Identification of genes realted to spcific properties of mammlian cells

为了确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了不同表型的细胞系，并对其进行了采样以进行微阵列分析。结合数据过滤和聚类算法对微阵列数据进行归一化处理。根据样本之间的差异表达水平、聚类技术和拟议的功能，识别了几个基因。用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对粘附性进行量化。 发现siat7e和lama4基因对HeLa细胞的黏附和形态有影响。在非锚定依赖的HeLa细胞中，降低II型糖基化唾液酸转移酶siat7e的表达导致更大的聚集和形态变化。当编码一种分泌型糖蛋白的lama4的表达增强时，在锚定非依赖的HeLa细胞中也可以看到类似的效果。在生长速度方面，两个不同的基因，一个编码线粒体组装蛋白，另一个编码与细胞周期蛋白依赖蛋白和丝裂原激活蛋白激酶序列同源的蛋白，被鉴定为可能的细胞生长促进因子。三个基因的临时专利已经提交，可能会扩大到包括其他基因。 在上述工作的基础上，我们决定集中研究MDCK细胞。这些依赖于锚定的细胞适合于病毒生产，但它们的生长特性影响病毒生产效率。通过将特定基因导入这些细胞，可以将细胞的生长特性从锚定依赖转变为锚定非依赖。目前正在对转化细胞的特性进行鉴定，并对其产生流感病毒的能力进行评估。

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. These anchorage depended cells are suitable for virus production but their growth properties affect the virus production efficiency. By transfecting these cells with specific gene it was possible to convert the growth properties of the cells from anchorage dependent to anchorage independent. The transformed cells are being characterized and evaluated for their ability to produce the influenza virus.