Single Cell Electroporation

描述(申请人提供)：电穿孔是一种在细胞膜上产生瞬时毛孔的技术。它主要用于转染，并应用于细胞悬液。单细胞电穿孔也用于转基因，但在单细胞上，通常是悬浮状态。这个项目解决了在不牺牲单细胞的情况下对单细胞进行分析化学的需要。由于单细胞电穿孔在细胞膜上产生了瞬时孔道，因此它是获取细胞质内容物样本的极佳方法。脱离上下文的细胞，例如自然贴壁细胞的悬浮液可能不代表它们的自然状态，因此该项目侧重于贴壁细胞和组织，而不是悬浮细胞。我们最近发现，培养中的贴壁细胞非常健壮。细胞即使在细胞质中失去了相当一部分低分子质量的溶质后仍能存活。我们还发现，我们可以控制单细胞电穿孔条件，以便细胞质中所需的低分子质量溶质的一部分，例如20%，通过瞬时毛孔扩散。这一观察结果为在不杀死单个细胞的情况下获取样本提供了基础。在这个项目中，我们将开发用于单细胞生化研究的重要工具。一种工具将能够以高空间分辨率灌流单个贴壁细胞，并同时电穿孔灌流的细胞。然后，我们可以详细地了解进入或离开单胞的溶质的质量传输率。另一种方法将被开发用于对培养的海马区组织中的单个细胞进行测量。它将被应用于一个与中风和类似事件相关的重要问题，在这些事件中，流向大脑某一区域的血液暂时失去。我们将建立这种方法来确定在海马区培养的单个神经元中重要的谷胱甘肽氧化还原系统的状态。这包括通过电穿孔和基于微流控的衍生化、分离和定量获得细胞质内容物。我们还将开发一种方法来降低星形胶质细胞通过缝隙连接相互交流的能力，这种方法是基于对产生缝隙连接的蛋白质的siRNA的焦点电穿孔。我们将验证这一假设，即在缺氧/葡萄糖剥夺后，相邻星形胶质细胞之间的溶质运输对于维持神经元谷胱甘肽水平是重要的。

DESCRIPTION (provided by applicant): Electroporation is a technique that creates transient pores in cell membranes. It is mostly used for transfection, and applied to suspensions of cells. Single-cell electroporation is also used for transfection but on single cells, typically in suspension. This project addresses the need to do analytical chemistry on single cells without sacrificing them. As single-cell electroporation creates transient ports in cell membranes, it is an excellent approach to obtaining samples of cytoplasmic contents. Cells taken out of their context, e.g. suspensions of naturally adherent cells may not be representative of their natural state, so the project focuses on adherent cells and tissues, not on suspended cells. We have recently found that adherent cells in culture are remarkably robust. Cells survive even after losing a significant fraction of the low-molecular weight solutes in the cytoplasm. We have also found that we can control single-cell electroporation conditions so that a desired fraction of the low-molecular weight solutes in the cytoplasm, e.g., 20%, diffuses through the transient pores. This observation provides the foundation for obtaining samples from single cells without killing them. In this project, we will develop significant tools for single-cell biochemical investigations. One tool will be able to perfuse single adherent cells with high spatial resolution and simultaneously electroporate the perfused cell. We can then learn in detail the mass transport rates for solutes entering or leaving single cells. Another method will be developed for making measurements on single cells in cultured hippocampal tissue. It will be applied to an important question related to stroke and similar incidents in which blood flow to a region of the brain is temporarily lost. We will establish this method for determining the status of the important glutathione redox system in a single neuron in a hippocampal culture. This includes obtaining cytoplasmic contents by electroporation and microfluidic-based derivatization, separation, and quantitation. We also will develop a means to diminish the astrocytes' ability to communicate with each other through gap junctions based on focal electroporation of siRNA for the protein that creates the gap junctions. We will test the hypothesis that solute transport between adjacent astrocytes is important for maintenance of neuronal glutathione levels following oxygen/glucose deprivation.