Generation of Transgenic Medaka Oryzias curvinotus (Nichols & Pope, 1927) Carrying a Cyan Fluorescent Protein Gene Driven by Alpha Actin Promoter

Abstract

The study aimed to produce fluorescent protein transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) as a novel strain of ornamental fish. These fish were produced by transferring a plasmid consisting of a fluorescent reporter gene and a strong promoter into one-cell stage embryos. For this purpose, myosin light chain 2, but not other promoters, was mainly used. The study also evaluated the stability of the transgenic medaka germline acquiring vivid fluorescent phenotypes via the transgenesis of the cyan fluorescent protein (CFP) gene under the control of O. curvinotus skeletal alpha-actin (OCacta) promoter. The pOCacta-CFP plasmid, containing a OCacta promoter and CFP reporter gene, was transferred into the one-cell stage of O. curvinotus embryos by a microinjection technique. As a result, 36 of 1386 microinjected O. curvinotus embryos exhibited CFP signals in their trunks. The expressed CFP signals in O. curvinotus embryos and adults were detected under a microscope using a green fluorescent protein (GFP) filter (450–490 nm wavelength), and blue LED light (400–450 nm wavelength). Five O. curvinotus founders showing clear CFP signals were selected and crossed with non-transgenic counterparts to produce subsequent generations. Among strains, the frequency of germline transmission from founder to F1 was highly variable. Only two of the five founders successfully pass the transgene to the F1 generation. At present, the progeny of subsequent generations is being produced and tested for the expression of CFP signals, and therefore, stable lines are ongoing.