Transgenic flies expressing the fluorescence calcium sensor cameleon 2.1 under UAS control
Transgenic flies expressing the fluorescence calcium sensor cameleon 2.1 under UAS control
复制标题
DOI：
10.1002/gene.10112
复制
发表时间：
2002-09-01
期刊：
影响因子：
1.5
通讯作者：
Buchner, E
中科院分区：
文献类型：
--
作者：
Diegelmann, S;Fiala, A;Buchner, E
使用传统荧光染料对细胞钙流入进行光学成像已发展成为一种常用工具。然而，传统染料的一个缺点在于其非特异性靶向。标记细胞的选择取决于染料的扩散特性和组织的解剖结构，而不是细胞的遗传特性或功能。克服这个问题的一个可能性是基因可编码的荧光蛋白，它允许光学记录细胞的生理反应，例如钙流入（回顾，参见Guerrero和Isacoff，2001）。这些探针中最先进的是钙传感器 Cameleon（Miyawaki 等，1997），这是一种由增强型青色荧光蛋白（ECFP）和增强型黄色荧光蛋白（EYFP）组成的蛋白质，两者都与钙调蛋白序列和钙调蛋白目标肽 M13 融合（Miyawaki 等，1997）。当在 440 nm 波长下激发时，钙流入会导致 EYFP 与 ECFP 发射的比率发生变化，这是钙依赖性构象变化的结果，导致 FRET（荧光共振能量转移）从 ECFP 到 EYFP（图 1）。 迄今为止，已经报道了 Cameleon 的几个版本。最初描述的 Cameleon 2.0（Miyawaki 等人，1997）在其排放特性中显示出对 pH 值的不利依赖性，这一副作用在 Cameleon 2.1 版本（Miyawaki 等人，1999）中得到了改进。最近，据报道，一种新颖的结构 Cameleon 6.1 可以提供更强的信噪比（Truong 等，2001）。 Cameleon 已被证明可在亚细胞水平的细胞培养中发挥作用（Foyouzi-Youssefi 等人，2000 年；Emmanouilidou 等人，1999 年）以及线虫的神经元和肌肉制备物（Kerr 等人，2000 年）。在 UAS 控制下使用 Cameleon 2.0 对果蝇幼虫神经肌肉接头进行光学记录也取得了成功（Reiff 等人，未发表的研究）。我们现在报道了在 UAS 启动子控制下表达 Cameleon 2.1 的 5 个转基因果蝇品系，从而利用果蝇的优势，将传感器定位到使用特定 Gal 4 品系的基因定义的细胞群。图 2 列出了每条飞线的确切 P 元素插入位置。通过蛋白质印迹分析，我们确定了 Cam 2.1/82 线，当由强全神经元 Gal 4 驱动程序驱动时，该线显示出大脑中最强的 Cameleon 2.1 表达
Optical imaging of cellular calcium influx using conventional fluorescence dyes has developed to a commonly used tool. However, one drawback of conventional dyes lies in its unspecific targeting. The selection of cells that are labeled depends on the diffusion properties of the dye and the anatomy of the tissue rather than on the genetic identity or function of cells. A possibility to overcome this problem came with genetically encodable fluorescence proteins that allow for the optical recording of physiological reactions of cells, eg calcium influx (for review, see Guerrero and Isacoff, 2001). The most advanced among those probes is the calcium sensor Cameleon (Miyawaki et al., 1997), a protein consisting of an enhanced cyan fluorescent protein (ECFP) and an enhanced yellow fluorescent protein (EYFP), both fused to a calmodulin sequence and the calmodulin target peptide M13 (Miyawaki et al., 1997). When excited at 440 nm wavelength, calcium influx induces a shift in the ratio of EYFP to ECFP emission as a result of a calciumdependent conformational change that leads to FRET (fluorescence resonance energy transfer) from ECFP to EYFP (Fig. 1).Several versions of Cameleon have been reported so far. The initially described Cameleon 2.0 (Miyawaki et al., 1997) showed in its emission properties an unfavorable dependence on the pH value, a side effect that was improved in the version Cameleon 2.1 (Miyawaki et al., 1999). Recently, a novel construct, Cameleon 6.1, has been reported to give stronger signal to noise ratio (Truong et al., 2001). Cameleon has been shown to work in cell culture on the subcellular level (Foyouzi-Youssefi et al., 2000; Emmanouilidou et al., 1999) and in a neuronal and muscular preparation of C. elegans (Kerr et al., 2000). Optical recordings at the larval neuromuscular junction of Drosophila using Cameleon 2.0 under UAS control has also been successful (Reiff et al., unpublished research). We now report five lines of transgenic flies expressing Cameleon 2.1 under the control of a UAS promotor, thereby exploiting Drosophila’s advantage of targeting the sensor to a genetically defined population of cells using specific Gal 4 lines. Figure 2 lists the exact P-element insert location for each fly line. By Western blot analysis we determined the line Cam 2.1/82 to show the strongest Cameleon 2.1 expression in the brain when driven by the strong panneuronal Gal 4 driver