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 these 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. 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). Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks and in bioreactors. 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. This work was expended to evaluate 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. 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. Additional work was done to understand how trasfection with a single gene can transform the MDCK cell from anchorage dependent to anchorage independent. This work demonstrated that a process called EMT (Epithelial-mesenchymal transition) is involve in this transformation. DNA microarray analysis on parental MDCK and siat7e-expressing MDCK cells revealed that many of the genes involved in the EMT were significantly differentially expressed between the cell lines. The hepatocyte growth factor (HGF) gene was subsequently identified using bioinformatics analysis on the gene expression data and was verified to be over-expressed in MDCK-siat7e cells on both the transcript and protein levels. Several other cellular functions that are part of the EMT process were found to be differentially expressed. The findings are being used to try and transform other anchorage dependent cells to grow in suspension. 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. 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. A combination of bioinformatics and experimental tools was used to predict and verify mmu-miR-466h anti-apoptotic targets. 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. 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. We are concentrating now on the mechanism of the miRNA activation which is linked to oxidative stress, in addition we are conducting high throughput using mimics of miRNA to identify microRNA responsible for the apoptotic process.
