这里介绍的研究是由宾夕法尼亚州的卫生署，埃利森医学，V和祖母绿基础支持。

1。表达的重组TC叔<ol><li>从六个新鲜板用的TEV裂解N -端含有合成TC叔质粒转化罗塞塔（DE3）pLysS中细胞接种6升（6 × 2升困扰锥形瓶包含在每1 ​​L 2YT肉汤）2YT媒体hexahistidine标签。 </li><li>细胞增长在37 ° C，而在220转的0.5-0.6一个外径600的摇晃。 </li><li>加入1 mM的IPTG诱导蛋白表达。 </li><li>降低温度至30 ° C，缓慢摇动至150转，让细胞表达蛋白质为4-5小时</li><li>离心收集细胞在4 ° C和30分钟3500转。 </li><li>每1升的细胞沉淀重悬在20毫升含25毫米的Tris，pH值7.5，10％的甘油，0.5 M的氯化钾，15毫米咪唑，5毫米β-巯基乙醇，0.1毫米苯磺酰氟（PMSF）和苯甲脒缓冲区。 </li><li>闪存冻结悬浮细胞缓缓滴入50毫升的塑料圆锥管充满液态氮，使每下降可能造成的影响，在液态氮冻结的细胞混合物。商店冷冻细胞在-80 ° C，直到准备蛋白质纯化。 </li></ol> 2。 TC叔的纯化<ol><li>细胞在室温下解冻，直到液体的一致性达到，然后将冰为超声金属烧杯。 </li><li>在30秒的时间间隔30秒的停顿，在约51 W的功率超声细胞在冰上2分钟。 </li><li>在20分钟18000转离心裂解细胞分开的可溶性和不可溶性蛋白组分，并小心取出上清液镍亲和层析。 </li><li>平衡NI - NTA（氮川三乙酸镍）含有的25毫米的Tris，pH值7.5，10％的甘油，0.5 M的氯化钾，15毫米咪唑，5毫米β-巯基乙醇，0.1毫米PMSF和苯甲脒缓冲列。 </li><li>在树脂负载上清，收集流过。 </li><li>列包含的25毫米Tris，pH值7.5，10％的甘油，氯化钾500毫米，15毫米咪唑，5毫米β-巯基乙醇，0.1毫米PMSF删除所有剩余的和非特异性结合蛋白的缓冲区清洗。 </li><li>镍NTA树脂使用15咪唑梯度洗脱TC叔- 300毫米，并收集3毫升组分进行SDS - PAGE分析。 </li><li>使用页面分析，以确定哪些分数包含TC叔蛋白，并集中在一个30 kDa的（微孔）过滤器Amicon这些分数。 </li><li>一个体积小于10毫升为进一步净化浓缩TC叔分数。 </li><li> TC叔是进一步纯化过的阳离子交换柱（HS波罗斯，美国应用生物系统公司）与含有25毫米的Tris，pH值7.5，10％的甘油，氯化钾500毫米，1毫米的DTT缓冲区预平衡。 </li><li>负载HS波罗斯列的蛋白质的结构和清洗，以消除所有蛋白质和核酸的污染物。 </li><li>洗净用含有500毫米700毫米，氯化钾，以消除任何剩余的污染物氯化钾缓冲列。 </li><li>洗脱用1 M KCl缓冲关闭HS波罗斯列TC叔蛋白质。此时应超过99％的纯蛋白质。 </li><li>删除任何蛋白质总量，通过一个Superdex - 200浆纱列（体积排阻色谱 - 通用电气医疗集团）的蛋白质，含有25毫米的Tris，pH值7.5，10％的甘油，氯化钾500毫米和1毫米TCEP缓冲区预平衡。 </li></ol> 3。赤拟谷盗虫文化<ol><li>的T.谷盗甲虫最初作为礼物送给布恩，博士，美国堪萨斯州立大学生物学部提供。 </li><li>启动T。