Name: a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation
Uploaded: 2016-08-08T14:00:00.000+00:00
Duration: 14 min 27 s
Description: Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

所有的爪蟾程序和研究是在遵守NRC指南实验动物第8版的管理和使用进行。协议由怀俄明州机构动物护理和使用委员会（保证＃A-3216-01）的大学批准。 1. X的准备蟾卵提取物（改编自27,28） <ol><li>总理的女X.蟾青蛙最少三天收蛋之前最多两个星期与一个100 IU注射孕马血清促性腺激素（PMSG）的。 </li><li>实验前一天，以每1/3倍马克的修改振铃（MMR）的青蛙1升在16℃，用注射绒毛膜促性腺激素（HCG）和地点的800 IU底漆青蛙。请参阅表1为所有缓冲区组成。 注意:有些青蛙不会下蛋或将为质量较差的蛋。一般为2 - 3青蛙被注入，以保证至少有一个青蛙将为sufficien好的鸡蛋质量T编号。平均青蛙将为足够的鸡蛋生产至少1毫升鸡蛋提取物。 </li><li>制备1升的1/3×MMR用冷的去离子蒸馏水（DDH 2 O的），500毫升缓冲液B，1升缓冲液C 500毫升缓冲液D，和100毫升缓冲液E. </li><li>转印的蛋的玻璃结晶皿（150×75毫米）。使用塑料巴斯德吸管用刀尖切断，农场出蓬松的白色蛋激活或裂解蛋。只保留具有鲜明的和平等的暗动物极和白色植物极蛋。 </li><li>制备13×51毫米离心管1毫升缓冲液E的补充有355微米松弛素D加入步骤1.7在使用前。 </li><li> Dejelly洗鸡蛋。 <ol><li>在一个适当大小的烧杯用冷的1/3×MMR简要洗鸡蛋，改变缓冲器3 - 4倍。 </li><li>用缓冲液B.此孵化取下鸡蛋果冻大衣从3采取任何地方 - 10分钟。由于阴天果冻大衣从卵子释放S，更改缓冲区。 注:Dejellying完成后倾斜和植物性极点朝下时，鸡蛋出现紧凑的菜的角落。鸡蛋是dejellying后脆弱得多，所以不要超过必要的和后续的洗涤更长孵育缓冲液B鸡蛋必须非常仔细地进行。 </li><li>鸡蛋洗净简要地3 - 缓冲C. 4的变化</li><li>鸡蛋洗净简要地缓冲D. 2变化</li><li>鸡蛋洗净简要地缓冲E. 2变化</li></ol></li><li>填充离心管，使用大口径玻璃吸管或塑料滴管鸡蛋。吸掉多余的缓冲区。收拾鸡蛋和在212 XG在吊桶式转子除去尽可能多缓冲越好，离心60秒，然后在478×g离心30秒。吸掉多余的缓冲区。 注意:在此步骤，以除去尽可能多的多余的缓冲液作为能够确保卵提取物最低限度稀释是很重要的。 </li><li>在摆动<html>桶转子，在真空下超速为在12,000 xg离心15分钟，4℃下粉碎鸡蛋打开并分离成不同的层，与细胞质是在中间琥珀色层。冰上取出离心管中，立即就位。 </li><li>收集提取物。 <ol><li>使用18号针头和注射器，穿刺离心管只在管的底部的暗着色层上方并去除中间琥珀色细胞质层。不收集任何顶部的脂质层。收集细胞质中一个适当大小的管中。 注意:通常，1青蛙产生至少1毫升提取物。 </li><li>补充胞浆1 / 1,000 19.7毫米的松弛素D的量，1 / 1,000 LPC股票的数量，和1/50的50倍能源结构的体积。轻轻翻转试管混合。不要过分吸管提取物。 </li><li>置于冰上提取物和最好的结果编制3小时内使用。 </li></ol></li></ol>_title"&gt; 2。Demembranated 爪蟾精子（改编自29）的制备注意:这里介绍的过程卷长达8睾丸。 <ol><li>除去0.05％苯佐卡因从4℃并温热至室温（在乙醇中制备的，并稀释到在青蛙罐水0.05％10％苯佐卡因库存）。麻醉牺牲男性的X. 30分钟-在室温下搅拌20 蟾在苯佐卡因溶液青蛙。通过检查和感胸部区域中，通过缺乏响应于机械性刺激的，和/或通过断头确保没有心跳的死亡。 </li><li>使沿腹部的U形切口。删除黄色脂肪体的质量tubulated。在肾脏的顶部，找到睾丸，这是椭圆形的浅粉色群众约1厘米的长度29。切除睾丸和滚他们在吸墨纸上以清除血液和其他组织。 </li><li>睾丸洗净缓冲中使用T.紧装杵讳言用1毫升缓冲睾丸T IN 1.5ml试管直到均匀（10冲程或更多）。离心5 - 10秒在微型离心收集上清，去除大块组织。重复一次离心，并收集上清液。 </li><li>传输含有精子的溶液到15毫升的塑料管中。增大音量共2毫升缓冲T.离心1411 XG 10分钟，在16℃，以沉淀精子。 </li><li>去除上清，在16℃下悬浮于500μl缓冲T.