A genetic resource for complex cell-cell interaction studies in Drosophila

为了研究发育、组织维持或复杂组织（如大脑）的高阶相互作用所涉及的过程，研究人员需要能够在时间和空间上操纵特定细胞的工具。这可以通过将调控元件（增强子）与感兴趣的基因组合来实现。在这种新的背景下，增强子将控制感兴趣的基因表达的时间和地点。由于生物体中有数千种不同的基因甚至更多的细胞，因此很难为每种特定的操作产生特定的增强子/感兴趣的基因组合。这个问题的一个巧妙的解决方案是使用二元表达系统，其将增强子与感兴趣的基因分开。增强子现在是称为“驱动”的控制单元的一部分，其调节DNA结合蛋白的表达，而感兴趣的基因与DNA结合蛋白的结合位点组合成称为“应答器”的功能单元。在激活增强子后，产生DNA结合蛋白，并结合到其与感兴趣的基因相邻的识别位点，并触发其表达。由于这种结构，驱动和应答元件可以在两个不同的动物品系中完全分离，并且只有当杂交在一起时，后代才会以所需的模式表达感兴趣的基因。这允许创建大量的驱动和响应集合，其可以自由组合以产生数千种不同的组合，而不需要建立永久的转基因动物。然而，复杂的细胞-细胞相互作用研究可能需要同时进行几种不同的操作，例如一个细胞中基因的上调和相邻细胞中另一个基因的下调。为了实现这一点，需要同时采用几种不同的二进制表达系统。到目前为止，只有三个二进制系统可用，这限制了并行操作的最大数量为三个。为了克服这一限制，我们开发了一种新的基于DNA结合蛋白（TALE）的二元表达系统，该系统可以被改变以识别不同的结合位点（VAS）。这种新的TALE-VAS系统有可能产生几乎无限数量的驱动器/响应器对。然而，要容易适用于其他研究人员，司机和响应者的基本资源是必要的。在这个项目中，我们建议生成这个基本资源，并进一步增强和修改TALE-VAS系统，以便为科学界提供一个有价值的和多功能的遗传工具。特别是，我们将确保所有TALE-VAS驱动程序-响应程序对完全无背景。此外，我们将修改TALE驱动程序，使它们可以被阻遏物关闭，具有更快的周转率，或与现有的分离技术一起工作，其中驱动程序可以被分离为两个组件，以实现更好的空间分辨率。我们还将提供一个新的分裂系统，将允许同时雇用许多分裂的司机同时。同时，我们将产生供体苍蝇，可用于转换现有的驱动程序，从最广泛使用的二进制表达系统，GAL 4-UAS系统，TALE-VAS系统。最后，我们将通过生成逻辑门（“开关”）来进一步扩展TALE-VAS系统的应用范围，所述逻辑门仅在若干条件为真时激活感兴趣的基因。例如，只有当基因-1和基因-2和基因-3表达时，或者如果基因-1和基因-2但不表达基因-3，才发生激活。使用这个系统，研究人员将能够操纵以复杂基因表达模式为特征的细胞群，并且难以以其他方式解决。总的来说，基本的TALE-VAS工具包将极大地造福于科学界，并使研究人员能够进行前所未有的复杂性的细胞间相互作用研究。

To study the processes involved in development, tissue maintenance, or higher order interactions of complex tissues such as the brain, researchers need tools that allow for the manipulation of specific cells in time and space. This can be achieved by combining a regulatory element (enhancer) with a gene of interest. The enhancer, in this new context, will then control when and where the gene of interest is expressed. Since there are thousands of different genes and even more cells in an organism, it would be very difficult to generate a specific enhancer/gene of interest combination for each particular manipulation. An elegant solution to this problem, is the use of binary expression systems which separate the enhancer from the gene of interest. The enhancer is now part of a control unit called "driver" which regulates the expression of a DNA binding protein while the gene of interest is combined with binding sites for the DNA binding protein into a functional unit termed "responder". Upon activation of the enhancer, the DNA binding protein is made and binds to its recognition sites next to the gene of interest and triggers its expression. Due to this architecture, driver and responder elements can be completely separated in two different animal lines and only when crossed together will the offspring express the gene of interest in the desired pattern. This allows for the creation of vast driver and responder collections which can be freely combined to generate thousands of different combinations without the need of establishing permanent transgenic animals. However, complex cell-cell interaction studies may require several different manipulations at the same time, e.g. the up-regulation of a gene in one cell and the down-regulation of another gene in the neighbouring cell. To achieve this, several different binary expression systems need to be employed simultaneously. To date, only three binary systems are available, which limits the maximum number of parallel manipulations to three. To overcome this limitation, we have developed a new binary expression system based on a DNA binding protein (TALE) that can be altered to recognise different binding sites (VAS). This new TALE-VAS system has the potential to generate an almost unlimited number of driver/responder pairs. However, to be readily applicable for other researchers, a basic resource of drivers and responders is needed. In this project, we propose to generate this basic resource and to further enhance and modify the TALE-VAS system in order to provide a valuable and versatile genetic tool for the scientific community. In particular, we will ensure that all TALE-VAS driver-responder pairs are completely background-free. Additionally, we will modify the TALE drivers so that they can be turned off by a repressor, have a faster turnover rate, or work with the existing split technology where the driver can be separated into two components to achieve better spatial resolution. We will also provide a new split system that will allow for the simultaneous employment of many split drivers simultaneously. In parallel, we will generate donor flies that can be used to convert existing drivers from the most widely used binary expression system, the GAL4-UAS system, to the TALE-VAS system. Finally, we will further extend the range of applications of the TALE-VAS system by generating logic gates ("switches") that will only activate the gene of interest if several conditions are true. For example, activation occurs only if gene-1 AND gene-2 AND gene-3 are expressed, or if gene-1 AND gene-2 but NOT gene-3 are expressed. Using this system, researchers will be able to manipulate cell populations that are characterised by complex gene expression patterns and are difficult to address otherwise. Taken together, the basic TALE-VAS toolkit will greatly benefit the scientific community and will enable researchers to perform cell-cell interaction studies of unprecedented complexity.