Hamada K, Tamaki K, Sasado T, Watai Y, Kani S, Wakamatsu Y, Ozato K, Kinoshita M, Kohno R, Takagi S, Kimura M
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
Mol Mar Biol Biotechnol. 1998 Sep;7(3):173-80.
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.
利用绿色荧光蛋白（GFP）的互补DNA，检测了青鳉β-肌动蛋白启动子作为转基因青鳉中一种遍在表达载体的活性。质粒pOBA-GFP包含青鳉β-肌动蛋白启动子和野生型GFP的cDNA，而pOBA-hGFP包含青鳉β-肌动蛋白启动子和突变型GFP的cDNA，其中第65位的苏氨酸被丝氨酸取代，并且密码子使用进行了人源化处理以促进在脊椎动物细胞中的翻译。将两种质粒的ApaI-SmaI片段在单细胞期显微注射到卵母细胞核或胚胎细胞质中。使用荧光立体显微镜从发育早期到孵化后1周检测基因表达。在早期胚胎、卵黄囊以及肌肉和表皮的小部分中检测到野生型GFP的表达。这种表达模式与我们之前研究中由青鳉β-肌动蛋白启动子驱动的大肠杆菌β-半乳糖苷酶报告基因（lacZ）的表达模式相似。突变型GFP在早期胚胎以及许多组织如表皮、血管、肌肉、脊索、鳍条、肠道、眼睛和卵黄囊中表达，并且荧光比野生型GFP强得多。因此，以突变型GFP作为报告基因证实了青鳉β-肌动蛋白启动子作为一种遍在表达载体的实用性。
