This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

所有的爪蟾实验在里尔第一大学的动物实验室根据欧盟委员会的准则（86/609 / EEC），实验室动物实验的规定执行。动物方案经当地机构审查委员会（科米特德Ethique连接实验安尼米尔北部 - 加来海峡，CEEA 07/2010）。 1.卵母细胞处理<ol><li>准备麻醉剂溶液：溶于1升的无菌水1克三卡因甲基磺酸粉末。 </li><li>切入女性爪蟾到这个解决方案，覆盖了烧杯，以避免逃逸，并等待大约45分钟的动物完全镇静剂（没有任何反应，一条腿捏）。 </li><li>用肥皂清洗青蛙和用自来水冲洗然后将其在清洁的铝箔回动物。 </li><li>夹紧腹部的侧部和在卵巢水平与钳皮肤。使大约1厘米长的切口，用剪刀用70％乙醇，使用前清洗。确保部分足够深，以达到潜在的腹壁，使卵巢组织中的几个卵母细胞发育的不同阶段被发现的切除。 </li><li>解剖卵巢裂片，在培养皿中（50毫米）与ND96介质（96毫摩尔NaCl，2mM的氯化钾冲洗4次，1毫氯化镁 ，1.8毫氯化钙 ，5mM的HEPES调节至pH 7.4，用NaOH为辅用50微克/ ml链霉素/青霉素，225微克/毫升丙酮酸钠，30微克/毫升大豆胰蛋白酶抑制剂，1微升/毫升四环素），除去血液和碎屑所有痕迹。 注：四环素允许最佳的保护和显微注射治疗后恢复良好。 </li><li>其存储在覆盖的培养皿在14℃下浸没在介质中，从而允许它们的保存一周。 </li><li>缝合腹壁并与兽医可吸收吨皮肤hread和缝合针（3或4针将是必要的）。 </li><li>放置在一个烧杯中的动物没有水和潮湿的皮肤用自来水直到蛙被再次移动。盖烧杯，以防止逃逸。 </li><li>通过使用下，用10倍的倍率在立体显微镜2对钳子的仔细分离中在5至10的卵母细胞群在介质卵巢瓣。在第六阶段选择的卵母细胞。他们可以通过我承认）的形状和颜色，用棕色色素动物极和黄色植物极有明确的皮带二）其规模优于1.2毫米直径10分开。 </li><li> 45分钟期间孵育（ 溶组织梭菌，溶解在ND96无四环素1毫克/毫升胶原酶A）中在缓慢搅拌下培养皿胶原酶溶液所选的卵母细胞，以促进卵母细胞defolliculation（胶原酶消化卵母细胞/滤泡细胞连接）。冲洗卵母细胞3或4次，ND96介质保持在14C。 注意：不要孵化的卵母细胞少于或多于45分钟，由于破坏卵母细胞表面的蛋白质，造成生存能力差的风险。胶原酶治疗可引起自发性GVBD。 </li><li>在3-4小时的中等孵化的卵母细胞。在双目放大镜删除滤泡细胞用细镊子，并保持它们在19℃的ND96网上平台。 </li></ol> 2.准备mRNA合成<ol><li>通过使用PME我酶酶切线性化5微克以下质粒。 注：pcDNA6.2 /含1599个氨基酸eIF4G1基因野生型（eIF4G1-WT; NM_198241）V5-DEST，包含eIF4G1显性基因（eIF4G1-DN）pcDNA6.2 / V5-DEST有突变c.2105T “G c.2106A&gt; C，c.2120-&gt; G，c.2122T&gt; A，2125T&gt; -和c.2126T”G在eIF4G1和eIF4E的相互作用在位置612到相应的蛋白质618的区域干扰互动betwe恩eIF4G1和eIF4E的8，pcDNA6.2 / C-EmGFP含氯霉素乙酰转移酶（GFP）。准备2的混合物为每个3质粒：第一包括PME I酶和第二不含酶作为对照。 </li><li>使用经典乙醇沉淀过程质粒DNA重悬它于20μl不含核酸酶的H 2 O的</li><li>使用分光光度计测定其浓度。浓度要优于150毫微克/微升转录的cRNA。 </li><li>运行1微升样本以及它们与5微升加样缓冲液在0.8％琼脂糖凝胶对照来验证质粒线性化。 </li><li>使用体外转录，并根据制造商的说明纯化的cRNA的试剂盒。转录的cRNA再悬浮于20μl不含核酸酶的H 2 O的样品可以贮存于-80℃下，如果需要的。 </li><li>准备含有0.2M MOPS（pH值7.0），20米MOPS 10X迁移缓冲液3M乙酸钠和10mM EDTA（pH8.0）中。消毒用0.45μm过滤溶液。库存保护免受光在RT以避免溶液氧化该溶液中。 </li><li>先后清洗电泳槽用NaOH，HCl和双过滤水的O彻底冲洗/ N，以避免RNA酶，防止RNA降解。 </li><li>投含有MOPS和甲醛1.5％的琼脂糖凝胶。温馨直至完全溶解琼脂糖，让它冷却至55℃。下通风橱中，添加0.1体积的MOPS 10X的，甲醛的6.6％和4微克溴化乙锭（10毫克/毫升）。倾通风柜下凝胶并等待凝胶变硬大约1小时。 注意：甲醛和溴化乙锭是有毒的。 </li><li>制备样品迁移在一个0.2毫升的试管用1微升的cRNA，甲醛的8.8％，甲酰胺的60％和0.1体积的MOPS 10X的。孵育3分钟，在70℃，把管冰浴10分钟。离心机样品几秒钟5000×g的。加入2微升凝​​胶上样缓冲液中。 注意：甲酰胺是有毒的。 </li><li>运行样品20分钟，在90V。浸泡在无核酸酶H 2 OO / N凝胶分析它来检查质量的cRNA之前脱色凝胶。 </li><li>确定的cRNA浓度使用分光光度计，并将它们存储在-80℃。 </li></ol> 3.显微注射合成的RNA和卵母细胞成熟刺激<ol><li>准备必要的设备，卵母细胞显微注射。使用微量拉马拉小玻璃毛细管。体视显微镜下，断绝毛细管的末端与镊子以创建一个平端。使用1ml注射器微孔0.45μm的过滤在相对的末端填充毛细管微量矿物油。安装微量到显微注射吸管。选择一个精确的微量校正由制造商提供良好的精度，适当的玻璃毛细管中使用时。 </li><li>执行注射defolliculation后1-2小​​时，需要延迟对卵母细胞的卵母细胞的恢复保持在14℃。使用培养皿的底部刮下用镊子创建用于卵母细胞的粘合表面和ND96介质填充它们。 </li><li>安排沿着在培养皿允许逐次注射的卵母细胞与毛细管微量的在45℃的角度的刮车道的卵母细胞。 </li><li>注入在卵母细胞赤道区域，着色动物区域下方样品在细胞质一个最佳扩散。插入毛细管尖端的上大约150-200微米深度只有最薄的部分。 </li><li>注入的cRNA 30纳克来自不同质粒在一个容积为60 NL在第一卵母细胞分别获得。注射后，等待5-10秒钟取出毛细管尖端，以避免样品逃生（ 图2A）之前。 注意：不要超过120 NL。作为注入慢慢地，你可以。建议用蒸馏水的控制。 </li><li>手动将培养皿到下一个卵母细胞注入。 注：确保尖端未通过产生一个小的脉冲出槽液后2〜3显微注射堵塞。 </li><li>在24孔培养板转移注入的卵母细胞（10卵母细胞/孔）装有3毫升新鲜ND96介质和离开他们在19℃。 </li><li>孵育在ND96的卵母细胞用孕酮（PG）（2微克/毫升）在19℃以触发细胞成熟（GVBD），15小时或分别从pcDNA6.2 / V5-DESTeIF4G1和pcDNA6所得聚腺苷酸化的cRNA的显微注射后4小时0.2 / C-EmGFP用作对照（图2A）。 注意：共注射荧光标记物与的cRNA是一个很好的工具，以确保的cRNA注入并保持在卵母细胞。执行使用了GFP标记感兴趣的cRNA这样的初步试验。 </li><li> Microinject如在＃3.5不同比率（1:3; 2：2; 3：1）的多聚腺苷酸化eIF4G1-WT和eIF4G1-DN的cRNA以忒ST的eIF4G1-WT的cRNA的突变表型（图2D）的翻译作用。 </li></ol> 4.胚泡破裂测定<ol><li>通过观察在黑色动物极用立体显微镜一个白点的存在下计数的PG刺激后的成熟卵母细胞的数目。重复计数每隔一小时，直至第二十四小时（图2B，2C）。 </li></ol> 5.免疫印迹（WB）分析<ol><li>均质化组的10卵母细胞通过来回运动用微量尖，在4℃在200μl在下列缓冲液：50mM HEPES，pH值7.4，500 mM氯化钠，0.05％SDS，5mM的MgCl 2的，1毫克/ ml牛血清白蛋白，10毫克/毫升亮肽素，10毫克/ ml抑肽酶，10mg / ml的大豆胰蛋白酶抑制剂，10毫克/毫升苄脒，1mM的PMSF，1mM的钒酸钠。 </li><li>离心样品15分钟以10,000×g离心以除去脂质（上层相）和膜（下层相）Fractions。收集细胞质部分，存储一个等分试样，以确定蛋白质浓度，并完成与的Laemmli或双Tris样品缓冲液，其余（1：1）。 注意：Bis-Tris凝胶和加载缓冲液具有更中性的pH，保护蛋白质水解</li><li>加热20微克的样品在95℃下进行10分钟。每个样品加载到丙烯酰胺凝胶的孔中。将凝胶在一个适当的缓冲罐。 </li><li> 1小时，在200五执行电泳</li><li>实现一个WB在TBS pH 8.0的液（Tris盐酸15mM的，氯化钠150mM的，吐温0.1％，含10％牛血清白蛋白）与山羊抗 - 极光激酶A /为Eg2（1：3000，2小时）或用兔抗-Aurora A /为Eg2-P（1：1000，O / N在4°C），鼠抗ERK2（1：3000，2小时），羊抗ERK2-P（Tyr204上）（1：3000，2小时），兔抗金属氧化物半导体（1：5000，4小时），兔抗GFP（1：3000，2小时）antibodies.Use抗体小鼠抗V5（1：10,000，2小时）和兔抗替代Rsk（1 ：1000，2小时）（作为上样对照）。 </li><li>洗刷该膜在TBS-Tween中孵育1小时，用3次，每次10分钟，或者抗小鼠或抗兔或抗山羊（IgM抗体）辣根过氧化物酶标记的二抗以1稀释：5000，1：7500和1 ：分别为5000。 </li><li>执行10分钟3次洗涤在TBS-Tween中并检测抗原 - 抗体复合物与高级的ECL检测系统。 </li></ol> 6.多聚腺苷酸含量<ol><li>每样条件5卵母细胞在1.5ml。用1毫升无核酸酶PBS 1X（pH值7.4）清洗。以下步骤将根据通风柜制成。 </li><li> RNA提取使用传统的有机过程（参见制造商的说明）。 </li><li>回收通过离心（10,000×g离心）5分钟的RNA在4℃。除去上清，空气干燥RNA的10分钟。 </li><li>于30μl不含核酸酶的H 2 O的重悬的RNA沉淀</li><li>取在一个新的管15微升样品，并添加85微升不含核酸酶的H 2 O的清理本身样品根据生产商的说明改善其在硅胶上的柱质量。洗脱使用30微升无核酸酶H 2 O的的RNA </li><li>运行1微升的样品，用5微升加样缓冲液在0.8％琼脂糖凝胶上以检查RNA的完整性。 </li><li>确定使用分光光度计RNA浓度。 </li><li>制备每种条件2连接混合物（即，eIF4G1-WT，eIF4G1-DN和H 2 O的控制）与以下试剂：10单位RNA连接酶，0.1体积的10X缓冲液，1mM ATP中，PEG8000 50％10％ 0.1底漆P1（5&#39;-P-GGTCACCTTGATCTGAAGC-NH2-3&#39;）11和使终体积到10微升用无核酸酶的 H 2 O的微克加在从卵母细胞无PG刺激获得的第一混合物的RNA和从在PG中获得的第二混合物的RNA刺激卵母细胞。 </li><li>孵育1小时，在37℃，然后20分钟在65℃下使酶失活。 </li><li>美国EA的cDNA反转录（RT）套件。准备RT混合物中，每管：0.1倍体积的10X RT缓冲液，0.1引物P2（5&#39;-GCTTCAGATCAAGGTGACCTTTTT）10，4毫米的dNTP混合，逆转录酶50单位和微克完全无核酸酶H 2 O的最终体积的10微升。加10微升先前获得的连接反应。执行对RT由生产所述的条件下。 </li><li>制备聚合酶链反应（PCR）混合物，每管：0.1体积缓冲器10X，1.5毫米氯化镁2，133μMdNTP混合液，0.2μM的特异性引物，0.2μM的引物P2，0.025 单位 Taq聚合酶，1μl的cDNA的和完整的无核酸酶H 2 O操作的50微升最终体积。执行的PCR在以下条件下：50℃，2分钟，95℃，10分钟，[95℃1分钟，56℃，30秒，72℃30秒] * 40个循环，72℃ 10分钟9。