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精神活动和行为，以及它们的变化如何导致神经和精神障碍。最近，我们研制出了脑弓转基因小鼠，可以方便地追踪这种回路。通过以一种新颖的方式使用Cre-lox重组系统，他们将中枢和周围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脑弓线追踪正常小鼠和人类疾病小鼠模型中的神经元连接，目前该线已在公共储存库中。尽管如此，脑弓方法目前在几个方面还不够完善。因此，我们建议产生、评估和分发第二代Brainbow型转基因株系，以绕过第一代的限制。(1)为了扩大脑弓方法可以应用到的细胞类型和发育阶段的范围，我们将使用其他调节元件产生新的系。(2)为了扩展光谱和克服光不稳定性，我们将测试新的荧光蛋白质，以及一种表位标记策略，该策略将使Brain弓能够跨越从深蓝色到远红色的光谱。(3)在迄今产生的脑弓线中，颜色被用来区分一个神经元和另一个神经元，但颜色的选择完全是随意的。我们将生成新的线条，在这些线中，颜色用于识别神经元类型以及区分类型中的单个神经元。(4)使用新的蛋白质和lox组合，我们将产生表达数百种色调的新品系。(5)我们将生成轴突和树突都被完全标记的线条，以及突触位置突出的线条。在4年的时间里，我们建议产生和测试大约200个转基因株系(40个构建物中的每个5个)。我们相信，该计划将绕过上面列举的许多(如果不是全部)限制。我们将公开所有的结果(包括积极的和消极的)和构造，并将构造存放在公共存储库中最有希望的行，这样它们就可以广泛分布。

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.