Construction Of Transformed Cultured Silkworm Cells And Transgenic Silkworm By φC31-att And Effects Of Pupal Development Of Bombyx Mori Using A Gal4/UAS Binary Transgenic System

The site-specific integrase system from the Streptomyces bacteriophage φC31is anefficient recombination system that uses an attP site in the phage genome and an attBsite in the host chromosome. In this study, the system has been used to transformcultured BmN cells and construct transgenic Bombyx mori individuals. Aplasmid,pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express agfp-zeocin fusion gene was co-transfected into cultured BmN cells, together with ahelper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the gfp-zeocin fusion gene wasdriven by an ie-1promoter with a φC31attB site upstream of the promoter. The helperplasmid encoded the φC31integrase, which was flanked by two nuclear localizationsignals. Expression of the gfp-zeocin fusion gene could be observed in transformed cells.The two plasmids were also transferred into silkworm eggs to obtain transgenicsilkworms. Successful integration of the fusion gene was indicated by the detection ofgreen fluorescence, emitted by the silkworms. Nucleotide sequence analysisdemonstrated that the attB site was cut and recombination between the attB andendogenous pseudo attP sites was performed by the φC31integrase in the culturedsilkworm cells and silkworm individuals.The pupal stage of the silkworm development is very important for production. Inthis study, we investiged the effects of egt gene transfer on the pupal development ofBombyx mori using a GAL4/UAS binary system. Hence, we construced the plasmidpiggyA3GFP-ie-neo-UAS-egt-polyA and piggyA3GFP-ie-neo-BmWCP4-Gal4-polyA.The expression vector BmWCP4-Gal4was transferred into strain Qingsong silkwormsovary by sperm-mediated gene transfer, and G9generation transgenic silkworms wereobstained by some screening and identification; the plasmidpiggyA3GFP-ie-neo-UAS-egt-polyA was transferred into strain Daozao silkwormsovary by sperm-mediated gene transfer, and G4generation UAS-egt transgenicsilkworms were obstained by some screening and identification. Then, it was studied that the egt gene expression could affect the development of the transgenic silkwormBmWCP4-Gal4and UAS-egt which was hybridized.