Identification of genes related to spcific properties of mammalian cells

为了鉴定与特定细胞特性功能相关的基因，在生物反应器中培养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。发现siat7e基因影响HeLa细胞的粘附和形态。在不依赖锚定的HeLa细胞中，降低II型膜糖基化唾液基转移酶siat7e的表达可导致更大的聚集和形态变化。基于上述工作，我们决定将重点放在适合流感病毒生产但不能在悬浮中生长的MDCK细胞上，因此不能用于疫苗的商业生产。通过转染人siat7e基因（ST6GalNac V），将锚定依赖性MDCK细胞转化为锚定非依赖性细胞。转化的细胞能够在生物反应器中产生病毒，这表明它们有能力取代目前基于鸡蛋的生产过程。作为概念验证，这项工作表明，通过使用适当的策略，可以从悬浮MDCK-siat7e细胞的可扩展培养中产生高产量的生物活性血凝素。

To identify 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 gene siat7e was 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. Based on the above work we decided to concentrate on MDCK cells that are suitable for influenza virus production but cannot grow in suspension and therefore are not being used for commercial production of the vaccine. The anchorage dependent MDCK cells were converted to anchorage independent cells by transfection with the human siat7e gene (ST6GalNac V). The converted cells were able to produce the virus in bioreactors demonstrating their capability to replace the current egg based production process. 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 transfection 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 involved 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 and was verified to be over-expressed in MDCK-siat7e cells. The findings are being used to try and transform other anchorage dependent cells to grow in suspension. Additional attempt to improve cellular properties of mammalian cells was based on identifying of microRNA that affect cells apoptosis. 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 microRNAs in CHO cells exposed to fresh or depleted media revealed up-regulation of miR-297-669 cluster in CHO cells subjected to depleted media. 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 miR-466h anti-apoptotic targets. In the next phase of this work we were able to understand the mechanism associated with the activation of the apoptosis cascade as a result of the nutrient depletion. This is especially important since changes in microRNA expression have been linked to the development of various diseases including cancer; however, the molecular events leading to these changes at different physiological conditions are not well characterized. We showed that the time-dependent activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation which was the result of glucose deprivation-induced oxidative stress. This oxidative stress caused the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity, reduced protein levels of HDAC2, and increased acetylation in miR-466h-5p promoter region which led to the activation of this miRNA. Based on this study and previous work, we suggest a possible role of miR-466h-5p (and miR 297-669 cluster) in the cells during toxic metabolites accumulation. Improved characterization of the molecular events that lead to the activation of miR-466h-5p may provide a better understanding of the relation between cellular environment and miRNA activation.