Identification of genes realted to spcific properties of mammlian cells

为了鉴定与特定细胞特性功能相关的其他基因，在生物反应器中培养具有不同表型的细胞系并取样进行微阵列分析。结合数据滤波和聚类算法对微阵列数据进行归一化处理。基于样本之间的差异表达水平、聚类技术和提出的功能，鉴定了几个基因。RT-PCR验证了这些基因的表达。使用RNAi（基因阻断）和质粒（基因增强）改变基因表达水平。使用细胞计数器和剪切流室来定量粘附。

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.