的特异性引物如下：金属氧化物半导体，GTTGCATTGCTGTTTAAGTGGTAA组蛋白样B4，AGTGACAAACTAGGCTGATATACT;细胞周期蛋白A1，CATTGAACTGCTTCATTTTCCCAG;细胞周期素B1：GTGGCATTCCAATTGTGTATTGTT 9。 </li><li>制备3％的琼脂糖凝胶。添加在每个PCR产物10微升样缓冲液。运行与10微升在110伏的样品的凝胶为最佳的迁移。后10和20分钟分析凝胶来观察的大小反映了尾多聚（A），因此核糖核酸成熟是一个先决条件，其翻译的长度的变化。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+recruitment+of+Xenopus+maternal+mRNAs+in+response+to+poly(A)+elongation+requires+initiation+factor+eIF4G-1.">Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.</a> Dev Biol. 206 (1), 1-14 (1999).</li><li>Wakiyama, M., Imataka, H., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+eIF4G+with+poly(A)-binding+protein+stimulates+translation+and+is+critical+for+Xenopus.oocyte+maturation.">Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus.oocyte maturation.</a> Curr Biol. 10 (18), 1147-1150 (2000).</li><li>Sheets, M. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+function+of+Mos:+the+control+switch+of+vertebrate+oocyte+meiosis.">Synthesis and function of Mos: the control switch of vertebrate oocyte meiosis.</a> Bioessays. 19 (1), 23-28 (1997).</li><li>Le Sommer, C., Lerivray, H., Lesimple, M., Hardy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+a+model+to+study+alternative+splicing+in+vivo.">Xenopus as a model to study alternative splicing in vivo.</a> Biol Cell. 99 (1), 55-65 (2007).</li><li>Au, S., Cohen, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis.oocytes+as+a+system+for+studying+nuclear+transport+of+viruses.">Microinjection of Xenopus laevis.oocytes as a system for studying nuclear transport of viruses.</a> Methods. 51 (1), 114-120 (2010).</li><li>Gotoh, T., Villa, L. 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The authors have nothing to disclose.

