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Time lapse nano-analysis of single cell components : Development of Harvesting, Identification and Injection Methods of Functional Molecules

单细胞成分的延时纳米分析：功能分子的收获、鉴定和注射方法的发展

基本信息

批准号：
15101004
负责人：
IKAI Atsushi
金额：
$ 67.81万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

Atomic force microscopy was used to develop the technology of nano-meter, nano-newton level manipulation of live cells and biological macromolecules such as proteins and DNA. The aim of this development effort is in "cell surgery" meaning that we would acquire the tools and means to monitor the time dependent process of the biochemistry of the interior of a living cell and to change the properties of the cell by inserting functional biomacromolecules into the cell. For this purpose we have done the following experiments and obtained the results as explained.1.Analysis of force curves of protein pull-out from the surface of a living cell.The modified AFM probes were used to pull out the membrane protein from the surface of live cells. Force curves obtained in such pulling experiments showed a long stretch of the cell membrane most likely corresponding to the lipid tether formation followed by eventual extraction of targeted protein. The characteristics of force curves were classified in … More to, 1) pulling membrane proteins without linkage with the cytoskeleton and other intracellular structures, and 2) pulling those with linkage to the cytoskeletons. The model experiments were performed on the red blood cell using specific lectins towards glycophorin A as the example of the former type, and to Band 3 an the example of the latter type of membrane proteins. In the former case of no linkage to cytoskeleton, force curves were characterized by the extension of a plateau force of about 70 pN ending with a single step jump to zero force level. Whereas, in the latter case, force curves were characterized by the appearance of multiple force peaks greater than 100 pN before and after the extension of plateau force. We proposed that the two types of force curves could be used to monitor the linkage status of membrane protein with the intracellular cytoskeletal structure.2.Extraction of mRNA and insertion of plasmid DNA.AFM probes were used to extract mRNA and other functional macromolecules from the cytoplasm of living cells. A probe that was pushed into a targeted locus of the cell was used as the starting material for RT-PCR and PCR amplification of the harvested mRNA on the probe. mRNA localization in a very limited position of a live cell can be routinely analyzed for its identity and quantification. This method will be useful for the time dependent monitoring of the production of specific proteins in different parts of a living cell. By using an AFM probe with adsorbed plasmid DNA, we showed that a number of cells could be transfected with the plasmid DNA having the gene of green fluorescent protein as a marker. Less

原子力显微镜被用来发展对活细胞和生物大分子(如蛋白质和DNA)的纳米、纳米牛顿水平的操纵技术。这项研究工作的目的是“细胞外科”，这意味着我们将获得工具和手段，以监测活细胞内部的生物化学过程的时间依赖过程，并通过将功能生物大分子插入细胞中来改变细胞的性质。为此，我们做了以下实验，得到了如下结果：1.活细胞表面蛋白质拔出的力曲线分析，用改进的AFM探针从活细胞表面拔出膜蛋白。在这样的拉力实验中获得的力曲线显示，细胞膜拉长，很可能对应于脂链的形成，然后最终提取目标蛋白。在…中对力曲线的特征进行了分类更重要的是，1)拉不与细胞骨架和其他细胞内结构相连的膜蛋白，以及2)拉那些与细胞骨架相连的膜蛋白。模型实验是用针对血糖素A的特异性凝集素在红细胞上进行的，以前者为例，以带3为例，作为后者的膜蛋白的例子。在前者没有与细胞骨架连接的情况下，力曲线的特征是大约70pN的平台力延伸，以一步跳到零力水平结束。而在后一种情况下，力曲线的特征是在高原力伸展前后出现大于100pN的多个力峰。我们认为这两种力曲线可以用来监测膜蛋白与细胞内骨架结构的连接状态。2.提取mRNA和插入质粒DNA。利用AFM探针从活细胞的细胞质中提取mRNA和其他功能大分子。将被推入细胞靶点的探针作为RT-PCR的起始材料，并对该探针上收获的mRNA进行PCR扩增。活细胞中一个非常有限的位置的信使核糖核酸的定位可以常规地进行分析，以确定其身份和进行定量。这种方法将有助于对活细胞不同部分特定蛋白质的产生进行随时间变化的监测。利用吸附了绿色荧光蛋白基因的质粒DNA的AFM探针，我们发现以绿色荧光蛋白基因为标记的质粒DNA可以被转染到多个细胞中。较少