谷盗文化28.5克面粉和1.5克的干面包师的酵母加入到容器中的一百年的成虫。 </li><li>商店甲虫在室温和文化在阴暗，潮湿的环境，为三到四个星期，以便繁殖和生长。放置一个烧杯中含有水甲虫的存储区域，以确保在潮湿的环境。 </li><li>收获为RNA提取所需的甲虫幼虫。让其余的幼虫成熟的成年人，让他们在相同的文化。 </li><li>每个月，转移到一个新的含有新鲜的面粉和酵母的容量瓶中，以补充他们的食物供应的最活跃的甲虫（不超过一百年）。 </li></ol> 4 T。谷盗总RNA提取<ol><li>收获20分离成虫和面粉/酵母培养他们从文化的甲虫幼虫（每个文化之间应该包含500-1000幼虫）。消除面粉或酵母颗粒研磨前甲虫。 </li><li>消毒板凳区，迫击炮和杵（以及考虑把手套）RNaseZap（Ambion公司，公司）使用此过程删除核糖核酸的所有痕迹，研磨前的甲虫。 </li><li>幼虫在液态氮研磨成细粉末使用迫击炮和杵和T同时保持液体氮 ，以确保粉末保持冻结，直到提取液加入到50 mL离心管ransfer的粉末。 </li><li>让多余的液体氮蒸发粉。液体氮蒸发后，均质200μL提取缓冲液中的甲虫粉（每20个甲虫幼虫），其中包含的Tris - HCl 25毫米，5毫米的β-巯基乙醇，1毫米EGTA，苯甲脒0.1毫米， 200毫米氯化钾，10 ％甘油，10毫米咪唑和20U RNasin，pH值7.5和孵化冰浴30分钟。 </li><li>离心20分钟15000转的匀浆液在4 ° C，收集上清和闪光灯冻结在二氮液体样品。如果有必要，样品可存放于-80 ° C过夜。 </li><li>使用的RNeasy试剂​​盒（QIAGEN）提取总RNA。 </li></ol> 5， 在体外重组，赤拟谷盗端粒酶<ol><li>混合使用50μLT 的 20微克重组他的标签TC叔谷盗总RNA结合缓冲液中含有的Tris - HCl 25毫米，200毫米氯化钾，10％甘油，5毫米β-巯基乙醇，10毫米咪唑，pH值7.5。 </li><li>孵育TC叔-两小时的总RNA混合物在22 ° T中的彗星谷盗裂解RNasin抑制剂，以防止RNA降解的存在。 </li><li>复杂的多Ni - NTA柱纯化​​，以去除裂解杂质和多余的RNA。 </li></ol> 6。端粒酶重复序列扩增法（TRAP）分析<ol><li>混合4微克镍- NTA纯化T。谷盗端粒酶在50μL伸长缓冲液含20毫米的Tris - HCl（pH值- 8.3），7.5毫米氯化镁 2，63毫米氯化钾，0.05％，0.01％BSA，1毫米EGTA吐温20，0.5毫米dNTPs，1μMTCAS - TS DNA引物（5&#39; - AAGCCGTCGAGCAGAGTC - 3&#39;），并孵育的混合物在30 ° C为60分钟，让重组T. TCAS - TS的DNA引物伸长率谷盗端粒酶。 </li><li>拉长的单链DNA（单链DNA）反应混合物中加入等体积的苯酚氯仿提取。摇动轻轻直到乳液均匀的混合物。 </li><li>五分钟12000转离心样品在4 ° C至分离污染物的单链DNA。 </li><li>样品的顶部的水层转移到1.5 mL Eppendorf管中，添加一个终浓度为10毫米NaOAc（pH值5.0），MgCl 2的1毫米，66.4％的乙醇，然后让样品沉淀-20隔夜° C。 </li><li>在三十分钟15000转的旋转样品在4 ° C颗粒沉淀的单链DNA。