Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish
Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish
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DOI：
10.1073/pnas.0704963105
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发表时间：
2008-01-29
影响因子：
11.1
通讯作者：
Kawakami, Koichi
中科院分区：
文献类型：
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作者：
Asakawa, Kazuhide;Suster, Maximiliano L.;Kawakami, Koichi
靶向基因表达是研究体内基因和细胞功能的有力手段。在果蝇中，P元件介导的Gal 4-UAS方法已成功地用于此目的。然而，在脊椎动物中还没有建立类似的方法。在这里，我们报告了一个有针对性的基因表达方法的发展在斑马鱼的基础上Tol 2转座元件及其应用的神经回路的功能研究。首先，我们开发了携带工程酵母Gal 4转录激活因子（Gal 4FF）的基因陷阱和增强子陷阱构建体和携带GFP或GFP基因下游Gal 4识别序列（UAS）的转基因报告鱼，并表明Gal 4FF可以通过斑马鱼中的UAS激活转录。其次，通过使用这种Gal 4FF-UAS系统，我们进行了大规模的筛选，并产生了大量在特定组织、细胞和器官中表达Gal 4FF的鱼类品系。最后，我们开发了转基因效应鱼携带破伤风毒素轻链（TeTxLC）基因下游的UAS，这是已知的阻断突触传递。我们将Gal 4FF鱼与UAS：TeTxLC鱼杂交，并分析了双转基因胚胎在触摸反应中的缺陷。从这个分析中，我们发现TeTxLC在大脑和脊髓的不同神经元群体中的靶向表达引起了触摸反应行为的明显异常。这些研究表明，我们的Gal 4FF基因陷阱和增强子陷阱的方法应该是一个重要的资源，在脊椎动物的神经元功能和行为的遗传分析。
Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.