Name: analysis of mrna nuclear export kinetics in mammalian cells by microinjection
Uploaded: 2010-12-04T13:00:00
Duration: 11 min 32 s
Description: Watch this Scientific Journal Video about Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection at JoVE.com

我们想感谢有用的意见和A.王尔德E.允许我们使用的各种设备的戈麦斯。这项工作是由一个由加拿大卫生研究院（FRN 102725）研究所授予法新社支持。

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	<li>Luo, M. J., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+is+required+for+rapid+and+efficient+mRNA+export+in+metazoans.">Splicing is required for rapid and efficient mRNA export in metazoans.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 14937-14942 (1999).</li><li>Bossie, M. A., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+macromolecules+between+the+cytoplasm+and+the+nucleus+in+yeast.">Movement of macromolecules between the cytoplasm and the nucleus in yeast.</a> Curr. Opin. Genet. Dev. 2, 768-774 (1992).</li><li>Farny, N. G., Hurt, J. A., Silver, P. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+HCMV+immediate+early+mRNA+expression+in+stably+transfected+fibroblasts.">Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts.</a> J. Cell. Sci. 115, 321-328 (2002).</li><li>Palazzo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+sequence+coding+region+promotes+nuclear+export+of+mRNA.">The signal sequence coding region promotes nuclear export of mRNA.</a> PLoS Biol. 5, e322-e322 (2007).</li><li>Valencia, P., Dias, A. P., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+promotes+rapid+and+efficient+mRNA+export+in+mammalian+cells.">Splicing promotes rapid and efficient mRNA export in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3386-3391 (2008).</li><li>Palazzo, A. F., Eng, C. H., Schlaepfer, D. D., Marcantonio, E. E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+stabilization+of+microtubules+by+integrin-+and+FAK-facilitated+Rho+signaling.">Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling.</a> Science. 303, 836-839 (2004).</li><li>Keller, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proper+alignment+of+the+microscope.">Proper alignment of the microscope.</a> Methods Cell Biol. 72, 45-55 (2003).</li><li>Mathews, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+chemical+modification+constraints+into+a+dynamic+programming+algorithm+for+prediction+of+RNA+secondary+structure.">Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292 (2004).</li><li>G&#246;rlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+sequence+receptor+has+a+second+subunit+and+is+part+of+a+translocation+complex+in+the+endoplasmic+reticulum+as+probed+by+bifunctional+reagents.">The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents.</a> J. Cell Biol. 111, 2283-2294 (1990).</li><li>Perales, R., Bentley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Cotranscriptionality":+the+transcription+elongation+complex+as+a+nexus+for+nuclear+transactions.">"Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions.</a> Mol. Cell. 36, 178-191 (2009).</li><li>Buratowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+through+the+RNA+polymerase+II+CTD+cycle.">Progression through the RNA polymerase II CTD cycle.</a> Mol. Cell. 36, 541-546 (2009).</li><li>Maniatis, T., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+network+of+coupling+among+gene+expression+machines.">An extensive network of coupling among gene expression machines.</a> Nature. 416, 499-506 (2002).</li><li>Carter, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+copy+number+estimation+using+a+mouse+whole-genome+oligonucleotide+microarray.">Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray.</a> Genome Biol. 6, 61-61 (2005).</li></ol>

