原GFP - Pem1李磊博士的礼物。这项工作是由从美国国立卫生研究院和埃利森医学基金会VG的赠款和支持

在这个协议中，我们描述了与染色体综合记者的方法构造 1,2双链断裂在体内诱导瞬时表达一种罕见的切割核酸内切酶的I - SCEI 3分析DNA DSB修复。综合检测分析染色体范围内DSB的修复提供了优势。然而，该协议要求延长细胞传代，这可能是问题时，与原电池的工作。 另一种方法是记者构造在体外消化与I - SCEI或HindIII位内切酶，然后转成线性DNA细胞染色体外检测。 4 exctrachromosomal检测协议是类似于下面描述的以下修改综合检测。对于染色体外检测省略记者结构的整合，而是合作与0.5微克NHEJ 记者在体外我SCEI或HindIII和0.1微克pDsRed2 - N1转染控制的线性质粒转染的细胞。对于人力资源检测联合转染2微克的人力资源报告质粒线性与我SCEI或HindIII和0.1微克pDsRed2 - N1。转染后3天用流式细胞仪分析细胞。染色体外检测允许分析DSB的修复初级分离，避免广泛的细胞传代，并促进多种细胞系的分析。 1。记者NHEJ能和人力资源结构 NHEJ记者卡带5包含了一个精心设计的3 kb的内含子从Pem1基因（GFP - Pem1）的绿色荧光蛋白基因。 Pem1内含子包含一个腺病毒介导的外显子，两侧为内切酶HindIII和我的SCEI诱导双链断裂（图1a）识别序列。我SCEI网站倒置方向。我SCEI有nonpalindromic识别序列，因此两个倒网站产生不兼容的DNA末端（图1c）。不相容的DNA末端的最佳模仿自然发生的DNA双链断裂。完整的NHEJ卡带是负的绿色荧光蛋白为腺病毒介导的外显子，扰乱了GFP的ORF。当我SCEI双链断裂的诱导，腺病毒介导的外显子和NHEJ能恢复功能的绿色荧光蛋白基因（图2）。本NHEJ记者的独特之处在于它可以检测广泛的NHEJ事件，因为内含子可以容忍的缺失和插入。 也是基于GFP - Pem1人力资源记者纸盒1（图1b）。在人力资源磁带，第一外显子的GFP - Pem1包含22 bp缺失与插入三个酶切位点，I-SceI/HindIII/I-SceI相结合。删除确保绿色荧光蛋白不能重组NHEJ事件。两个I - SCEI网站倒方向，使我SCEI消化叶不相容的两端（图1c）。其次是一个promoter-less/ATG-less第一外显子和内含子的GFP - Pem1 GFP - Pem1的第一个副本。完整的构造是GFP阴性。当一个DSB诱导的I - SCEI消化功能的绿色荧光蛋白基因重组GFP - Pem1第一外显子突变的两个副本之间的分子内或分子间的基因转换。由于绿色荧光蛋白基因的第二个副本是缺乏的第一个ATG密码子和第二外显子，交叉或单链退火不会恢复GFP活动。这种设计可实现基因转换的独家发现，这是在哺乳动物细胞中的主要人力资源的途径（图3）。 2。记者整合到基因组构造<ol><li>制作高质量的质粒NHEJ或HR记者准备。为了达到最佳效果，请使用Qiagen公司的远藤免费包。在260/280 nm处测量吸光度质粒的浓度和纯度。 </li><li> 10微克的记者质粒线性化消化50 U NheI在50μL（总体积的反应）为6小时37 ° C培养箱（不使用干块，以防止蒸发）。净化消化解决方案Qiagen公司QiaexII包，到20μL10毫米的Tris - HCl pH值8.0洗脱DNA的DNA。测量DNA的浓度和纯度在260/280 nm的吸光度。通常情况下，你将失去20％至30％的DNA在纯化过程中。确认连同未消化的记者构造，凝胶上运行一个小等份（2μL）的产量和消化。纯化的线性记者构造，可以储存在20 ° C </li><li>在准备进行转染，使细胞的最佳生长状况。如果从一个冷冻小瓶开始，分裂细胞转染前的两倍。 ，如果从一盘的融合细胞开始，他们一次分裂。 </li><li>增长70-80％汇合时（通常是两天，如果镀5X10 5 cells/100毫米钢板，正常的人成纤维细胞）的细胞。它是带入对数生长期的细胞的最佳转染效率的关键。积极日益增长的文化包含在M期细胞（被视为附着在表面的圆形细胞）。 </li><li> TRANSFECT的细胞，使用Amaxa Nucleofector以下制造商的建议。对于成纤维细胞，使用NHDF的解决方案和计划U20。用于转染，收获两个100毫米板（〜2 × 10 6细胞）的细胞。这是不超过trypsinize细胞的关键。停止胰蛋白酶尽快脱离板细胞的80％。在NHDF溶液重悬细胞。细胞是敏感NHDF解决方案，因此，最大限度地减少孵育时间，并轻轻混合细胞。转染的细胞，用0.5微克的线性记者构造。这些转的条件下，当正常的人成纤维细胞在一个单拷贝记者整合的结果构造为广大的integrants。 </li><li>选择加入1毫克/毫升geneticin（G418）24小时转染后的细胞与染色体综合记者结构。继续选择7-10天。 </li><li>挑选G418抗性克隆（你可以选择单独的殖民地或池抗性克隆）。约5个100毫米板展开的文化。冻结三个板块供日后使用，其余的两个板块展开所需的细胞数（通常是10个100毫米板足够的五个转染）。 </li><li>当细胞达到70-80％汇合（两天后电镀5X10 5％100毫米板为成纤维细胞的细胞），我SCEI表达质粒转染细胞准备。 </li></ol> 3。 DSB的瞬时表达我的SCEI内切酶的诱导<ol><li>转染〜2 × 10 6细胞（两个板块）对数增长与文化的表达质粒转染效率使用Amaxa Nucleofector（使用成纤维细胞NHDF解决方案和U20程序的控制和0.1微克pDsRed2 - N1（Clontech公司）的混合物5微克的I - SCEI ）。 </li><li>同时准备三个校准控制为流式细胞仪trasfecting与（i）〜2 × 10 6细胞5微克的EGFP表达质粒;（二）5微克pDsRed2 - N1;（三）5微克的控制质粒，并没有表达出荧光蛋白（我们使用的是HPRT小基因质粒编码）。 </li><li>成长为4天的细胞提供足够的时间表达GFP和DsRed的蛋白质。 </li><li> （可选）三天后转验证转染GFP和DsRed的蛋白的表达，荧光倒置显微镜检测GFP和DsRed的过滤器的效率。 </li></ol> 4。流式细胞仪分析了GFP +和DsRed的+细胞<ol><li>四天后，转染收获我的SCEI转染细胞和对照细胞。在500μL的PBS重悬的细胞和细胞转移流式细胞仪管。在这个阶段，关键是收获的所有细胞，并有圆形细胞。为了实现这一目标，建议使用较长的胰蛋白酶消化时间或更强的胰蛋白酶。确认，99％的细胞是从板块分离，并通过显微镜下观察细胞的圆形。保持避光冰细胞（盖用铝箔的冰水桶）。它可以存储在冰上几个小时的细胞。最好的结果，分析后，立即收获细胞。 </li><li>校准与GFP，DsRed的，与阴性对照流式细胞仪。调整电压和色彩补偿，以包括所有在分析荧光的细胞（图4）。请记住，将实验样品的荧光强度比对照低​​。 </li><li>分析我的SCEI转染细胞的流式细胞仪。我们每次治疗至少20,000细胞计数。为了防止潜在干扰GFP和荧光DsRed的建议是，保持低于25％％的GFP +或DsRed的+细胞。如果DsRed的+或GFP +细胞的百分比较高，减少pDsRed2 - N1或I - SCEI质粒量。在我们的实验中，我们只需要调整的金额pDsRed2 - N1质粒，但不是我SCEI质粒。 </li><li> ％对应的GFP +细胞的DNA DSB修复的效率和DsRed的+细胞的百分比表示的转染效率。 DsRed的+细胞的GFP +细胞的比例，计算DNA DSB修复的相对效率。 </li><li>修复结束路口可测序测试修复的准确性。记者构造含有大肠杆菌原产地复制和卡那霉素抗性基因（图1）。这使抢救消化基因组DNA，用EcoRI酶，重新环，并到主管的大肠杆菌细胞转化结构。获救的质粒酶切和测序分析。 </li></ol> 5。代表性的成果典型的流式细胞仪的控制转染和NHEJ能和人力资源实验结果显示在图4和5。我SCEI转染细胞含有integrat荧光强度和荧光细胞的数量将会降低ED NHEJ能和人力资源结构（图5）相比，对照组（图4）由于在绿色荧光蛋白在这些细胞中DsRed的构造的拷贝数的差异。在实验中的每个单元格只包含一个集成的记者兴建的副本，从而成功修复的事件将重组绿色荧光蛋白基因在细胞的一小部分。 0.1微克，人成纤维细胞pDsRed2 - N1转染提供了大约1％细胞的质粒拷贝。 NHEJ能和人力资源的效率的GFP + / DsRed的+细胞的比例计算。 NHEJ是一个比人力资源更有效地在人类细胞中的过程2。在人类成纤维细胞的NHEJ效率通常是0.6-1.3，和人力资源的效率是0.05-0.3。由于DSB的效率是衡量一个比例的GFP + / DsRed的+我的SCEI和DsRed2 - N1质粒的混合物细胞的变化可能会影响结果。质粒的质量也可能通过改变转染效率影响的结果。因此，要取得一致的测量，重要的是在一项实验中使用相同的质粒组合。此外，DSB修复效率的影响类型的细胞，细胞周期，染色体定位和综合结构。 <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="图1" /> 图1。记者构造NHEJ（A）和HR（二）分析DNA DSB修复。 （三）不兼容的DNA两端两个倒我SCEI网站消化所产生的SD，拼接捐助; SA，剪接受体;阴影广场，聚腺苷酸化的网站;新/假名，单一的ORF由两个启动子控制：SV40赋予新霉素抗性哺乳动物。细胞和β-内酰胺酶在大肠杆菌赋予kanamicyn阻力;大利，E.大肠杆菌临市局原产地复制。 <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="图2" /> 图2。记者NHEJ分析 ，在此兴建构建绿色荧光蛋白基因处于非活动状态，但经诱导的DSB成功NHEJ构建成为GFP + 。下面显示的是一个由NHEJ记者修复产品。 <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="图3" /> 图3。记者构建的人力资源，通过基因转换分析 。基因转换（GC）后诱导的DNA双链断裂，人力资源由I - SCEI重组活跃的绿色荧光蛋白基因。如下图所示是主要的DNA修复途径修复产品记者：GC，过境超过（X - OVER），单链退火（SSA）的，和NHEJ。 X - OVER修复产品是分子内重组，exramolecular重组将给予同样的产品，但位于一条染色体上。基因表达活跃的绿色荧光蛋白（GFP）是由绿色表示。 <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="图4" /> 图4。流式细胞仪分析参数的校准（一）成纤维细胞与pHPRT质粒转染，不包括汽车荧光细胞作为阴性对照。 （二）成纤维细胞转染pEGFP质粒，用于设置浇注的GFP +细胞（绿色区域）。 （三）成纤维细胞转染pDsRed2 - N1质粒DsRed的+细胞（红色区域）设置的门控。 <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="图5" /> 图5。典型NHEJ（A）和HR（二）在正常的人成纤维细胞与染色体整合的记者构造的分析结果。