离心机的颗粒在1411 XG 10分钟。根据需要清理的体细胞和血红细胞的沉淀一次或两次重复此步骤。 </li><li>重悬在室温下在100μl缓冲液T与300微升缓冲液S.孵育沉淀5分钟。 注:缓冲S包含溶血卵磷脂，它负责demembranation。 </li><li>加入缓冲的R三卷（准备使用前新鲜）。倒置管一次。离心在1411 xg离心15分钟，在16＃176℃。 </li><li>悬浮于400μl缓冲液R中的沉淀，然后在1411 xg离心16℃添加额外的2毫升缓冲液R.离心机15分钟。 </li><li>悬浮颗粒在50微升缓冲T. </li><li>用稀释9微升缓冲T. 1微升demembranated精核应用此稀释血球。计数薄S形精子核数目在一个大的4×4网格，并乘以100号，以获得每微升精子细胞核股票的浓度。 </li><li>稀释股票每微升100000精核与缓冲器T，导致200X的股票。准备3 - 5微升等分。闪冻在液氮中，并储存于-80℃。 </li></ol> 3.核大会<ol><li>补充100微升卵提取物与1.5微升35.5毫酮（终浓度为0.5 mM），6微升20倍钙储备溶液（最终浓度〜0.6毫摩尔），和0.7微升200X精子（终浓度〜700精核/微升）。缩放反应体积向上或向下是必要的。反转或轻轻拍打至混合。 注:摘自中期循环进入间期为核组装一个更强大和可靠的平台。 </li><li>孵育在16水浴 - 20℃90分钟。通过每15分钟轻轻拍打，以确保核留在提取暂停混合。 </li><li>通过准备一个壁球如下显示器在45分钟的核组装的进度。 <ol><li>吸取2微升提取物的载玻片上，加入2微升核修复。 </li><li>与22毫米2盖玻片叠 ​​加。图像上使用水，油，或空气20的落射荧光显微镜 - 100X目标和一个DAPI过滤。 注:核可以通过亮场成像的可视化，但更容易被荧光显现。 </li><li>在-80℃，使用立即细胞核或闪存冷冻等份在液氮和存储。 注:添加4％甘油的核PRIOR冻结有助于保持其完整性。冻结核仍普遍可行的，有能力核进口的，但是冻结过程可能会导致中断或结构更脆弱的核。核高达冻结​​一年后即可使用。 </li></ol></li></ol> 4. 十的研制蟾胚胎提取物<ol><li>诱发女性X.蟾青蛙在1.1和1.2描述产卵。 </li><li>隔离男性睾丸X.在2.2中描述蟾青蛙。淹没补充有50IU / ml青霉素和链霉素的在L15媒体睾丸中的玻璃35×10毫米的培养皿。储存在4℃下最多两个星期。 </li><li>收集和卵子受精。 <ol><li>通过举办青蛙在一个可重复使用的玻璃培养皿中收集新鲜鸡蛋和施加温和的压力，下背部泄殖腔附近的促进产蛋。挤压青蛙太硬会造成损伤，导致臃肿的腹部和REQuiring安乐死29。 </li><li>道具的菜在大约45°角，让鸡蛋的菜边收集，去除所有多余的缓冲区，补充足够的1/3倍MMR勉强盖住鸡蛋。收集15分钟内卵子受精。 </li><li>使用紧密配合杵浸渍并在1.5ml试管用高盐500微升改性巴特的盐水（高盐MBS）均匀睾丸的1/4。浸软睾丸添加到蛋和允许受精在室温下搅拌15发生 - 20分钟，然后淹没鸡蛋1/3×MMR。 注意:睾丸效能随着储存时间而减小，因此，可以利用该已存储了超过一个星期睾丸当需要为成功受精更睾丸组织。 </li><li>除了睾丸粉碎后的30分钟 - 通过检查暗色素动物极收缩，并通过与他们的动物极胚胎的旋转朝上，通常约20施肥确认秒。期望第一小区裂解以90分钟内发生。 </li><li>使用大口径玻璃吸管努力去除死皮（白色和浮肿或蛋黄和色素分布不均匀），裂解，或者未受精的卵子（ 即未切割），因为它们会迅速导致死亡邻国胚胎。 </li><li>任何地方从1 - 2.5小时受精后，胚胎dejelly在2 - 3％半胱氨酸溶解在1/3点¯x孕产妇死亡率和10 N KOH调节pH值至7.9。执行是每3两缓冲区的改变 - 4分钟。彻底清洗胚6 - 101/3×MMR的简要变化以除去半胱氨酸的所有痕迹。 注:Dejellied胚胎有卵黄膜和不作为dejellied鸡蛋一样脆弱。 </li></ol></li><li>使用大口径玻璃吸管，健康受精（ 即，切除）的胚胎转移到含有新鲜的1/3×MMR培养皿，并允许他们发展到在RT所需阶段，或者，如果发展较慢是优选的，16℃。继续以去除死或LYSED胚胎缺乏第一师或蓬松的白色外观的指示。 </li><li>通过检查胚的几乎完全封闭并且通过参考材料24进行比较，以提供分级的胚胎的附图验证胚胎的阶段。 注意:胚胎在16℃下培养将达阶段11.5 - 在约12小时12。 </li><li>孵育阶段11.5 - 12胚在补充有0.5mM的放线菌酮在RT 1小时新鲜的1/3×MMR，在后期相间逮捕胚胎。 </li><li>与大口径玻璃或塑料吸管至少15（优选30个或更多）相间被捕胚胎离心管转移。 