Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
Transgenic Res. 2013 Apr;22(2):435-44. doi: 10.1007/s11248-012-9653-8. Epub 2012 Sep 16.
The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5'-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.
当建立转基因动物时，精确控制靶基因的时空表达至关重要，而引入荧光标记蛋白的基因对于加速分子水平的研究是不可避免的。为此，我们构建了一种新型的双启动子表达载体，用于两个独立的报告基因，绿色荧光蛋白（GFP）和红色荧光蛋白（mCherry）。它们的表达受两个不同的组织特异性启动子的控制，例如来自肌球蛋白轻链 2 基因的斑马鱼心肌特异性启动子（cmlc2）和牙鲆骨骼肌特异性启动子（myl2），并且它们呈头对头排列。将双启动子表达载体微注射到牙鲆的受精卵中后，发育中的鱼胚胎和产生的转基因鱼系在全身（骨骼肌）显示强烈的 GFP 信号，而在心脏（心肌）显示 mCherry 信号。然而，在心脏中观察到微弱的 GFP 信号，表明骨骼肌启动子存在渗漏。为了提高双启动子表达的严格性，我们在两个启动子的边界处插入了两个鸡衍生的绝缘子，例如串联重复的核心序列（250 bp）的 cHS4（5'-高敏感位点-4 鸡β-珠蛋白绝缘子）。带有绝缘子的双启动子表达载体现在确保了在转基因鱼系中严格的组织特异性表达。因此，我们带有绝缘子的双启动子表达系统与传统的 IRES 和融合报告基因系统兼容，并且将是产生转基因鱼的替代方法。