翻译是一个涉及许多人类疾病，包括几种神经退行性疾病的病理生理机制。例如，在帕金森氏病的几份报告建议与遗传性基因突变8,12,13相关的翻译的损害。 一些细胞模型可用来研究翻译。这里，为了研究在eIF4G1的突变，作为显性负突变减少eIF4G1和eIF4E的伙伴8之间的相互作用的平移后果，在非洲爪蟾卵母细胞中。该模型具有的优点是简单，因为它仅由一...

蛋白是细胞生命的较大规模的生物体的基本要素，因此。它们确保大部分的细胞功能，包括结构，运输，反应催化，调节，基因表达等。它们的表达是翻译的允许的mRNA转化成蛋白质的复杂机制的结果。翻译进行各种控制，以适应并根据该小区需要调节基因表达，发育和分化，衰老，生理应力或病理表现期间。 翻译分为3个阶段（起始，延伸和终止），并提出3开始翻译系统，以满足这些需求： 通过内部核糖体进入段（IRES）的结构和总量控制的独立翻译增强（帽依赖性，依赖帽CITE）。 大多数真核生物基因的翻译在帽DEPE经由 7-甲基-5&#39;-三磷酸帽在作为在蛋白质合成的识别特征ndent方式。此帽结合eIF4E的，的eIF4F复杂与eIF4G1和eIF4A的成分。合并有其他合作伙伴，如聚（一）结合蛋白（PABP）的eIF2-GTP-MET-tRNA的满足，这些翻译起始因子允许环化表达，提高其辅助形式43S复杂，直到AUG起始密码子识别 1。这个事件对应于翻译起始即翻译的第一步骤的结束。 章独立翻译为紧张的条件下诱导，例如细胞增殖和凋亡下必需的蛋白质使用的基因编码。此机制涉及的二级结构在mRNA的5&#39;非翻译区（UTR）称为IRES，eIF4G1的羧基末端与eIF4A和43S复合物有关。该43S启动前C中的结合omplex到IRES启动而不需要eIF4E的因素2,3的帽依赖性翻译。 最后，另一种转换机制仍然没有得到很好的理解支持这种依赖帽的翻译活动强调的条件下，通过 CITE位于mRNA的UTR 4内的结构。 通过翻译通过起始步骤不同的这些各种模式，翻译在细胞内环境稳定的重要作用，并在这些过程中的一个的任何改变从而将影响生物体有小到大规模的效果。事实上，起始是限速步骤理事mRNA的正确翻译流程成蛋白质，因此许多控制和调节点5的目标。无论是对于后者，或这些过程的组成部分，如果一个结果是有缺陷的，它会扰乱在细胞内建立平衡，从而有可能导致病理康迪系统蒸发散。在此背景下，翻译因子的突变已经参与了一些障碍，包括神经退行性疾病，如`白质脑病与消失的白色matter&#39;（eIF2B1-5亚基）6，沃尔科特，Rallison综合征（用于PERK EIF2AK3基因编码）7，潜在帕金森氏病（eIF4G1 p.R1205H）8。因此重要的是要进行的细胞和分子这些突变蛋白的研究，以增加对疾病的发展和对翻译起始的一般过程我们的知识。 为了进行这些研究，有必要选择最适当的模式来观察这些突变的后果非洲爪蟾卵母细胞特别适合由于他们的生理生化特征：生理同步（阻挡在细胞周期的阶段G2）蛋白质合成的高容量（200-400毫微克/天/卵母细胞），高数提取OOC的从同一动物ytes（800-1000的卵母细胞/孔）和细胞尺寸（1.2-1.4毫米直径），这便于它们的操作。 爪蟾卵母与合成mRNA的显微注射能够容易地进行剖析翻译步骤。这种观点认为它提出的其他优点。由于减数分裂进展和翻译后核显微注射（〜24小时）的速度， 非洲爪蟾卵母细胞代表一个快速系统相比，重组蜂窝系统（从大肠杆菌中提取，小麦胚芽或兔网织红细胞...），其中的mRNA是翻译具有减小翻译速率和以较低的速度。所以，在与mRNA引入一个突变的影响将是迅速地观测和容易地研究了几个卵母细胞。 爪蟾卵母的另一个优点是，产妇的mRNA是潜在的，蛋白质翻译孕酮刺激前阻止。因此，加用孕激素是控制翻译归纳的很好的手段。细胞质polyadenylation不会发生卵子发生过程中。它开始时在一时间顺序孕酮刺激的卵母细胞的卵母细胞成熟和持续贯穿早期发展，并且可以用于研究不同步骤的翻译。 MOS mRNA的多聚腺苷酸化是最先出现，并与极光A /为Eg2，组蛋白样B4基因属于类如查尔斯沃思等人定义的“早熟” 的基因。（2004）9。平移感应“迟到”表达，如细胞周期蛋白A1和细胞周期素B1左右出现的生发泡破裂（GVBD）的时间。金属氧化物半导体的mRNA编码的丝氨酸/苏氨酸 - 蛋白激酶。它的翻译是至关重要的，因为它导致了MAP激酶级联反应，间接地激活卵母细胞成熟。事实上，响应于孕酮，MOS mRNA的聚腺苷酸化是通过涉及极光A /为Eg2调节蛋白和其它RNA结合蛋白与第一个过程增强MOS mRNA的3&#39;UTRË。这种增加的MOS mRNA的聚腺苷酸化导致增加MOS蛋白质水平，从而激活MEK1。此过程介导的胞外信令调节的激酶2（ERK2）（图1）的活化。然后，该信号级联可以触发成熟M相促进因子，通过细胞周期蛋白B和的Cdc2激酶形成的复杂，并最终导致在减数分裂恢复。 因此，在非洲爪蟾卵母细胞 ，产妇的mRNA如马鞍山的研究可以方便地使用他们的高效聚腺苷酸化有几个端点，以测试他们译的几个MOS信号元件，其中也包括GVBD率的测定翻译。这个系统是有趣的，因此要评估翻译起始因子突变的第一个后果，而没有转染效率新的mRNA转录或干扰，问题往往与eukary发生耳细胞研究。 这里，协议被建立，其中突变eIF4G1的mRNA显微注射在爪蟾卵母细胞和母体mRNA翻译进行测试。在在GVBD进展的缺陷，金属氧化物半导体的mRNA聚腺苷酸化是用于进程必需通过卵母细胞减数分裂细胞周期和为早期和晚期的mRNA类随后翻译的存在下确定。磷酸极光A /为Eg2和ERK还研究确认的MOS deregulation.Thus的结果， 爪蟾卵母代表来分析不同步骤mRNA翻译的一个简单的方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tricaine methane sulphonate</td>
			<td>Sandoz</td>
			<td>MS-222</td>
			<td rowspan="8">oocytes handling</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Moria</td>
			<td>Dumont MC40</td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>Sigma</td>
			<td>781</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Tetracyclin</td>
			<td>Sigma</td>
			<td>T7660</td>
		</tr>
		<tr>
			<td>Veterinarian absorbable Vicryl thread</td>
			<td>Johnson&#38;Johson Intl</td>
			<td>JV1205</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche diagnostics</td>
			<td>10103586001</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolabs</td>
			<td>R0560S</td>
			<td rowspan="14">Preparation of synthetic mRNA</td>
		</tr>
		<tr>
			<td>DNAse/RNAse free H2O</td>
			<td>Life Technologies</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Merck</td>
			<td>6268</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sigma</td>
			<td>02854</td>
		</tr>
		<tr>
			<td>Nano Drop</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE 10X</td>
			<td>Eppendorf</td>
			<td>0032006.507</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10 mg/mL</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE&#174; Kit</td>
			<td>Ambion by Life Technologies</td>
			<td>AM1344</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fluka</td>
			<td>03609</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Merck</td>
			<td>1.04003.1000</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