注:15胚胎足以产生大约20微升提取物。根据需要扩展。 </li><li>添加辅以1微升LPC股票1毫升鸡蛋裂解液（ELB）。轻轻颠倒洗胚胎，允许胚胎下降到管的底部，并删除缓冲器。重复此洗涤步骤两次。&lt;/ LI&gt; <li>于500μlELB加上0.5μl的LPC库存，5微升19.7毫酮和5微升35.5毫细胞松弛素的D悬浮胚胎（抑制肌动蛋白聚合）。离心在200 xg离心在台式微量1分钟。 </li><li>删除多余的缓冲区，并用杵彻底地粉碎胚胎。在离心机吊桶式转子在10000 XG 10分钟和16℃。 </li><li>从与200μl的移液管尖的顶端穿刺脂质层。用干净的200μl枪头，去掉中间的细胞质琥珀色层到合适大小的管。 </li><li>用1/50补充胚胎提取物的50倍能源结构中的体积（以提供一个ATP再生系统），1/100 35.5毫环己酰胺，1/500 19.7毫细胞松弛素的D量，和1 / 1,000的量的体积LPC股票。 </li><li>如3.3所描述的可视化在提取物中的内源性的胚胎的细胞核制备南瓜。 注意:胚胎提取可以与冷冻<html>在这一点上的内源性核。分装，以减少冷冻/解冻循环并储存在-80℃。 </li><li>从胚胎提取除去细胞核，稀释提取物用等体积ELB含1/50的50×能源结构中的体积，1/100 35.5毫环己酰胺的体积，1/500 19.7毫细胞松弛素D中的体积，并1 / 1,000 LPC股票的音量。离心机在17000 XG 15分钟吊桶式转子在16℃。 </li><li>收集上清小心，以避免在顶部的任何剩余的脂质。在管的底部不会干扰沉淀细胞核。如3.3中所述，以保证大多数核已被删除准备一个南瓜。使用提取新鲜或分装和储存在-80℃。 </li><li>热灭活对于对照实验中，热30胚胎提取物 - 提取物的100微升等分试样在56℃，使用热循环仪30分钟。允许热灭活的提取物返回到在使用前室温。 </li></ol>_title"&gt; 5。核试验萎缩和免疫<ol><li>在1.5ml管150微升预先装配核在1ml ELB - 通过稀释25隔离在卵提取物组装核。离心机在台式微量离心机1600 xg离心3分钟，4℃。删除缓存，小心不要打扰核的颗粒脆弱在试管的底部。 </li><li>悬浮核在胚胎提取等于5.1使用卵提取物的原体积的体积。使用ELB或控制反应热灭活提取物，适当的。 </li><li>轻轻敲击管，打破了颗粒和悬浮细胞核。在室温下孵育90分钟，轻轻拍打管以混合每15 - 30分钟。注意:大多数核萎缩出现的第一个30分钟内。 </li><li>通过准备一个壁球监测核萎缩的进展。 <ol><li>吸取2微升提取物的载玻片上，加入2微升核修复。 </li><li>覆盖有22毫米2 </s达&gt;盖玻片。图像上的落射荧光显微镜。 </li></ol></li><li>轻按管悬浮核之前，为了加入500微升由ELB的固定液，15％甘油和2.6％多聚甲醛。立即倒置，并将管在旋转器上在室温下15分钟。 </li><li>通过准备舾装15毫升圆底塑料衬垫（设计，可根据要求提供原理图）圆底玻璃管降速管。加入5毫升核气垫缓冲。下降12mm的圆形盖玻片入管，应确保其平放在塑料插入件的顶端。 </li><li>轻轻层含有上使用宽孔移液管尖端核衬垫的顶部固定核溶液。离心机的水平转头在1000 XG 15分钟和16℃。 </li><li>使用吸气器除去所有的缓冲液中，该塑料插入件拉至管的顶部。搭配一双细镊子取出的盖玻片，小心注意盖一侧滑其上细胞核纺丝。在冷的甲醇修复交（储存在-20℃）在RT 5分钟。 </li><li>盖玻片核面朝上转移到塑料薄膜石蜡衬的大塑料培养皿中的薄片。将湿的一次性湿巾沿盘，以防止脱水的一面。 </li><li>补充水分与500微升PBS-NP40的（1X PBS加0.1％NP40）和抽吸后5盖玻片核 - 10秒。 </li><li>仔细层75微升的PBS-3％牛血清白蛋白（BSA）的上盖玻片。允许阻止在室温下1小时或过夜，在4℃。 </li><li>除去封闭溶液，并用一级抗体孵育（ 例如，mAb414针对核孔复合物，1:1,000）在PBS-3％BSA中稀释，在室温下或4℃过夜1小时。用PBS-NP40五个立即改变清洗。 </li><li>用在PBS-3％BSA中稀释，在室温1小时的二级抗体孵育。用PBS-NP40五个立即改变清洗。 </li><li>孵化用10微克/毫升的Hoechst稀释在PBS-3％BSA在室温下5分钟。用PBS-NP40洗五次。删除所有多余的缓冲区。 </li><li>安装盖玻片到5微升防褪色的安装介质的幻灯片。用透明指甲油密封。图片立即用20一落射荧光显微镜 - 在4℃100X目标或商店后成像。 注意:如前面所述23进行量化。 </li></ol>

