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

用于果蝇复杂细胞间相互作用研究的遗传资源

• 批准号: BB/V018477/1
• 负责人: Scott Waddell
• 金额: $ 81.48万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV018477%2F1
• 关键词: genetic; resource; complex; cell; interaction

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.

为了研究涉及发育、组织维持或复杂组织（如大脑）的高阶相互作用的过程，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）与感兴趣的基因结合来实现。在这种新的环境下，增强子将控制目标基因的表达时间和地点。由于生物体中有数千种不同的基因和更多的细胞，因此很难为每种特定操作生成特定的增强子/感兴趣的基因组合。这个问题的一个优雅的解决方案是使用二进制表达系统，它将增强子与感兴趣的基因分开。增强子现在是一个称为“驱动”的控制单元的一部分，该单元调节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结合成一个称为“应答器”的功能单元。当增强子被激活后，DNA结合蛋白被合成并结合到目标基因旁边的识别位点并触发其表达。由于这种结构，驱动因子和响应因子可以在两个不同的动物系中完全分离，只有当杂交在一起时，后代才能以期望的模式表达感兴趣的基因。这允许创建庞大的驱动程序和响应程序集合，可以自由组合以产生数千种不同的组合，而无需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，在一个细胞中上调一个基因，而在邻近细胞中下调另一个基因。为了实现这一点，需要同时使用几种不同的二进制表达式系统。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，该系统可以改变以识别不同的结合位点（VAS）。这个新的TALE-VAS系统有潜力产生几乎无限数量的驾驶员/应答者对。然而，为了方便地适用于其他研究人员，需要一个基本的驱动程序和响应程序资源。在这个项目中，我们建议产生这个基本资源，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通用的遗传工具。特别是，我们将确保所有TALE-VAS驱动程序-响应器对完全无背景。此外，我们将修改TALE驱动器，使它们可以通过抑制因子关闭，具有更快的周转率，或者与现有的拆分技术一起工作，其中驱动器可以分成两个组件，以实现更好的空间分辨率。我们还将提供一个新的拆分系统，允许同时雇用许多拆分驱动程序。同时，我们将产生供体果蝇，可用于将现有驱动程序从最广泛使用的二进制表达系统GAL4-UAS系统转换为TALE-VAS系统。最后，我们将通过生成逻辑门（“开关”）进一步扩展TALE-VAS系统的应用范围，该逻辑门只会在几个条件成立时激活感兴趣的基因。例如，只有表达基因1、基因2和基因3，或者基因1和基因2但不表达基因3时，激活才会发生。利用这个系统，研究人员将能够操纵以复杂的基因表达模式为特征的细胞群，否则很难处理。总的来说，基本的TALE-VAS工具包将极大地造福科学界，并将使研究人员能够进行前所未有的复杂的细胞-细胞相互作用研究。