IDENTIFYING BRAIN WIRING MECHANISMS BY GENE TRAPPING

哺乳动物脑发育研究中的一个主要挑战是鉴定指导脑复杂布线模式组装的细胞外配体和受体。 最近修改的基因陷阱技术在胚胎干细胞中，“分泌陷阱”，允许系统捕获的基因编码的配体和受体，并在这些基因中产生突变的小鼠，提供了一个重要的工具，阐明这些基因的功能。 然而，研究大脑线路的问题是，由于难以追踪神经元的轴突投射，因此很难识别实验扰动导致的线路变化。 特别是，它通常是非常困难的，以确定在突变小鼠的布线缺陷，无论是自发产生的突变或基因靶向或插入诱变产生的。我们已经设想了一个进一步修改的基因陷阱技术，现在应该大大促进识别和表征的受体参与布线哺乳动物神经系统。 在我们的方法中，一种组织化学轴突标记物被靶向于正常表达被捕获基因的神经元。 通过简单的组织化学染色，可以直接观察这些神经元的连接，并直接观察纯合突变动物中的布线缺陷。 通过这种方式，可以快速评估被困基因在大脑布线中的作用。同样重要的是，该方法也将同时导致在不同轴突群体中表达组织化学标记物的小鼠库的产生，这将为有兴趣阐明哺乳动物大脑中神经元连接的正常模式的研究人员提供重要工具。这里提出的要求是支持使用这种修饰的载体分离大量分泌陷阱ES细胞系。 建议每年将这些品系中的80个通过种系进行表达和表型分析，以识别参与神经系统布线的那些，并提供在不同神经元群体中表达轴突标记的小鼠资源，供神经科学界使用。 我们希望这种方法将有助于大大加快发现指导哺乳动物神经系统布线的机制的步伐。

A major challenge in the study of mammalian brain development is to identify the extracellular ligands and receptors that direct the assembly of the complex wiring pattern of the brain. A recent modification of the gene trap technique in embryonic stem cells, the "secretory trap", allows the systematic trapping of genes encoding ligands and receptors, and the generation of mice harboring mutations in these genes, providing an important tool to elucidate the functions of these genes. However, the problem with studying brain wiring is that it can be extremely difficult to identify changes in wiring resulting from experimental perturbation because of the difficulty of tracing the axonal projections of neurons. In particular, it is usually extremely difficult to identify wiring defects in mutant mice, whether the mutation arose spontaneously or was generated by gene targeting or insertional mutagenesis. We have conceived a further modification of the gene trap technique that should now dramatically facilitate the identification and characterization of receptors involved in wiring the mammalian nervous system. In our method, a histochemical axonal marker is targeted to neurons that normally express the trapped gene. This enables direct visualization of the connections made by these neurons, and direct visualization of wiring defects in homozygous mutant animals, through a simple histochemical stain. In this way the role of the trapped gene in brain wiring can be rapidly assessed. Of equal importance, and of great benefit to the Neuroscience community at large, the method will also simultaneously result in the generation of a bank of mice expressing the histochemical marker in different populations of axons, which will provide an important tool for researchers interested in elucidating the normal pattern of neuronal connections in the mammalian brain. A request is made here to support the isolation of a large number of secretory trap ES cell lines using this modified vector. It is proposed that 80 of these lines per year will be put through the germ line for expression and phenotypic analysis to identify those involved in wiring the nervous system, and to provide a resource of mice expressing the axonal marker in different populations of neurons for use by the Neuroscience community. We expect that this method will help greatly accelerate the pace of discovery of mechanisms that direct the wiring of the mammalian nervous system.