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	<li>Conklin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+and+nuclear+size.">Cell size and nuclear size.</a> J. Exp. Embryol. 12, 1-98 (1912).</li><li>Wilson, E. B. <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 Cell in Development and Heredity. , 727-733 (1925).</li><li>Levy, D. L., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+size+is+regulated+by+importin+alpha+and+Ntf2+in+Xenopus.">Nuclear size is regulated by importin alpha and Ntf2 in Xenopus.</a> Cell. 143 (2), 288-298 (2010).</li><li>Chan, Y. H., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+properties+of+cell+and+organelle+size.">Scaling properties of cell and organelle size.</a> Organogenesis. 6 (2), 88-96 (2010).</li><li>Walters, A. D., Bommakanti, A., Cohen-Fix, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+nucleus:+Factors+and+forces.">Shaping the nucleus: Factors and forces.</a> J Cell Biochem. 113 (9), 2813-2821 (2012).</li><li>Webster, M., Witkin, K. L., Cohen-Fix, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+up+the+nucleus:+nuclear+shape,+size+and+nuclear-envelope+assembly.">Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly.</a> J Cell Sci. 122 (Pt 10), 1477-1486 (2009).</li><li>Edens, L. J., White, K. H., Jevtic, P., Li, X., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+size+regulation:+from+single+cells+to+development+and+disease.">Nuclear size regulation: from single cells to development and disease.</a> Trends Cell Biol. 23 (4), 151-159 (2013).</li><li>Jevtic, P., Edens, L. J., Vukovic, L. D., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+and+shaping+the+nucleus:+mechanisms+and+significance.">Sizing and shaping the nucleus: mechanisms and significance.</a> Curr Opin Cell Biol. 28, 16-27 (2014).</li><li>Levy, D. L., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+intracellular+scaling.">Mechanisms of intracellular scaling.</a> Annu Rev Cell Dev Biol. 28, 113-135 (2012).</li><li>Vukovic, L. D., Jevtic, P., Edens, L. J., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+mechanisms+and+functions+of+nuclear+size+regulation.">New insights into the mechanisms and functions of nuclear size regulation.</a> Int Rev Cell Mol Biol. 322, 1-59 (2016).</li><li>Jevtic, P., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+size+scaling+during+Xenopus+early+development+contributes+to+midblastula+transition+timing.">Nuclear size scaling during Xenopus early development contributes to midblastula transition timing.</a> Curr Biol. 25 (1), 45-52 (2015).</li><li>Hara, Y., Iwabuchi, M., Ohsumi, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranuclear+DNA+density+affects+chromosome+condensation+in+metazoans.">Intranuclear DNA density affects chromosome condensation in metazoans.</a> Mol Biol Cell. 24 (15), 2442-2453 (2013).</li><li>Zink, D., Fischer, A. H., Nickerson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+structure+in+cancer+cells.">Nuclear structure in cancer cells.</a> Nat Rev Cancer. 4 (9), 677-687 (2004).</li><li>Jevtic, P., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+nuclear+size+regulation+in+model+systems+and+cancer.">Mechanisms of nuclear size regulation in model systems and cancer.</a> Adv Exp Med Biol. 773, 537-569 (2014).</li><li>Chow, K. H., Factor, R. E., Ullman, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+envelope+environment+and+its+cancer+connections.">The nuclear envelope environment and its cancer connections.</a> Nat Rev Cancer. 12 (3), 196-209 (2012).</li><li>Blom, J. H., Ten Kate, F. J., Schroeder, F. H., van der Heul, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometrically+estimated+variation+in+nuclear+size.+A+useful+tool+in+grading+prostatic+cancer.">Morphometrically estimated variation in nuclear size. A useful tool in grading prostatic cancer.</a> Urol Res. 18 (2), 93-99 (1990).</li><li>Dey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+nucleus:+morphology+and+beyond.">Cancer nucleus: morphology and beyond.</a> Diagn Cytopathol. 38 (5), 382-390 (2010).</li><li>Cross, M. K., Powers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+about+cancer+from+frogs:+analysis+of+mitotic+spindles+in+Xenopus+egg+extracts.">Learning about cancer from frogs: analysis of mitotic spindles in Xenopus egg extracts.</a> Dis Model Mech. 2 (11-12), 541-547 (2009).</li><li>Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M., Rapoport, T. 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+class+of+membrane+proteins+shaping+the+tubular+endoplasmic+reticulum.">A class of membrane proteins shaping the tubular endoplasmic reticulum.</a> Cell. 124 (3), 573-586 (2006).</li><li>Chan, R. C., Forbes, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts.">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li><li>Edens, L. J., Levy, D. L., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus Development. , 325-345 (2014).</li><li>Levy, D. L., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Scaling+Problems+and+Solutions+in+Amphibians.">Biological Scaling Problems and Solutions in Amphibians.</a> Cold Spring Harb Perspect Biol. 8 (1), a019166 (2016).