Generation and validation of inducible Cre driver lines by enhancer trapping

描述（由申请人提供）：小鼠中用于在时间和空间维度上操纵基因表达的Cre驱动系的有限可用性严重阻碍了生物医学研究进展。研究人员需要这些细胞系才能在正常小鼠中进行基因过表达、基因错误表达和基因沉默实验，以及在突变生物体中进行基因拯救实验。尽管某些基因的确定的调控元件原则上可用于驱动Cre表达并控制空间维度，但用于表达天然基因的调控元件众多、复杂且难以分解成单个元件。此外，在小鼠脑中在非常有限的空间域中表达的基因非常少，使得经典敲入方法预计不能提供精确的表达空间特异性。另一种由研究其他模式生物（特别是果蝇）的研究人员开发的策略是增强子捕获。在该策略中，携带可促进感兴趣基因表达的最小启动子的DMA区段被随机整合到转基因动物的基因组中，使得任何随机整合子篡夺附近增强子的活性，并且这导致感兴趣基因根据附近增强子的决定簇在时间和空间上表达。这些策略已经用数千种增强子捕获系填充果蝇研究领域，所述增强子捕获系在生物体的几乎任何组织和细胞类型中表达Gal 4或诱导型Gal 4衍生物，并且这些试剂是无数发现的基础。我们在这里提出了一个类似的策略在小鼠。我们将在未来5年内构建和验证超过2500个新的增强子陷阱线在小鼠中携带诱导形式的Cre。我们预计，这些细胞系中的1%到5%将在时间和空间上在大脑中显示特定的Cre表达。该项目将在贝勒医学院进行，该学院以其对小鼠遗传学的重视和卓越而闻名。该项目将包括在果蝇中开发新的和可诱导的增强子陷阱载体并使用这些载体进行脑表达模式大屏幕的研究人员，构建携带Cre诱导型的新增强子陷阱载体的杰出分子生物学家，领导构建数百种新小鼠转基因/敲除模型的小鼠遗传学家，以及使用高通量、基于机器人的工作站进行RNA原位杂交和其他类型的组织学程序对小鼠大脑中的基因表达进行组织学分析的专家。与首先定义适当的调控元件和/或将Cre敲入定义的基因的策略相比，使用基于随机增强子捕获的方法获得提供实验者对表达以及时间和空间的控制的新Cre驱动系的效率将大得多。这是从果蝇遗传学中得到的教训，我们有充分的理由期待它在老鼠身上得到应用。

DESCRIPTION (Provided by Applicant): The limited availability of Cre driver lines in the mouse for manipulating the expression of genes in the dimensions of both time and space has severely hampered biomedical research progress. These lines are required for investigators to be able to perform gene overexpression, gene misexpression, and genesilencing experiments in the normal mouse as well as gene-rescue experiments in mutant organisms. Although defined regulatory elements of some genes could, in principle, be used to drive Cre expression with control over the spatial dimension, the regulatory elements that are used to express native genes are numerous, complex, and difficult to dissect into individual elements. In addition, there are very few genes that are expressed in the mouse brain in a very limited spatial domain, such that classical knock in approaches are not anticipated to provide precise spatial specificity on expression. An alternative strategy developed by investigators working with other model organisms, particularly Drosophila, is enhancer trapping. In this strategy, a DMA segment carrying a minimal promoter that can promote the expression of a gene of interest is integrated into the genome at random in transgenic animals, so that any random integrant usurps the activity of a nearby enhancer and this causes the gene of interest to be expressed in time and space according to the determinants of the nearby enhancer. Such strategies have populated the Drosophila research field with thousands of enhancer trap lines that express Gal4 or inducible Gal4 derivatives in virtually any tissue and cell type of the organism and these reagents are the basis for uncountable discoveries. We propose here, a similar strategy in the mouse. We will construct and validate over the next 5 years more than 2500 new enhancer trap lines in the mouse that carry inducible forms of Cre. We anticipate that between 1 and 5 percent of these lines will show specific Cre expression in the brain in both time and space. The project will be performed at the Baylor College of Medicine, known for its emphasis on and excellence in mouse genetics. The project will include investigators that have developed new and inducible enhancer trap vectors in Drosophila and used these to perform large screens for brain expression patterns, outstanding molecular biologists to construct new enhancer trap vectors carrying inducible versions of Cre, mouse geneticists that have led in the construction of hundreds of new mouse transgenic/knock-out models, and experts in the histological analysis of gene expression in the mouse brain using high-throughput, robotically-based workstations for RNA in situ hybridization and other types of histological procedures. The efficiency of obtaining new Cre driver lines offering experimenter control over expression and time and space will be much greater using the random enhancer trapping-based approach compared to strategies for first defining appropriate regulatory elements and/or knocking Cre into defined genes. This is the lesson learned from Drosophila genetics and there is every reason to expect it to hold in the mouse.