Single Cell Electroporation

描述（由申请人提供）：广泛的长期目标：开发和应用控制，干扰和询问功能单细胞的工具。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称为“电穿孔”的现象-使用电场在细胞膜上产生微小的瞬时孔。具体目标：1。单细胞电穿孔的定量和定性理解。确定控制膜孔隙率、孔重新密封动力学、孔径和单细胞细胞活力的实验参数和细胞特性。测试诱导膜孔隙率取决于某些细胞特性以及所施加的场的假设。2.受体的细胞内滴定和酶活性的测量。结合联合收割机微流控方法和单细胞电穿孔，以获得细胞内物质（主要是酶和受体）与外部应用的细胞不渗透配体和底物之间相互作用的热力学和动力学数据。为阐明细胞内信号网络动力学提供手段。3.器官型海马培养物中的局灶性电穿孔和电泳分析。其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。电穿孔大鼠器官型海马培养物的小区域（低于100 μ m），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，以相同的空间分辨率测定脑组织中所有细胞中的保护性化学物质谷胱甘肽的细胞内浓度。健康相关性：负责对药物、癌症和压力反应的细胞过程通常依赖于蛋白质、DNA和RNA的复杂相互作用网络。所描述的工具使研究人员能够以定量的方式更深入、更轻松地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.