</li><li>Edens, L. J., Levy, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cPKC+regulates+interphase+nuclear+size+during+Xenopus+development.">cPKC regulates interphase nuclear size during Xenopus development.</a> J Cell Biol. 206 (4), 473-483 (2014).</li><li>Nieuwkoop, P. D., Faber, 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> Normal Table of Xenopus laevis (Daudin). , (1967).</li><li>Newport, J., Kirschner, 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+major+developmental+transition+in+early+Xenopus+embryos:+II.+Control+of+the+onset+of+transcription.">A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription.</a> Cell. 30 (3), 687-696 (1982).</li><li>Newport, J., Kirschner, 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+major+developmental+transition+in+early+Xenopus+embryos:+I.+characterization+and+timing+of+cellular+changes+at+the+midblastula+stage.">A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30 (3), 675-686 (1982).</li><li>Maresca, T. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts.">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li><li>Hannak, E., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+mitotic+spindle+assembly+and+function+in+vitro+using+Xenopus+laevis+egg+extracts.">Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.</a> Nat Protoc. 1 (5), 2305-2314 (2006).</li><li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+extracts.">Cell cycle extracts.</a> Methods Cell Biol. 36, 581-605 (1991).</li><li>Khan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+alterations+in+nuclear+structure+predict+prostate+carcinoma+distant+metastasis+and+death+in+men+with+biochemical+recurrence+after+radical+prostatectomy.">Quantitative alterations in nuclear structure predict prostate carcinoma distant metastasis and death in men with biochemical recurrence after radical prostatectomy.</a> Cancer. 98 (12), 2583-2591 (2003).</li><li>Tan, P. H., Goh, B. B., Chiang, G., Bay, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+nuclear+morphometry+with+pathologic+parameters+in+ductal+carcinoma+in+situ+of+the+breast.">Correlation of nuclear morphometry with pathologic parameters in ductal carcinoma in situ of the breast.</a> Mod Pathol. 14 (10), 937-941 (2001).</li><li>Zeimet, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+ploidy,+nuclear+size,+proliferation+index+and+DNA-hypomethylation+in+ovarian+cancer.">DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer.</a> Gynecol Oncol. 121 (1), 24-31 (2011).</li><li>Theerthagiri, G., Eisenhardt, N., Schwarz, H., Antonin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleoporin+Nup188+controls+passage+of+membrane+proteins+across+the+nuclear+pore+complex.">The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex.</a> J Cell Biol. 189 (7), 1129-1142 (2010).</li><li>Wuhr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+upper+limit+to+mitotic+spindle+length.">Evidence for an upper limit to mitotic spindle length.</a> Curr Biol. 18 (16), 1256-1261 (2008).</li><li>Loughlin, R., Wilbur, J. D., McNally, F. J., Nedelec, F. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Katanin+contributes+to+interspecies+spindle+length+scaling+in+Xenopus.">Katanin contributes to interspecies spindle length scaling in Xenopus.</a> Cell. 147 (6), 1397-1407 (2011).</li><li>Wilbur, J. D., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+scaling+during+Xenopus+development+by+kif2a+and+importin.">Mitotic spindle scaling during Xenopus development by kif2a and importin.</a> eLife. 2, e00290 (2013).</li><li>Kieserman, E. K., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+size+scaling+in+Xenopus.">Mitotic chromosome size scaling in Xenopus.</a> Cell Cycle. 10 (22), 3863-3870 (2011).</li><li>Maresca, T. J., Freedman, B. S., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H1+is+essential+for+mitotic+chromosome+architecture+and+segregation+in+Xenopus+laevis+egg+extracts.">Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts.</a> J Cell Biol. 169 (6), 859-869 (2005).</li><li>MacCallum, D. E., Losada, A., Kobayashi, R., Hirano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISWI+remodeling+complexes+in+Xenopus+egg+extracts:+identification+as+major+chromosomal+components+that+are+regulated+by+INCENP-aurora+B.">ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B.</a> Mol Biol Cell. 13 (1), 25-39 (2002).</li><li>Goehring, N. W., Hyman, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+growth+control+through+limiting+pools+of+cytoplasmic+components.">Organelle growth control through limiting pools of cytoplasmic components.</a> Curr Biol. 22 (9), R330-R339 (2012).</li><li>Du Toit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development:+Honey,+I+shrunk+the+nucleus.">Development: Honey, I shrunk the nucleus.</a> Nat Rev Mol Cell Biol. 15 (10), 633 (2014).</li><li>Buchner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+protein+kinase+C+in+the+regulation+of+cell+growth+and+in+signalling+to+the+cell+nucleus.">The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus.</a> J Cancer Res Clin Oncol. 126 (1), 1-11 (2000).</li><li>Mochly-Rosen, D., Das, K., Grimes, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C,+an+elusive+therapeutic+target.">Protein kinase C, an elusive therapeutic target.</a> Nat Rev Drug Discov. 11 (12), 937-957 (2012).</li></ol>

