Advancing tools for inducing gene activity in the CNS.

描述（由申请人提供）：这项为期2年的R21申请的目的是通过产生可诱导工具来扩展Flp-FRT位点特异性重组酶系统（由我的实验室在小鼠中开创）的能力，该工具将允许在胚胎和成年小鼠中产生时间控制的遗传修饰;特别是，可以与组成型或诱导型Cre-loxP基试剂平行使用的工具。这种产生的双重重组酶能力目前不可用，并且将代表重要的进步，允许工程化连续基因修饰作为模拟多步发育或成人疾病过程的手段，或者同时基因修饰作为模拟多基因疾病的手段。这种先进的能力，暂时调节重组将是特别重要的研究过程中发生在胚胎晚期和成人阶段，这些过程最受限制的标准（非诱导）条件的方法。所提出的实验集中于产生两种类型的诱导Flp工具，一种广泛活跃，另一种组织特异性。这些实验来自我们先前的结果，表明Flp变体Flpe在小鼠中与Cre一样有效，并且来自其他人的工作，表明类固醇受体与Cre融合能够对重组进行时间调节。在目标1中，我们将向小鼠中引入广泛诱导的Flpe转基因，该转基因能够在胚胎和成年生活中的选定时间在不同组织的细胞中催化遗传变化。在目标2中，我们将向小鼠中引入能够在神经管中以空间和时间调节的方式介导遗传修饰的组织特异性诱导型Flpe转基因。这些小鼠将允许遗传修饰的特定子集的细胞谱系，其区别在于它们的时间出现从背神经管。此外，它们将作为其他组织特异性诱导型Flpe转基因的原型。 虽然所提出的工具将提供新的能力来描绘过程的发展和许多器官系统的健康，我们特别感兴趣的中心脑干谱系，特别是那些谱系，相继出现的菱形唇区域的背后脑。我们计划将所提出的工具与Cre-loxP-containing试剂一起应用，以操纵“晚出生”的菱形唇谱系，这对小脑前传入系统（一组对运动控制至关重要的不同核团）和延髓中缝系统（一个稳态控制中心，其中发育异常与婴儿猝死综合征有关）的形成至关重要。

DESCRIPTION (provided by applicant): The objective of this 2-year R21 application is to extend the capabilities of the Flp-FRTsite specific recombinase system (pioneered in mice by my laboratory) by generating inducible tools that will permit the generation of temporally controlled genetic modifications in embryonic and adult mice; notably, tools that can be used in parallel with either constitutive or inducible Cre-loxP-based reagents. This resulting dual recombinase capability is not presently available and would represent an important advance, permitting the engineering of either successive gene modifications as a means to model multistep developmental or adult disease processes or simultaneous gene modifications as a means to model multigene disorders. This advanced capability to temporally regulate recombination will be especially important for study of processes occurring at late embryonic and adult stages, those processes most limited by standard (non-inducible) conditional approaches. The proposed experiments focus on generating two types of inducible Flp tools, one broadly active and the other tissue-specific. These experiments follow from our previous results showing that the Flp variant, Flpe, is as effective in mice as Cre, and from the work of others showing that steroid-receptor fusions with Cre enable temporal regulation of recombination. In aim 1, we will introduce into mice widely inducible Flpe transgenes capable of catalyzing genetic changes in cells of diverse tissues at chosen times in embryonic and adult life. In aim 2, we will introduce into mice a tissue-specific inducible Flpe transgene capable of mediating genetic modifications in both a spatially and temporally regulated fashion in the neural tube. These mice will permit the genetic modification of specific subsets of cell lineages, distinguished by their temporal emergence from the dorsal neural tube. Moreover, they will serve as a prototype for other tissue-specific inducible Flpe transgenics. While the proposed tools will provide new capabilities for delineating processes critical to the development and health of many organ systems, our particular interest centers on brainstem lineages, in particular, those lineages that emerge successively from the rhombic lip region of the dorsal hindbrain. We plan to apply the proposed tools in conjunction with Cre-loxP-containing reagents to manipulate "late-born" rhombic lip lineages critical to the formation of both the precerebellar afferent system, a set of diverse nuclei essential for motor control, and the medullary raphe system, a center for homeostatic control in which developmental abnormalities have been implicated in sudden infant death syndrome.