USEFULNESS OF THE MEDAKA BETA-ACTIN PROMOTER INVESTIGATED USING A MUTANT GFP REPORTER GENE IN TRANSGENIC MEDAKA (ORYZIAS-LATIPES)

The activity of the medaka beta-actin promoter as a ubiquitous express ion Vector in transgenic medaka was examined using complementary DNA o f the green fluorescent protein (GFP). Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while p OBA-hGFP contained the medaka beta-actin promoter and cDNA of the muta nt GFP in which serine was substituted for threonine at position 65 an d codon usage was humanized to promote translation in vertebrate cells . The ApaI-SmaI fragment of both plasmids was microinjected into the n uclei of oocytes or the cytoplasm of embryos at the one-cell stage. Th e gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching. The express ion of the wild-type GFP was detected in early embryos, in the yolk sa c and in small portions of the muscle and epidermis. This expression p attern was similar to that of the Escherichia coli beta-galactosidase reporter gene (lacZ), driven by the medaka beta-actin promoter, which was examined in our previous studies. The mutant GFP was expressed in early embryos and in many tissues such as the epidermis, blood vessels , muscle, notochord, fin ray, gut, eyes, and yolk sac, and the fluores cence was much stranger than that of the wild-type GFP. Thus, the usef ulness of the medaka beta-actin promoter as a ubiquitous expression ve ctor was confirmed using the mutant GFP as a reporter gene.