The authors have nothing to disclose.

Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink...

细胞器的尺寸通常比例与电池的尺寸，这已被也许是最好的核大小的与细胞大小1-10缩放记录。这是胚胎发育和细胞分化，当这两种细胞和核大小显着减少经常观察到11,12时尤其如此。此外，改变的核尺寸是在癌症诊断和预后13-17的关键参数。有助于核大小调控机制在很大程度上是未知的，部分是由于复杂性和核的结构和功能的基本性质。这里所描述的方法被开发作为核大小缩放体外测定是适合于生物化学操作和核大小调控机制澄清。 非洲爪蟾卵提取物是一个行之有效的制度来概括和研究在体外复杂的细胞过程</em&gt;背景。这些提取物已经透露了几个细胞过程，包括装配和有丝分裂纺锤体，内质网的功能，以及核18-22新的基础资料。向萃取系统的一个主要优点是，X。蟾卵提取物代表几乎未稀释的细胞质的组合物可以容易地改变，例如通过加入重组蛋白或免疫耗竭的。此外，一个是能够通过采用处理，否则可能会在体内上下文致死操纵基本过程。蛋提取过程的变型允许从X的提取物的隔离蟾胚胎而不是卵，这些胚胎提取物也同样适合于生物化学操作23。在X.蟾发展，单细胞受精胚胎（约1毫米直径）经历一系列十二快速细胞分裂（阶段1 - 8），以产生几千50μ米直径和较小的小区，达到一个发育阶段称为midblastula过渡（MBT）或阶段8月24日至26日。 MBT的特点是合子转录，细胞迁移，异步细胞分裂，获取间隙相的，并建立无稳态大小，而不是连续的核扩张为在预MBT胚胎的发作。从第4阶段到原肠胚形成（阶段10.5 - 12），从个人细胞核的体积超过10倍11减小。 在这里，我们的目标是，以确定负责发育进程中这些削减核大小的机制。该方法是先在组装核X.蟾卵提取物和从卵胞浆/提取那些核隔离。然后，这些核悬浮在细胞质中晚期原肠期胚胎。孵育期后，从卵提取物晶核成为在后期胚胎提取物小。我们的理由是竟被这d是用于识别存在于晚期胚胎有助于发展核大小缩放细胞质成分的有用试验。使用这种测定法，再加上在体内验证，我们表明，蛋白激酶C（PKC）有助于在十核大小发育削减蟾 23。

