Köster R W, Fraser S E
Division of Biology and Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Dev Biol. 2001 May 15;233(2):329-46. doi: 10.1006/dbio.2001.0242.
Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels. Furthermore, the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells. We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos. In comparison to conventional expression vectors, Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels. The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells. The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP. The combinatorial nature of the expression system greatly facilitates changing the tissue-specificity, the transgene expressed, or the cell compartment-specific GFP reporter, making it simpler to address a gene's function in different tissues as well as its cell biological consequences.
由于在可检测水平表达感兴趣转基因的细胞频率较低，通过注射质粒DNA进行异位表达在斑马鱼胚胎中很少使用。此外，质粒注射异位表达的镶嵌性质需要直接检测表达转基因的细胞。我们使用转录激活因子Gal4-VP16来放大活斑马鱼胚胎中的转基因表达。与传统表达载体相比，Gal4-VP16放大表达导致在可检测水平表达转基因的细胞数量显著增加。Gal4-VP16激活因子和Gal4-VP16依赖性转基因可以置于单个表达载体上。使用组织特异性调控元件，我们表明Gal4-VP16依赖性转基因的表达可以可靠地限制在肌肉、脊索或神经组织中。此外，Gal4-VP16可以驱动来自同一构建体的两个或更多转基因的表达，从而在几乎所有表达细胞中同时共表达这两个基因。报道的表达系统不仅在斑马鱼胚胎中有效，而且在非洲爪蟾胚胎、鸡、小鼠和人类培养细胞中也有效，因此适用于多种脊椎动物。转基因表达的高频率以及一个以上转基因的连锁共表达开启了通过用荧光报告基因GFP标记来实时轻松监测单个表达转基因细胞行为的可能性。表达系统的组合性质极大地促进了改变组织特异性、所表达的转基因或细胞区室特异性GFP报告基因，使得更易于研究基因在不同组织中的功能及其细胞生物学后果。
