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 expression vector in transgenic medaka was examined using complementary DNA of the green fluorescent protein (GFP). Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while pOBA-hGFP contained the medaka beta-actin promoter and cDNA of the mutant GFP in which serine was substituted for threonine at position 65 and codon usage was humanized to promote translation in vertebrate cells. The ApaI-SmaI fragment of both plasmids was microinjected into the nuclei of oocytes or the cytoplasm of embryos at the one-cell stage. The gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching. The expression of the wild-type GFP was detected in early embryos, in the yolk sac and in small portions of the muscle and epidermis. This expression pattern 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 fluorescence was much stronger than that of the wild-type GFP. Thus, the usefulness of the medaka beta-actin promoter as a ubiquitous expression vector was confirmed using the mutant GFP as a reporter gene.
Hamada
K
K
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
Tamaki
K
K
Sasado
T
T
Watai
Y
Y
Kani
S
S
Wakamatsu
Y
Y
Ozato
K
K
Kinoshita
M
M
Kohno
R
R
Takagi
S
S
Kimura
M
M
D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009990:Oryzias / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins