Identification of genes realted to spcific properties of mammlian cells

Diabetes, Digestive, Kidney Diseases

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Abstract

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. [unreadable] [unreadable] 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.