METHODS FOR MAPPING GENE EXPRESSION IN THE CNS

人类大脑由大约1000亿个神经元组成，这些神经元精确地组织成网络，产生诸如情感、学习和记忆以及感知等认知现象。不幸的是，对于许多被认为存在于中枢神经系统中的神经元细胞类型，实际上没有独特的标记。事实上，我们也不知道到底存在多少种不同的细胞类型。我们的长期目标是通过阐明区域-或细胞类型-基因表达的特定模式-大脑的分子解剖来量化大脑区域和神经元细胞类型的多样性。为了实现这一目标，我们的目标是(1)开发在确定的大脑区域内绘制基因表达的方法，(2)开发确定脊椎动物中枢神经系统中神经元身份和标记基因的方法。第一个目标包括改进我们开发的一种新的mRNA扩增程序，使人们能够从少量组织中构建cDNA文库，从而使原始mRNA长度的表示与由未扩增的RNA制成的最佳cDNA文库相当。在DNA微阵列杂交中使用这种改进的mRNA扩增程序，还将开发用于绘制CNS小区域内基因表达的方法。第二个目标包括在异质神经元群体中开发定义神经细胞类型和细胞类型特异性标记基因的方法。这将通过使用微阵列来表征单个细胞的基因表达模式来完成。将开发方法来扩增单细胞的mRNA含量以杂交到微阵列，并将开发统计方法来分析和比较来自多个实验的单细胞微阵列杂交数据。这些后一种方法将允许定义神经元细胞身份以及基于复杂基因表达模式的新标记基因。我们绘制中枢神经系统基因表达图谱的方法将使研究人员能够识别在健康和患病组织的小脑区域功能中发挥作用的基因和基因变异。此外，我们鉴定标记基因的方法最终将使单个神经元类型被分离或靶向研究。

The human brain comprises approximately 100 billion neurons precisely organized into networks that give rise to cognitive phenomena such as emotion, learning and memory, and perception. Unfortunately, there are virtually no unique markers for the many neuronal cell types thought to exist in the CNS. Indeed, we also do not know how many different cell types actually exist. Our long-term goal is to quantitate the diversity of brain regions and neuronal cell types by elucidating region- or cell type- specific patterns of gene expression - the molecular anatomy of the brain. In order to achieve this goal, we aim (1) to develop methods for mapping gene expression within identified brain regions, and (2) to develop methods for defining neuronal identity and marker genes in the vertebrate CNS. The first aim includes improving a novel mRNA amplification procedure we have developed that enables one to construct cDNA libraries from small amounts of tissue, such that the representation of original mRNA lengths is comparable to the best cDNA libraries made from unamplified RNA. Methods will also be developed for mapping gene expression within small regions of the CNS using this improved mRNA amplification procedure in DNA microarray hybridizations. The second aim includes developing methods for defining neuronal cell types and cell-type specific marker genes in heterogeneous neuronal populations. This will be done by using microarrays to characterize gene expression patterns of single cells. Methods will be developed to amplify the mRNA content of single cells to hybridize to microarrays, and statistical methods will be developed for analyzing and comparing single-cell microarray hybridization data from multiple experiments. These latter methods will allow the definition of neuronal cell identities as well as novel marker genes based upon complex gene expression patterns. Our methods to map gene expression in the CNS will enable researchers to identify genes and gene variants that are playing a role in the function of small brain regions in both healthy and diseased tissue. In addition, our methods to identify marker genes will eventually allow individual neuronal types to be isolated or targeted for study.