<table><tbody><tr><td>Alexa Fluor 568 Donkey anti-mouse IgG</td><td>Molecular Probes</td><td>A10037</td><td></td></tr><tr><td>ATP disodium salt</td><td>Sigma Aldrich</td><td>A2383</td><td></td></tr><tr><td>Benzocaine</td><td>Sigma Aldrich</td><td>E1501</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A3059</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C3306</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>J2-21M</td><td></td></tr><tr><td>Centrifuge rotor</td><td>Beckman</td><td>JS 13.1</td><td></td></tr><tr><td>chymostatin</td><td>Sigma Aldrich</td><td>C7268</td><td></td></tr><tr><td>creatine phosphate disodium</td><td>Calbiochem</td><td>2380</td><td></td></tr><tr><td>cycloheximide</td><td>Sigma Aldrich</td><td>C6255</td><td></td></tr><tr><td>cytochalasin D</td><td>Sigma Aldrich</td><td>C8273</td><td></td></tr><tr><td>disposable wipes (kimwipes)</td><td>Sigma Aldrich</td><td>Z188956</td><td></td></tr><tr><td>L-cysteine</td><td>Sigma Aldrich</td><td>W326306</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma Aldrich</td><td>F8775</td><td></td></tr><tr><td>Glass crystallizing dish (150 x 75 mm)</td><td>VWR</td><td>89090-662</td><td></td></tr><tr><td>Glycerol</td><td>Macron</td><td>5094-16</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>Hoechst - bisBenzimide H 33342 trihydrochloride</td><td>Sigma Aldrich</td><td>B2261</td><td></td></tr><tr><td>HCG - Human Chorionic Gonadotropin&amp;nbsp;</td><td>Prospec</td><td>hor-250-c</td><td></td></tr><tr><td>L15 Media</td><td>Sigma Aldrich</td><td>L4386</td><td></td></tr><tr><td>leupeptin</td><td>Sigma Aldrich</td><td>L2884</td><td></td></tr><tr><td>Lysolecithin</td><td>Sigma Aldrich</td><td>L1381</td><td></td></tr><tr><td>mAb414</td><td>Abcam</td><td>ab24609</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>EMD</td><td>MX0045-2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M9397</td><td></td></tr><tr><td>Maltose</td><td>Sigma Aldrich</td><td>M5885</td><td></td></tr><tr><td>NP40</td><td>BDH</td><td>56009</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicillin + Streptomycin</td><td>Sigma Aldrich</td><td>Pp0781</td><td></td></tr><tr><td>pepstatin</td><td>Sigma Aldrich</td><td>P5318</td><td></td></tr><tr><td>PIPES</td><td>Sigma Aldrich</td><td>P6757</td><td></td></tr><tr><td>Plastic paraffin film (parafilm)</td><td>Sigma Aldrich</td><td>P7793</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Mallinckrodt</td><td>70100</td><td></td></tr><tr><td>KOH</td><td>Baker</td><td>5 3140</td><td></td></tr><tr><td>PMSG - Pregnant Mare Serum Gonadotropin</td><td>Prospec</td><td>hor-272-a</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>BP328</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>SX0720-1</td><td></td></tr><tr><td>NaOH</td><td>EMD</td><td>SX0590</td><td></td></tr><tr><td>Pestle</td><td>Thomas Scientific</td><td>3411D56</td><td></td></tr><tr><td>Round bottom glass tubes, 15 ml</td><td>Corex</td><td>8441</td><td></td></tr><tr><td>Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)</td><td>ThermoFisher</td><td>A10037</td><td></td></tr><tr><td>sucrose</td><td>Calbiochem</td><td>8550</td><td></td></tr><tr><td>thermal cycler</td><td>Bio-Rad</td><td>T100</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>L8-80M</td><td></td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman</td><td>SW 50.1</td><td></td></tr><tr><td>Vectashield (anti-fade mounting medium)</td><td>Vector</td><td>H-1000</td><td></td></tr></tbody></table>