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

荧光NHEJ和HR记者分析提供定量的方式，分别测量每个DSB修复途径在体内。检测是非常敏感，流式细胞仪能够可靠地检测到10个GFP +细胞在20000细胞。检测可用于测量通过检测的GFP +细胞在几分钟之内或双链断裂2诱导后小时的外观在“实时”的修复事件改编。此外，分析的GFP +细胞不依赖于其他细胞的增殖，使DSB的修复将在不同的细胞周期的各个阶段进行分析后，与各种药物的治疗， <s...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
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analysis of dna double-strand break (dsb) repair in mammalian cells

DNA双链断裂是最危险的DNA损伤，可能会导致遗传信息和细胞死亡的大量流失。使用两个主要途径：非同源末端连接（NHEJ）和同源重组（HR）的细胞修复DNA双链断裂。 NHEJ能和人力资源的扰动往往与过早衰老和肿瘤的发生相关，因此重要的是有一个定量的方式测量每个DSB修复途径。我们的实验室已开发出荧光记者构造，使敏感和NHEJ能和人力资源的定量测量。是基于一个精心设计的绿色荧光蛋白含有一种罕见的切割我SCEI内切酶，DNA双链断裂感应识别位点的基因构造。开始构造的GFP负，作为额外的外显子的绿色荧光蛋白基因灭活，或突变。我SCEI诱导休息NHEJ或HR的成功修复，恢复功能的绿色荧光蛋白基因。流式细胞仪计数GFP阳性细胞的数量提供NHEJ或人力资源效率的定量测量。

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

本文介绍了基于GFP的荧光<em&gt;在体内</em&gt;分析，分别量化同源重组和非同源末端在哺乳动物细胞中加入。

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K 次观看.  University of Rochester. 本文介绍了基于GFP的荧光在体内分析，分别量化同源重组和非同源末端在哺乳动物细胞中加入。

视频: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

科研

生物学

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

游离成年哺乳动物元DNA的细胞核内内微量注射

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

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

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

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 potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

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 ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

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 ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

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

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.