Basic Research for Development of Expression Cloning Vectors Using Insect Cell Culture Systems

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In order to establish basic technologies required for development of eukatyotic gene expression cloning vector systems using cultured insect cells, I have constructed an expression cloning plasmid vector which expressed foreign genes under control of the helper virus for the baculovirus vector system and another expression cloning plasmid vector which coexpresses puromycin resistant (pac) gene for the Drosophila S2 cell system, and obtained following results.1. For the baculovirus vector system, the expression cloning plasmid vector pBMIHpa (ca. 5.8 kbp) containing the polyhedrin promoter was constructed and, after inserting foreign genes, cotransfected to the silkworm cells (BmN4) with the baculovirus DNA.Although the foreign gene expression was detected in the cotransfected cells, it was impossible to clone the cells because of the cell lysis by virus multiplication.2. For the S2 cell system, the green fluorescent protein (GFP) gene was inserted to the expression cloning plasmid vect … More or pDhsp(pac^+) coexpressing the pac gene as a selectable marker or another plasmid vector pDhsp(pac-) lacking the pac gene. The GFP fluorescence intensity in the transformed S2 cells, which were obtained by transfection with pDhspGFP(pac^+), was remarkably higher than that in another transformed S2 cells, which were obtained by cotransfection with pDhspGFP(pac^-) and pDhsp(pac^+), demonstrating that the use of coexpression plasmid vector can effectively enhance the sensitivity in the detection of the target gene expression. Detectable levels of beta-galactosidase activity were observed in transformed S2 cells by cotransfection of the LacZ gene expressing plasmid pDhspLacZ(pac^-) and pDhsp(pac^+) at 1 : 1 or 1 : 10 ratio, but not at 1 : 100 ratio, suggesting that the limit number of clone for screening the cotransfected S2 cells should be between 10 to 100 clones.According to the results obtained in this research, I will further investigate the development of efficient screening methods of transformed insect cells. Less