspase-3/7 activation. Microarray comparison of known mouse and rat microRNAs in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells subjected to depleted media. Mmu-miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation in depleted media was confirmed with qRT-PCR. Since microRNAs suppress mRNA translation, we hypothesized that up-regulated mmu-miR-466h inhibits anti-apoptotic genes and induces apoptosis. A combination of bioinformatics and experimental tools was used to predict and verify mmu-miR-466h anti-apoptotic targets. 8708 predicted targets were obtained from miRecords database and narrowed to 38 anti-apoptotic genes with DAVID ncbi annotation tool . Several genes were selected from this anti-apoptotic subset based on nucleotide pairing complimentarity between the mmu-miR-466h seed region and 3 UTR of the target mRNAs. qRT-PCR analysis revealed reduced mRNA levels of bcl2l2, dad1, birc6, stat5a and smo genes in CHO cells exposed to depleted media. The inhibition of the mmu-miR-466h increased the expression levels of those genes and resulted in increased cell viability and decreased caspase-3/7 activation. The up-regulation of mmu-miR-466h in response to nutrients depletion causes the inhibition of several anti-apoptotic genes in unison. This suggests the pro-apoptotic role of mmu-miR-466h and its capability to modulate the apoptotic pathway in mammalian cells. Are any products or services commercially available or being developed that have arisen from the research in this project? Yes. Two provisional were submitted: 1) Composition and methods for modifying cellular properties. 2)compositions and methods for vaccine and virus production.
