Name: analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells
Uploaded: 2011-05-31T12:00:00
Description: Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

这项工作是由一个从（GM070736）国家卫生研究院授予阁下Fölsch。 SF昂是支持的A * STAR研究生奖学金，RS康是由细胞和分子基础的疾病培训计划（GM8061）的支持

1。质粒DNA的分离<ol><li>内毒素游离DNA，根据制造商的协议，使用一个Sigma - Aldrich公司内毒素maxiprep套件准备。该套件为我们的，因为它可靠从DNA制剂中删除任何内毒素。与细胞核的DNA注入内毒素会导致细胞死亡。 </li><li>添加100μL酚/ chlorofom /异戊醇（25:24:1），以孤立的DNA，涡和13,000 RPM的Eppendorf离心1分钟旋转。上层水相转移到一个新的管，加100μL氯仿/异戊醇（24:1），涡旋和上面一样旋转。转移?...

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	<li>Mellman, I., Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 9 (11), 833-833 (2008).</li><li>Rodriguez-Boulan, E., Kreitzer, G., Musch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature reviews. 6 (3), 233-233 (2005).</li><li>Ang, A. L., F&#246;lsch, H., Koivisto, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 163 (2), 339-339 (2003).</li><li>Kroschewski, R., Hall, A., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 1 (1), 8-8 (1999).</li><li>Moskalenko, S., Henry, D. O., Rosse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 4 (1), 66-66 (2002).</li><li>Schuck, S., Gerl, M. J., Ang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traffic (Copenhagen, Denmark). 8 (1), 47-47 (2007).</li><li>Nokes, R. L., Fields, I. C., Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of cell biology. 182 (5), 845-845 (2008).</li><li>Collins, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cell.">Molecular cell.</a> 12, 1064-1064 (2003).</li><li>Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science (New York, N.Y. 279 (5350), 509-509 (1998).</li><li>Scales, S. J., Pepperkok, R., Kreis, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 90 (6), 1137-1137 (1997).</li><li>Keller, P., Toomre, D., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature cell biology. 3 (2), 140-140 (2001).</li></ol>

一个成功的显微注射实验的最关键步骤的质量和纯度的DNA和你的细胞的极性。没有极化的细胞，注射控制将已经mistargeted VSVG和实验，不能使用。如果质量差的DNA，DNA可能会堵塞导致较差或没有所需的蛋白质的表达在所有的注射针头。另外，最好是用已知的导致高表达水平，如pRKV的表达质粒。 如果你想要测试的其他货物的蛋白质排序，温度变化协议可以被修改。例如，低密度...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>加热阶段AXIOVERT 200显微镜</td><td>卡尔蔡司公司</td><td>自定义顺序</td></tr><tr><td> Injectman NI2 Femtojet显微</td><td> Eppendorf公司</td><td>自定义顺序</td></tr><tr><td> Femtotips二（显微注射针） </td><td> Eppendorf公司</td><td> 930000043 </td></tr><tr><td> Microloader技巧</td><td> Eppendorf公司</td><td> 930001007 </td></tr><tr><td>清除12毫米Transwell小过滤器支持</td><td>康宁中光学</td><td> 3460 </td></tr><tr><td>无内毒素质粒maxiprep套件</td><td> Sigma - Aldrich公司</td><td> NA0400 </td></tr></tbody></table>

analyzing the function of small gtpases by microinjection of plasmids into polarized epithelial cells

上皮细胞分化成生化和功能上截然不同的心尖和基底域心尖域面临的&#39;自由&#39;的表面和基底外侧膜与基板和邻近细胞接触的质膜。这两种膜域是分开的紧密连接，形成一个扩散屏障。心尖，基底极化可以成功详文化Madin Darby狗肾细胞（MDCK），如上皮细胞接种在高密度聚碳酸酯过滤 ​​器和培养1 2数天。如RalA，Cdc42的，Rab8，Rab10和Rab13 3 4 5 6 7 Ras超家族小GTP酶的数组是受细胞极性的建立和维护。所有GTP酶一样，这些蛋白质之间的一个无效的GDP结合状态和积极的GTP结合状态的周期。核苷酸结合区域的特定的突变与此骑自行车的干扰8。例如，Rab13T22N是永久锁定在国内生产总值的形式，并因此被称为“显性负”，而Rab13Q67L再也不能水解GTP，从而锁定在“显性活跃” 状态 7 。要分析其功能，在细胞中都占主导地位的消极和主导的GTP酶活跃等位基因通常表示在高水平干扰9内源性蛋白的功能。一种优雅的方式来实现的过度表达水平高，在很短的时间介绍质粒编码有关的蛋白质直接进入极化细胞过滤器支持采用显微注射技术种植的原子核。这往往是记者质粒共注射编码质膜是专门排序根尖或基底域的受体结合。常用的分析货物分拣基底域A货是一个温度敏感等位基因的水泡性口炎病毒糖蛋白（VSVGts045）10。 ，这种蛋白质不能正确折叠在39 ° C，因此将保留于内质网（ER），而利益调节蛋白在细胞质中装配。转向至31 ° C将允许VSVGts045正确折叠，离开的ER和旅游质膜 11 。这追逐通常是在放线菌酮的存在，以防止进一步的蛋白质的合成，导致清洁结果。在这里，我们详细描述了显微注射到极化细胞和随后的孵化，包括温度变化，使一个基底排序所涉及的监管蛋白质的综合分析质粒的程序。

Watch this Scientific Journal Video about Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells at JoVE.com

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

本文详细介绍了在极化上皮细胞，利用显微注射技术的小GTP酶的过度表达和分析所涉及的程序。

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Research

JoVE Journal

Biology

Microinjection of Zebrafish Embryos to Analyze Gene Function

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One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

科研

生物学

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

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A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

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Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

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In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

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Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

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Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

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In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

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A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...