显微注射是一个强大的工具，可以用来研究一些不同的细胞过程。不同于传统的细胞转染，显微注射，使研究人员有效地引入一个非常狭窄的时间内细胞的细胞核的核酸。因此，一个可以执行的动力学分析，如确定mRNA的出口，mRNA的本地化和蛋白质的合成率。此外，由于实验的时间表是相对短暂的，可以公开，而不会产生多效性细胞在短的时间跨度内对有毒化合物。此外，显性负的蛋白质，如抑制或刺激化合物，可以合作的核酸注射。最后，microinjections避免转染试剂的要求，这往往是细胞毒性，并可能扰乱不同的细胞功能。 基因与质粒DNA注射选择其中核酸注入将取决于众多的因素。质粒DNA注射后，RNA聚合酶II不仅合成的mRNA，但也新兵蛋白因子新生成绩单 12,13 。由于这些因素的管理事件，如mRNA加工，出口核和细胞质定位，质粒DNA注射液可用于评估转录是如何加上下游工序。虽然可能会耦合到核出口14转录，我们发现注入基因是出口略快于在体内转录的mRNA 6。这种情况的原因目前尚不清楚，但这个结果可能表明，新合成的mRNA由RNA聚合酶II的出现，它可能被捆绑起来，延缓核出口物。 与mRNA注射有一定的优势。首先，研究人员并没有使用RNA聚合酶抑制剂，这可能会影响细胞如何调节整体的RNA代谢。其次，在RNA可以在体外进行修改，在注射之前。例如，可以合成的mRNA与不同的5&#39;帽类似物或聚（A）尾巴长度，以评估这些特性如何影响的核出口 6 。第三，注射RNA的确切数额，可以粗略估计。上文所述的协议之后，我们估计约20,000至50,000分子相比总数的成绩单，在一个典型的哺乳动物细胞（40万至85万分子 ）15时，在每个细胞核注入，这是比较小的。与mRNA注射的主要缺点是，在显微注射过程中，一些进入细胞质注射液泄漏是不可避免的。由于直接注入细胞质中的mRNA（A.宫，未发表的观察）长期稳定，格外小心，必须采取选择量化的细胞。一般来说，我们分析细胞&gt; 90％的注射液在细胞核内，作为评估OG -葡聚糖的分布。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Injection buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>140 mM KCl and 10 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Oregon Green 488-70kD Dextran</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D7173</td>
<td>Stock of 10mg/ml in injection buffer can be stored for longs periods of time at -80&deg;C</td>
</tr>
<tr>
<td>Borosilicate capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>1B100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Gel loading pipette tips for loading injection needles</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>022351656</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Eclipse Ti-S</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige Group</td>
<td>NT-88-V3MSH</td>
<td>&nbsp;</td>
<tr>
<td>Syringe for injection</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>512311</td>
<td>&nbsp;</td>
<tr>
<td>PBS Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4</td>
</tr>
<tr>
<td>SSC Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>150 mM NaCl, 15 mM sodium citrate, pH 7.4</td>
</tr>
<tr>
<td>4% Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15686</td>
<td>Stock of 37% Paraformaldehyde is diluted in PBS</td>
</tr>
<tr>
<td>0.1% Triton X-100 (Surfact-Amps)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>28314</td>
<td>Stock of 10% Triton X-100 is diluted in PBS</td>
</tr>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>F84-1</td>
<td>&nbsp;</td>
<tr>
<td>Dextran sulfate</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>BP1585-100</td>
<td>&nbsp;</td>
<tr>
<td>E. coli tRNA</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R1753</td>
<td>&nbsp;</td>
<tr>
<td>Vanadyl riboside complex (VRC)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R3380</td>
<td>&nbsp;</td>
<tr>
<td>Whatman filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>&nbsp;</td>
<tr>
<td>Vibration control table</td>
<td>&nbsp;</td>
<td>Kinetic Systems</td>
<td>5702E-3036-21</td>
<td>&nbsp;</td>
<tr>
<td>Fluoromount G</td>
<td>&nbsp;</td>
<td>Southern Biotech</td>
<td>0100-20</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;-amanitin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A2263</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-374-12</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum Grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>695400008</td>
<td>&nbsp;</td>
<tr>
<td>T7 RNA Polymerase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0251S</td>
<td>&nbsp;</td>
<tr>
<td>Poly(A) Polymerase</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1350</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>100mg/ml dextran sulfate, 5mM VRC, 150mM NaCl, 15mM sodium citrate, 0.5mg/ml E. coli tRNA, 60% formamide</td>
</tr>		</tbody>
		</table>
		</div>

analysis of mrna nuclear export kinetics in mammalian cells by microinjection

在真核生物中，信使核糖核酸（mRNA）的转录在细胞核中，必须进入细胞质访问翻译机器出口。虽然被广泛地研究在爪蟾卵母细胞和基因听话的生物，如酵母2 和派生的果蝇S2细胞系3 mRNA的核出口，很少有研究已经在哺乳动物细胞中进行。此外，只能推断哺乳动物体细胞中的mRNA出口动力学间接 4,5 。为了测量核出口动力学哺乳动物组织培养细胞中的表达，我们已经开发了一种采用显微注射的力量 ， 再加上在荧光原位杂交（ FISH）检测。这些实验都被用来证明，在哺乳动物细胞中，大多数的mRNA出口在剪接依赖性6,7，或在地，需要特定的RNA序列，如信号序列的编码区（SSCR）6 。在这个实验中，细胞显微注射，无论是在体外合成的mRNA或含有目的基因的质粒DNA 。显微注射细胞培养不同时间点，然后固定和RNA的亚细胞定位是评估使用鱼。相反转，转录发生后几个小时的核酸此外，DNA或mRNA显微注射可以快速表达，可以生成精确的动力学数据。

Watch this Scientific Journal Video about Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection at JoVE.com

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

在这里，我们描述了一个实验，采用显微注射的力量，再加上用荧光<em&gt;原位</em&gt;杂交为了准确地测量哺乳动物体细胞中的mRNA的核出口动力学。

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

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

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显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

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We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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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 ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

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The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

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The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...