Single Cell Electroporation

描述(由申请人提供)：广泛的、长期的目标：开发和应用控制、干扰和审问功能单一细胞的工具。开发这些工具来了解组织对氧化应激的反应。这些工具是基于一种被称为“电穿孔”的现象--利用电场在细胞膜上制造微小的、瞬时的小孔。具体目标：1.对单细胞电穿孔的定量和定性认识。确定控制单个细胞的膜孔隙率、孔再密封动力学、孔大小和细胞存活率的实验参数和细胞特性。验证了诱导膜孔隙率取决于某些细胞特性和外加电场的假设。2.细胞内受体滴定和酶活性测定。结合微流控方法和单细胞电穿孔，以获得细胞内物种之间相互作用的热力学和动力学数据，主要是酶和受体，以及外部应用的细胞无关配体和底物之间的相互作用。提供了阐明细胞内信令网络动力学的方法。3.海马器型培养中的焦点电穿孔和电泳谱分析。技术目标是开发改变生物过程的工具，并以良好的空间分辨率定量测量组织中这些改变的结果。电穿孔小区域(低于100？m)的大鼠海马器型培养，以研究对氧化应激的反应，从而更好地防御应激源。在芯片上开发在线毛细管电泳法，以相同的空间分辨率测定脑组织中谷胱甘肽的细胞内浓度，谷胱甘肽是所有细胞中的一种保护性化学物质。与健康相关：负责对药物、癌症和压力做出反应的细胞过程通常依赖于蛋白质、DNA和RNA组成的复杂的相互作用网络。所描述的工具使研究人员能够更深入、更轻松地以定量的方式研究这些关键的细胞事件。

DESCRIPTION (provided by applicant): Broad, Long Term Objectives: Develop and apply tools that control, perturb and interrogate functioning single cells. Develop these tools for understanding a tissue's response to oxidative stress. These tools are based on a phenomenon called 'electroporation' - the creation of tiny, transient pores in the cell membrane using an electric field. Specific Aims: 1. Quantitative and qualitative understanding of single-cell electroporation. Determine the experimental parameters and cellular properties that control the membrane porosity, pore resealing dynamics, pore size, and cell viability of single cells. Test the hypothesis that the induced membrane porosity depends on certain cellular characteristics as well as the applied field. 2. Intracellular titration of receptors and measurement of enzyme activity. Combine microfluidic methods and single-cell electroporation in order to obtain thermodynamic and kinetic data on interactions between intracellular species, mainly enzymes and receptors, and externally applied, cell impermeant ligands and substrates. Provide means for elucidation of intracellular signaling network dynamics. 3. Focal electroporation and electrophoretic analysis in organotypic hippocampal cultures. The technical aim is the development of tools to alter biological processes and measure quantitatively the results of those alterations in tissue with good spatial resolution. Electroporate small (below 100 ¿m) regions of rat organotypic hippocampal cultures to study the response to oxidative stress leading to better defense against stressors. Develop on-line capillary electrophoresis on a chip to determine the intracellular concentration of glutathione, a protective chemical in all cells, in brain tissue at the same spatial resolution. Health relatedness: Cellular processes responsible for response to drugs, cancer, and response to stress often rest on complex interacting networks of proteins, DNA and RNA. The tools described empower researchers to study in more depth and with more ease these key cellular events in a quantitative way.