倒出使用移液器的解决方案，然后添加200μl70％乙醇的样品从样品其余NaOAc和MgCl 2的洗。以相同的速度和温度重新颗粒样品，离心五分钟的样品。 </li><li>倒出的乙醇溶液使用移液器和删除允许Eppendorf管中，在室温下孵育三分钟乙醇剩下的痕迹。在样品中的乙醇会干扰PCR扩增步骤。 </li><li>在50μlPCR反应缓冲液含10毫米的Tris - HCl（pH为8），50 mM的氯化钾， 氯化镁 2 2毫米，100μMdNTPs（dATP的液重悬的单链DNA颗粒（含端粒酶拉长产品和TCAS - TS底漆）， dGTP和dTTP），10μM[32 P] dCTP，1微米TCAS - CX引物（5&#39; - GTGTGACCTGACCTGACC - 3&#39;）和1.25 U Taq DNA聚合酶。 </li><li> PCR扩增端粒酶伸长产品（94 ° C为30秒，50 ° C 30秒，和72℃1分钟29周期），使他们12％的聚丙烯酰胺凝胶上可见。 </li><li>前运行的Tris -硼酸- EDTA（TBE）的运行缓冲液含有44.5毫米三，硼酸44.5毫米，1毫米EDTA，pH值8三十分钟之前运行的PCR样本的12％的凝胶。经过预运行和装载凝胶PCR反应，在185伏特运行凝胶为1.5-2小时，在4℃或冰上°。 </li><li>干凝胶和使用磷光体成像板，可视化的活动。 </li></ol> 7。代表性的成果： 例如体积排阻色谱纯化的TC叔后如图2所示。集中净化后的蛋白质12％SDS聚丙烯酰胺凝胶上运行，被证明是99％以上的纯（图2A）。还提供了体积排阻色谱（S200）是（图2B），以证明其纯度和纯化蛋白质样品总量不足。 5毫克后，所有已完成的纯化步骤蛋白产量通常是获得虽然最终产量可能有所不同。 代表TRAP法一个重组的T。谷盗端粒酶是如图3所示，米切尔等。12 。可以看出，但在1巷，端粒酶产品分步进行的阶梯他们是缺席的RNase处理端粒酶和泳道2和3分别叔的活性中心突变体（D251A）样品。 <img src="/files/ftp_upload/2799/2799fig1.jpg" alt="图1"/> 图1。参与重组的重组TC叔步骤的流程图。首先，重组TC叔过在大肠杆菌中表达大肠杆菌 。的蛋白质是由镍，阳离子交换和体积排阻色谱纯化。的T.谷盗甲虫在内部培养和它们的幼虫是用于分离的总RNA。纯化的TC叔和总RNA，然后混合重组端粒酶的RNP复杂，并随后陷阱检测是用来确认的RNP复杂，是一个积极的端粒酶。 <img src="/files/ftp_upload/2799/2799fig2.jpg" alt="图2"/> 图2。体积排阻色谱和TC叔蛋白SDS - PAGE分析。 （一）12％SDS聚丙烯酰胺凝胶的TC叔蛋白后体积排阻色谱。 TC叔蛋白（70 kDa）的SDS - PAGE凝胶上的迁移速度更快，因为它具有很高的等电点（PI - 9.8）（B）尺寸排除色谱TC叔蛋白（Superdex S200）。 <img src="/files/ftp_upload/2799/2799fig3.jpg" alt="图3"/> 图3。 在体外的陷阱检测重组赤拟谷盗端粒酶适应米切尔等12 。巷1：野生型TC叔和总RNA分离出甲虫幼虫。巷2：野生型TC叔和RNase处理幼虫总RNA和细胞提取物。巷3：活性部位突变TC叔（D251A）和甲虫幼虫总幼虫RNA。

