Microfluidic Techniques for the Molecular and Functional Analysis of Gene Express

实体肿瘤中的癌症干细胞最近被分离出来，似乎是导致 疾病的生长和传播。肿瘤中干细胞群体的存在对 癌症的诊断和治疗，因为必须以这些癌症干细胞为靶点才能实现治愈。 初步证据表明，癌症干细胞有许多差异表达的基因。 细胞及其非致瘤后代。这些基因中的许多被认为在基础癌症中起作用。 功能包括增殖、存活、自我更新和对标准疗法的抵抗。揭开面纱 白血病、乳腺癌中表面均一的干细胞群体的真正功能分层 和其他实体癌症是一个挑战，需要在单细胞水平上进行新的测量。 根据克拉克和魏斯曼研究小组最近的发现，我们认为 正常干细胞以及癌症干细胞在表观遗传水平上受到显著调控。表观遗传 基因表达的调节部分是由组蛋白的翻译后修饰控制的，如 以及DNA的甲基化，两者都会导致染色质结构的改变。这些修改 用染色质免疫沉淀(CHIP)法检测。表观遗传学分析是必要的 补充基因表达分析以了解正常和肿瘤干细胞的控制 自我更新，发现新的治疗靶点。由地震实验室开发的微流控工具 单细胞基因表达(AIM1)、芯片(AIM 2)和体外细胞高通量培养(AIM 3)将使 Clarke和Weissman实验室将对稀有的、纯化的 来自模型鼠和原发人类癌症或肿瘤异种移植的细胞。这些分析都将使用微流控技术 已经在地震实验室开发和验证的平台或已投入商业使用的平台 可用。在这个项目过程中，还将根据需要设计新的系统。微流控表观遗传学 基因分析将允许对高度纯化的、同质的、稀有的细胞群体进行研究 以前无法使用标准技术访问。微流控细胞培养平台将允许发现 肿瘤干细胞体外培养的适宜条件及新的治疗靶点

Cancer stem cells in solid tumors have been recently isolated and appear to be primarily responsible for the growth and spread of the disease. The presence of a stem cell population in a tumor has implications for the diagnosis and treatment of cancer, as it is these cancer stem cells that must be targeted to achieve a cure. Preliminary evidence demonstrates that there is a host of genes differentially expressed by the cancer stem cells and their non-tumorigenic progeny. Many of these genes are thought to play a role in essential cancer functions including proliferation, survival, self renewal and resistance to standard therapeutics. Uncovering the true functional stratification of the superficially uniform population of stem cells in leukemia, breast cancer and other solid cancers is a challenge which requires new kinds of measurements at the single-cell level. Based on recent discoveries by the Clarke and Weissman groups, we believe that gene expression in normal stem cells, as well as cancer stem cells is significantly regulated at the epigenetic level. Epigenetic regulation of gene expression is in part controlled by the posttranslational modification of histone proteins, as well as methylation of DMA, both of which result in the alteration of chromatin structure. These modifications are examined using Chromatin immunoprecipitation (ChIP) assays. Epigenetic analysis is a necessary complement to gene expression analysis in order to understand the control of normal and cancer stem cell self-renewal and discover new therapeutic targets. The microfluidic tools developed by the Quake lab for single cell gene expression (aim1), chip (aim 2) and high throughput in vitro cell culture (aim 3) will allow the Clarke and Weissman laboratories to perform gene expression and epigenetic analyses on rare, purified cells from model mice and primary human cancer or cancer xenografts. These assays will all use microfluidic platforms which have already been developed and validated in the Quake laboratory or are commercially available. New systems will also be designed as needed in the course of this project. Microfluidic epigenetic and genetic assays will allow the study of highly purified, homogeneous, rare cell populations that were previously inaccessible with the standard techniques. Microfluidic cell culture platforms will allow finding appropriate conditions to culture cancer stem cells in vitro and test new therapeutic targets