		</tr>
		<tr>
			<td>Gel Doc Imager</td>
			<td>Biorad</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Pipet with 100 8&#34; Glass Capillaries</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-510-X</td>
			<td rowspan="4">microinjection</td>
		</tr>
		<tr>
			<td>Replacement Glass 8&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-210-G8</td>
		</tr>
		<tr>
			<td>0,45 &#181;m filter</td>
			<td>Millipore</td>
			<td>SLHA025NB</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P0130</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td rowspan="32">Western Blot</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Biorad</td>
			<td>161-0301</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>A3294</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4612</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L8511</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
		</tr>
		<tr>
			<td>benzamidine</td>
			<td>Sigma</td>
			<td>B6506</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr>
			<td>sodium vanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Biorad</td>
			<td>161-0737</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td>
			<td>Life Technologies</td>
			<td>NP0323BOX</td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis, mini-Protean TGX</td>
			<td>Biorad</td>
			<td>456-1036 and -1096</td>
		</tr>
		<tr>
			<td>horizontal semi-dry blotting system</td>
			<td>W.E.P. Compagny</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Biorad</td>
			<td>161-0719</td>
		</tr>
		<tr>
			<td>Hybond ECL Membrane</td>
			<td>Amersham Life Science</td>
			<td>10401180</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20846-292</td>
		</tr>
		<tr>
			<td>Ponceau Red (0,5%)</td>
			<td>Sigma</td>
			<td>P3504</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Life Technologies</td>
			<td>A11122</td>
		</tr>
		<tr>
			<td>anti-V5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-58052</td>
		</tr>
		<tr>
			<td>anti-Eg2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-27884</td>
		</tr>
		<tr>
			<td>anti-Eg2-P</td>
			<td>Cell Signaling</td>
			<td>C39D8</td>
		</tr>
		<tr>
			<td>anti-ERK2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1647</td>
		</tr>
		<tr>
			<td>anti-ERK2-P (Tyr204)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7976</td>
		</tr>
		<tr>
			<td>anti-Rsk</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-231</td>
		</tr>
		<tr>
			<td>anti-mos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86</td>
		</tr>
		<tr>
			<td>anti-mouse horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
		</tr>
		<tr>
			<td>anti-rabbit horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2812</td>
		</tr>
		<tr>
			<td>anti-goat horseradish peroxidase labeled secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2378</td>
		</tr>
		<tr>
			<td>Advanced ECL Detection System</td>
			<td>Amershan Life Science</td>
			<td>RPN2135</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td rowspan="11">polyadenylation assay</td>
		</tr>
		<tr>
			<td>TRIZOL (Qiazol Lysis Reagent)</td>
			<td>Qiagen</td>
			<td>79306</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>31998-8</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>278475-1L</td>
		</tr>
		<tr>
			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr>
			<td>RTL buffer&#160;</td>
			<td>Qiagen</td>
			<td>79216</td>
		</tr>
		<tr>
			<td>RNAse free DNAse set</td>
			<td>Qiagen</td>
			<td>79254</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Eurogentec</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1&#160;</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
		</tr>
		<tr>
			<td>High capacity c-DNA Reverse Transcription kit</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4368813</td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>Life Technologies</td>
			<td>10342020</td>
		</tr>
	</tbody>
</table>