<ol>
	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

这里介绍的方法可以为大量生产的T 的催化亚基谷盗可溶性活性形式，结构和生化研究端粒酶叔。 TC叔过度表达的方法是敏感微妙的变化，在温度或从上述这些细胞密度和蛋白表达水平可以显着地影响。具体来说，我们发现，诱导蛋白表达的细胞之前，在600 nm的光密度达到0.5，或降低温度至30 ° C诱导前，在蛋白质产量显着减少。 在净化方面，TC叔蛋...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>产品编号</th></tr><tr><td>罗塞塔（DE3）pLysS中细胞</td><td> Novagen公司</td><td> 70956 </td></tr><tr><td> 2YT肉汤</td><td> Teknova </td><td> Y0215 </td></tr><tr><td>经IPTG </td><td>黄金生物技术</td><td> I2481C </td></tr><tr><td> MISONIX Sonicator 3000 </td><td> Qsonica，LLC。 </td><td></td></tr><tr><td> ÄKTA净化FPLC </td><td> GE生命科学</td><td></td></tr><tr><td> NI - NTA Superflow树脂</td><td> Qiagen公司</td><td> 30410 </td></tr><tr><td> Amicon超- 15离心过滤装置</td><td> Millipore公司</td><td> UFC903008 </td></tr><tr><td> POROS 50 HS强阳离子交换包装</td><td>美国应用生物系统公司</td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ强阳离子交换包装</td><td>美国应用生物系统公司</td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300体积排阻柱</td><td> GE生命科学</td><td> 17-5175-01 </td></tr><tr><td>酚：氯仿：异戊10MM三，pH值8，为1mM EDTA的25:24:1 </td><td>西格玛</td><td> P3803 - 100ML </td></tr><tr><td> RNaseZap </td><td> Ambion公司</td><td> AM9780 </td></tr><tr><td>重组RNasin核糖核酸酶抑制剂</td><td> Promega公司</td><td> N251B </td></tr><tr><td>的RNeasy迷你包</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td> DNA寡核苷酸</td><td>综合DNA技术</td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

努力找出端粒酶的催化亚基，叔，足够数量结构研究，已会见了十余有限的成功。在这里，我们目前的重组赤拟谷盗叔（TC叔）的分离和重组的积极 T.方法谷盗端粒酶的核糖核蛋白（RNP）复合物在体外 。 端粒酶是一个专门的反转录酶1，增加了短的DNA重复序列，称为端粒，线性染色体 2，有利于保护他们从终端到终端的融合和退化的3&#39;端。短段DNA复制，失去了在3号染色体的结束，如果没有端粒酶，细胞继续分裂，直至最终达到他们海弗利克极限4。此外，端粒酶在大多数体细胞在成人的 5中处于休眠状态，但活跃于癌细胞，促进细胞不死7。 最小的端粒酶由两个核心部分组成：蛋白亚基（叔），其中包括酶的催化亚基和一个完整的RNA组分（TER），其中包含的模板叔使用合成端粒8,9。在2008年之前，只有个别端粒酶域的结构已经解决了10,11。在这一领域的一个重大突破来自活跃的 12晶体结构测定，T的催化亚基谷盗端粒酶，TC叔1。 在这里，我们目前生产大量的活性，可溶性TC叔结构和生化研究，并重组端粒酶活性检测端粒酶在体外的RNP复杂的方法。使用的实验方法概述如图1所示。

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

努力找出端粒酶的催化亚基，叔，足够数量结构研究，已会见了十余有限的成功。在这里，我们目前的重组赤拟谷盗叔隔离的方法（<em&gt; TC</em&gt;叔）和重组活动<em&gt; T。谷盗</em&gt;端粒酶的核糖核蛋白（RNP）复合物<em&gt;在体外</em&gt;。

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

in-vitro-reconstitution-of-the-active-t-castaneum-telomerase

Research

JoVE Journal

Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

11.6K 次观看.  University of Pennsylvania. 努力找出端粒酶的催化亚基，叔，足够数量结构研究，已会见了十余有限的成功。在这里，我们目前的重组赤拟谷盗叔隔离的方法（ TC叔）和重组活动 T。谷盗端粒酶的核糖核蛋白（RNP）复合物在体外。

视频: In vitro Reconstitution of the Active T. castaneum Telomerase

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

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 ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

端粒长度和端粒酶活性;阴阳细胞衰老

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

前体mRNA底物的3&#39;末端切割：用无细胞体系中的RNA加工反应分析<em&gt;在体外</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

重组β-catenin的降解<em&gt;爪蟾</em&gt;卵提取物

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...