a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation

在细胞生物学中的一个基本问题是细胞和细胞器的尺寸是如何监管。人们早已认识到，核的大小通常与该单元的大小时，在这两种细胞显着减少和核大小发生磅秤，尤其是在胚胎发生期间。核大小调控机制在很大程度上是未知的，并且可以是相关的癌症，其中改变的核的大小是关键的诊断和预后参数。 体内接近识别核大小调节由核函数的必要性和复杂性是复杂的。这里描述的研究核大小控制的体外方法考虑在核大小爪蟾发展过程中发生的正常减少的优点。首先，细胞核被组装在十蟾卵提取物。然后，这些晶核分离并再悬浮于细胞质从晚期胚胎。经过30 - 90分钟的潜伏期，核表面积20下降 - 60％，提供了有益的试验，以确定目前在后期阶段的胚胎，有助于发展核大小缩放细胞质成分。这种方法的主要优点是相对设施与该卵和胚胎提取物可生化操纵，从而允许调节核大小新型蛋白质和活动的识别。正如任何体外方法中， 在体内系统的结果的验证是重要的，和X的显微注射蟾胚胎特别适用于这些研究。

核的大会卵提取物 此协议的第一步是准备X.蟾卵提取物（协议1）和demembranated精子细胞核（协议2）。然后，这些试剂用于组装细胞核从头 （协议3）。 图1示出了一些有代表性的数据。钙的添加驱动减数分裂逮捕卵提取物成相间，和放线菌酮保持在间被捕的提取物。由30 - 反应开始45分钟?...

Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

无细胞测定中使用<em&gt;非洲爪蟾</em&gt;胚胎提取研究核的大小调节机制。

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

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Research

JoVE Journal

Biology

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

8.3K 次观看.  University of Wyoming. Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

视频: A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

生物化学

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

生物学

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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 &#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 ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

无细胞测定中使用

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利用共聚焦分析

无细胞爪卵萃取物微乳液细胞周期振荡的重建

制备及分馏爪蟾卵提取

在使用分馏爪蟾卵提取物的体外核大会

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

生产

重组β-catenin的降解

无细胞化验，研究染色质解聚在有丝分裂的结束