Comprehensive gene expression analysis using microarray for metabolic engineering of silkworm cell lines

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For the establishment of insect cell expression systems useful for the protein production, comprehensive gene expression pattern analyses in lepidopteran insect cell lines and the development of gene manipulating techniques to alter metabolic properties such as translational modification rationally were conducted in this research project, and the following results were obtained.1.Comparative gene expression analyses were performed comprehensively between two Bomby mori-derived cell lines, BmN4 and BoMo, using a cDNA microarray as well as between BmN4 cells cultured in different media using an oligoDNA array, and genes showing cell line-specific or medium-specific increases in the expression level were identified. Among them, the medium-susceptible gene promoters are useful for developing technologies controlling the protein production by medium components.2.Mammalian-like N-glycosylation property and medium-dependent AcNPV susceptibility were revealed in an Antheraea pernyi-derived AnPe cell line. Comparative gene expression analysis was performed comprehensively between AnPe cells cultured in different media by differential display, and medium-susceptible transcripts were identified. By developing methods to control the expression of genes encoding these transcripts, high AcNPV-susceptible AnPe cells for the production of glycoproteins with mammalian-like N-glycans will be established.3.A piggyBac vector containing B.mori actin A3 promoter for heterologous gene expression and Drosophila melanogaster heat shock protein 70 promoter-driven puromicin resistance gene for selection of transformed cells was constructed for efficient transformation of B.mori cell lines. Using this vector, a glycoprotein, which had not been produced as a membrane-bound form by baculovirus expression vector, was successfully produced, suggesting the piggyBac vector can be used effectively for metabolic engineering of not only B.mori but also other lepidopteran insect cell lines.