Next Generation Brainbow Transgenes for Neural Circuit Analysis

神经科学的一个中心目标是绘制哺乳动物大脑中的神经回路，以了解它们如何解释心理活动和行为，以及它们的改变如何导致神经和精神疾病。最近，我们开发了Brainbow转基因小鼠，这有助于追踪这种电路。通过以一种新的方式使用Cre-lox重组系统，他们将中枢和外周神经系统中的单个神经元着色为100种不同的色调之一。许多研究人员计划使用Brainbow线，现在在一个公共存储库中，用于追踪正常小鼠和人类疾病小鼠模型的神经元连接。尽管如此，Brainbow方法目前在几个方面还不完善。因此，我们建议产生，评估和分发第二代Brainbow转基因品系，规避第一代的限制。(1)为了扩大Brainbow方法可以应用的细胞类型和发育阶段的范围，我们将使用其他调控元件产生新的细胞系。(2)为了扩大光谱并克服光不稳定性，我们将测试新的荧光蛋白，以及一种表位标记策略，使Brainbow能够跨越从深蓝色到远红色的光谱。(3)在迄今为止生成的Brainbow线中，颜色用于区分一个神经元与另一个神经元，但颜色选择完全是任意的。我们将生成新的线条，其中颜色用于识别神经元类型以及区分类型内的单个神经元。(4)使用新的蛋白质和脂氧合酶组合，我们将产生新的品系，其中表达了数百种色调。(5)我们将生成轴突和树突乔木都被完全标记的线，并且还生成突触位点被突出显示的线。在4年的过程中，我们建议产生和测试大约200个转基因株系（40个构建体中的每一个5个）。我们相信，这一计划将规避许多，如果不是所有的限制以上列举。我们将使所有结果（包括正面和负面）和构造公开可用，并且存款在公共存储库中构造最有希望的行，因此它们可以广泛分布。

A central aim of neuroscience is to map neural circuits in the mammalian brain, in order to learn how they account for mental activities and behaviors, and how their alterations lead to neurological and psychiatric disorders. Recently, we developed Brainbow transgenic mice, which facilitate tracing of such circuits. By using the Cre-lox recombination system in a novel way, they color individual neurons in both central and peripheral nervous systems in one of-100 distinct hues. Numerous investigators plan to use Brainbow lines, now in a public repository, for tracing neuronal connectivity in normal mice and in mouse models of human disease. Nonetheless, the Brainbow method is presently imperfect in several respects. We therefore propose to generate, evaluate and distribute second-generation Brainbow transgenic lines that circumvent limitations of the first generation. (1) To expand the range of cell types and developmental stages to which Brainbow methods can be applied, we will generate new lines using other regulatory elements. (2) To expand the spectrum and overcome photoinstability, we will test new fluorescent proteins, as well as an epitope tagging strategy that will allow Brainbow to span the spectrum from deep blue to far red. (3) In the Brainbow lines generated so far, colors are used to distinguish one neuron for another, but color choice is totally arbitrary. We will generate new lines in which colors serve to identify neuronal type as well as to distinguish individual neurons within a type. (4) Using new proteins and lox combinations, we will generate new lines in which many 100s of hues are expressed. (5) We will generate lines in which both axonal and dendritic arbors are fully labeled, and also generate lines in which synaptic sites are highlighted. Over the course of 4 years, we propose to generate and test approximately 200 transgenic lines (5 from each of 40 constructs). We believe this program will circumvent many if not all of the limitations enumerated above. We will make all results (both positive and negative) and constructs publicly available, and deposit constructs the most promising lines in a public repositories, so they can be broadly distributed.