Novel Gene Expression Assay in Living Neuronal Subtypes

描述(申请人提供)：在许多疾病中，细胞亚群的功能被破坏，例如帕金森氏病中含有多巴胺的神经元选择性死亡，或在健康细胞中变成癌症的细胞亚群。人体组织是复杂的不同细胞类型的混合物，每种细胞都有自己的基因表达模式，以及伴随而来的生物学功能和对疾病的脆弱性。比较扮演不同生物学作用或具有不同疾病脆弱性的细胞中的差异基因表达谱，将解释基因表达是如何产生这些差异的。当前技术的局限性阻碍了这一重要目标的实现。我们需要基因表达标记物来识别细胞亚群，以针对这些细胞进行单独的生物学研究。其他分析需要许多细胞，如生化测试和确定整个基因表达谱的测试。因此，我们还需要技术来收集许多表达亚型标记的细胞。我们在这里建议结合两种成熟的技术来寻找细胞亚型的基因表达标记，并根据基因表达收集细胞种群。神经元的亚型通常通过其特征形状来识别，因此寻找神经元亚型标记的技术将把细胞的形状与其基因标记的表达联系起来。这将通过基因枪同时传递荧光染料和仅在其目标mRNA存在的情况下发光的分子来实现。染料将填充神经元以照亮其形状，而基因表达标记将指示其目标基因是否在该细胞中表达。然后，这些标记可以被用来针对个别研究的细胞亚型。此外，组织中标记和未标记细胞的混合物可以使用荧光激活的细胞分类成组来分离和分类。这种单个亚型细胞纯化群体的收集将使生化和基因表达谱研究成为必要，以了解基因表达如何导致每个细胞亚型的独特生物学功能或功能障碍。这些新技术的发展将彻底改变基因表达如何产生生物差异的研究。

DESCRIPTION (provided by applicant): In many diseases the function of subsets of cells is disrupted, e.g. the selective death of dopamine-containing neurons in Parkinson's disease, or the subsets of cells that become cancerous amidst healthy cells. The tissues of the body are complex heterogeneous mixtures of cell types, each with its own gene expression pattern, and concomitant biological function and vulnerability to disease. Comparing the differential gene expression profiles in cells that play different biological roles or have different vulnerability to disease would elucidate how gene expression generates those differences. The limitations of current technologies prevent the achievement of this important goal. We need gene expression markers to identify subpopulations of cells to target those cells for individual biological study. Other assays require many cells, such as biochemical tests and tests to determine an entire gene expression profile. Therefore, we also need techniques to collect many cells that express a subtype marker. We propose here to combine two established technologies to find gene expression markers of subtypes of cells, and to collect populations of cells based on gene expression. Subtypes of neurons are often identified by their characteristic shapes, therefore the technology to find markers for neuron subtypes will relate the shape of a cell to its expression of the gene marker. This will be accomplished by gene gun delivery of fluorescent dye simultaneously with molecules that fluoresce only in the presence of their target mRNA. The dye will fill the neurons to illuminate their shapes, while the gene expression marker will indicate whether its target gene is expressed in that cell. These markers can then be used to target subtypes of cells for individual study. Furthermore, the mixture of marked and unmarked cells in a tissue can be dissociated and sorted using fluorescence-activated cell sorting into groups. This collection of purified populations of cells of individual subtypes will enable the biochemical and gene expression profile studies necessary to understand how gene expression leads to the unique biological function or dysfunction of each cell subtypes. The development of these novel techniques will revolutionize the study of how gene expression generates biological differences.