xenopus laevis as a model to identify translation impairment

蛋白质的合成是一个基本过程的基因表达影响不同的生物学过程特别是适应环境条件。引发步骤，其中涉及核糖体亚基在mRNA起始密码子，参与起始因子包括eIF4G1的组装。在翻译这个限速步骤的缺陷都与不同的疾病。为了研究这种放松管制的潜在后果， 非洲爪蟾卵母细胞构成具有高程度的保护与人类基本的细胞和分子机制的一个有吸引力的模式。此外，在母细胞成熟，卵母细胞转录抑制和所有必需的蛋白从先前存在的，母体衍生的mRNA翻译。这种廉价的模型使外源基因，成为一种有效的翻译完美地融为一体。这里描述了一种协议，用于使用保存位置评估翻译用感兴趣的因子（此处eIF4G1）编产妇的mRNA是第一个被聚腺苷酸化，并在卵母细胞成熟作为生理读数翻译。起初，合成出的mRNA 通过体外转录感兴趣的质粒（此处eIF4G1）的卵母细胞和卵母细胞成熟的动力学通过胚泡破裂检测注入被确定。所研究的母体mRNA靶是丝氨酸/苏氨酸 - 蛋白激酶MOS。其多聚腺苷酸化和随后的翻译与所述MOS信号级联涉及卵母细胞成熟的蛋白质的表达和磷酸化研究在一起。当前协议的变化，提出翻译缺陷也提出来强调其普遍适用性。在光新兴的证据表明，异常蛋白合成可能参与神经障碍的发病机制，这样的模式提供了机会，轻松地评估这个障碍，并确定新的目标。

爪蟾卵母细胞和确定所述卵母细胞百分比GVBD的24小时的PG刺激（ 图2B，2C）后动力学成熟： 为了研究所述eIF4G1-DN突变的平移后果， 在非洲爪蟾给PG响应蟾卵母细胞显微注射的cRNA eIF4G1-DN进行比较eIF4G1-WT和其它控制条件（H 2 O的，GFP）。该控制使评估卵母细胞显微注射的发生不论eIF4G1过表达的注射的cRNA或性质。 10卵?...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

JoVE Journal

Biology

Xenopus laevis as a Model to Identify Translation Impairment

10.6K 次观看.  INSERM UMR-S1172, CHRU Lille, University of Lille 1 and Lille 2. Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

视频: Xenopus laevis as a Model to Identify Translation Impairment

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

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 ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

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 ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...