Activity of the medaka translation elongation factor 1alpha-A promoter examined using the GFP gene as a reporter.
The translation elongation factor 1alpha (EF-1alpha) is known to have several isoforms, which are expressed in a tissue- and stage-specific manner. Two genes encoding EF-1alpha exist per haploid genome in the medaka. In the present study, the promoter activity of the 5'-flanking region of the medaka EF-1alpha-A gene, an isoform of EF-1alpha, was characterized using transgenic techniques. First, using CAT gene as a reporter, it was revealed that about 1.8 kbp 5'-flanking sequence from the transcription initiation site of EF-1alpha-A was sufficient for high-level promoter activity. Second, the green fluorescent protein (GFP) gene fused to this region was introduced into medaka eggs using the microinjection method. Three germline transgenic individuals (one male and two female) were mated with non-transgenic medaka to obtain F1 offspring. In the case of embryonic and adult F1 transgenic individuals, GFP fluorescence was observed in almost all the tissues examined (e.g. kidney, liver, heart, gill, ovary, and testis), except for the skeletal muscle. In the case of F2 transgenic embryos derived from F1 transgenic males and non-transgenic females, the fluorescence was observed from the early gastrula stage. On the other hand, in the case of F2 transgenic embryos derived from F1 transgenic females and non-transgenic males, the fluorescence was observed even at the 1-cell stage, suggesting that this region is transcriptionally active during oogenesis. The usefulness of the EF-1alpha-A promoter as a tool for introducing foreign proteins into oocytes is discussed.
Kinoshita
M
M
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan. kinoshit@kais.kyoto-u.ac.jp
Kani
S
S
Ozato
K
K
Wakamatsu
Y
Y
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D009990:Oryzias / Q000235:genetics*; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic
D017931:DNA Primers; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins