This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

所有的实验程序都经弗吉尼亚机构动物护理和使用委员会的大学。 注意: 图1示出的实验步骤的示意图。 1.准备卵母细胞<ol><li>前总理X.蟾女性，150ü孕马血清促性腺激素（PMSG）大约提前一个星期的卵母细胞隔离。注射1毫升150单位/毫升PMSG到背部淋巴囊有1毫升无菌注射器用29克针。 </li><li>准备注入卵母细胞和卵母细胞动物帽检测解决方案。 <ol><li>制备的Ca ++ / Mg ++的-free OR2（OR2-）:82 mM氯化钠，2.5mM的氯化钾，1.5mM的钠2 HPO 4，50mM的HEPES pH7.2的。 </li><li>准备OR 2:OR2-加1毫米氯化钙2和1毫米氯化镁2。 </li><li>制备OCM:60％的Leibovitz L15，0.4毫克/毫升BSA，100μg/ ml的庆大霉素，pH值7.8。 </li><li>预削1×MBS:88 mM氯化钠，1mM的氯化钾，0.7毫米氯化钙2，1mM的MgSO 4干燥，5mM的HEPES（pH值7.8），2.5毫碳酸氢钠。 </li><li>准备3％聚蔗糖在1X MBS。 </li><li>制备1×NAM:110 mM氯化钠，2mM的氯化钾，1毫米的Ca（NO 3）2，1mM的MgSO 4干燥，0.1毫摩尔EDTA，1毫碳酸氢钠 ，2毫的Na 3 PO 4，pH 7.4中。 </li></ol></li><li>麻醉雌性在3-氨基苯甲酸乙酯甲磺酸盐在0.1倍的MBS 0.03％溶液（MS222）通过将蛙在溶液中（第一溶解在10ml 95％乙醇0.3克MS222，然后稀释在1升0.1×MBS） 10 - 15分钟或直到反应迟钝。 <ol><li>检查麻醉完成后转动麻醉青蛙到它的后面，以确保它不会响应（不完全麻醉的青蛙四肢移动或转动身体以上）。 </li></ol></li><li>手术通过穿过皮肤和体壁与手术刀片腹部切口，分离卵巢组织钳分离卵巢片段和剪刀。将卵巢碎片进入OR2-。收身的墙上用3-0丝线在一个24mm弯针，并分别与相同缝合关闭皮肤。允许女性的恢复在1克/升鱼缸盐水。 </li><li>用细镊子，撕裂卵巢组织成小（10 - 20卵母细胞）件，转移至新鲜OR2-。 </li><li>在OR2- 2.0毫克/毫升胶原酶甲Defolliculate卵巢片段轻轻搅动在摇床上1小时，转移到新鲜胶原酶和搅拌一小时。 </li><li>洗卵母细胞的10倍OR 2 [含Ca ++ / Mg ++的]，舍弃小卵母细胞。 </li><li>在OCM洗卵母细胞的两倍，并转移到新鲜OCM。在目视检查隔离第六阶段的卵母细胞通过尺寸和丢弃未成熟（较小）的卵母细胞:第六阶段的卵母细胞是大于未成熟卵母细胞即使色素沉着动物半球和大约1.2-1.4毫米的直径。 </li><li>卵母细胞保持在18 - 20°C IñOCM注射前。注意:琼脂糖包被的培养皿中，可以使用最小化的卵母细胞的粘附抛出。 </li></ol> 2.注射图书馆成绩单<ol><li>制备定向cDNA文库15使用RNA在发展的适当阶段（例如，神经板级14 10）萃取。购买的cDNA文库，或者使用下面的四个步骤每概览​​商品化试剂盒构造之一。 <ol><li>通过使用产品以富集多聚（A）+ RNA（见表材料）和下列制造商的说明分离约5微克的mRNA。注意:要使用的转录物产生的cDNA文库可以被限制为一个诱导的组织（如神经板，以下所需组织的显微切割），或者可以是从全胚胎在特定阶段。 </li><li>产生的cDNA用市售试剂盒，按照制造商的指示。 </li><li>结扎大约20纳克的cDNA到vectoř附带市售试剂盒或其他合适的载体中。合适的质粒载体包括用于mRNA稳定性如5&#39;β珠蛋白和3多聚（A）序列（PCS2 + pTnT，pCS105）序列。 </li><li>转换连接入使用供应商推荐的协议，用于热休克转化能力的细菌细胞。 注意:可替换地，开放阅读框（ORFeome）克隆的集合是可​​商购，并且可以用于产生转录物用于注射。 </li></ol></li><li>制备的10 3的质粒的10池10 4复杂性（10 4〜10 5的总的复杂性）。这代表10板的1000 - 每套1殖民地。 <ol><li>板库培养到10 15厘米LB-氨苄青霉素板，成长12 - 18小时在37℃，并收集从每个用一个玻璃吊具温和的压力菌落在7ml LB. </li><li>从0.5毫升准备甘油股票（添加到0.2毫升无菌甘油并储存在-20℃＃730; C）中，并用剩余的6.5 ml至与标准制备DNA市售的DNA miniprep试剂依照制造商的指示。 </li></ol></li><li>线性合并的质粒DNA（1.0 - 2.0微克）用合适的限制性酶消化16在37℃，1 - 1.5小时。隔离用苯酚/氯仿提取，随后用乙醇沉淀和再悬浮在每所使用的RNA聚合酶试剂盒的规格水的线性化的DNA。合成有义RNA用市售RNA聚合酶试剂盒按照生产商的指示。 </li><li>制备约20微米的直径的针显微注射（使用针拔出器与玻璃毛细管）;衡量一个复合式显微镜与校准的目镜测微尺针尖。注:针拔出器设置必须根据经验确定生产针用细尖，这样当用细镊子破碎产生期望的针尖大小。 </li><li>准备（粘土推入在培养皿底部）均匀一层黏土衬里35×10毫米，平行的凹槽显微注射期间举行的地方卵母细胞培养皿中;产生的凹槽与融合在火焰尖端商场探头或巴斯德吸管。作为一种替代粘土，准备做的压痕的弹性模 17琼脂糖菜肴。转移的卵母细胞用宽口移液管至3％聚蔗糖在1X MBS（约2毫升）中的粘土内衬菜肴行。 </li><li>用微量注射器，填补针1毫微克/ NL RNA和调整平衡，产生轻微的正压力（以防止制订卵母细胞胞浆内）。 </li><li>注入的卵母细胞，在赤道区域大约20 NL核糖核酸。允许1小时的注入卵母细胞留在菲-MBS，然后轻轻地转移到1X MBS。在20℃之前动物帽测定24小时 - 孵育8。 </li></ol> 3.动物帽化验<ol><li>准备3/4倍不结盟运动和获得细镊子，发圈​​，弯曲盖玻片碎片，黏土衬里的DISHES杯状凹陷举行的卵母细胞。 （ - 2毫米×2 - 4 mm产品大约1），并穿过火焰直到边缘抛光和下垂，产生弯曲片通过分解盖玻片成小片段，使弯曲盖玻片片段。 </li><li>施肥X.蟾卵18通过在体外或自然交配和文化的原肠胚阶段-在0.1倍MBS（10 11小时受精后在室温）之前逐步排序;收集中期原肠胚（阶段11 - 11.5）10胚胎。 </li><li>转移到胚的培养皿大约半满与3/4×NAM。使用两对细镊子，从原肠胚取出施肥（卵黄）膜。 </li><li>卵母细胞转移到3/4倍NAM（约2毫升）的粘土成荫的菜肴和固定在粘土个人的印象，产生的印象，以适应单个胚胎的凹槽中如上所述。 </li><li>转移胚胎到粘土内衬菜切动物帽关克使用两对细镊子的astrulae。小心分离的动物帽外胚层，而不是只赤道组织18。 </li><li>放在每个卵母细胞与动物帽接触卵母细胞的内表面形式的动物用半球动物帽。 8小时或更长的时间 - 通过将弯曲的玻璃盖玻片片段并施加向下的压力施加到玻璃，压扁外胚层作为盖玻片接触粘土（动物帽可以保持与暴露于卵母细胞为6的表面内层开放保持重组体一起）。 注意:可替换地，将动物帽成粘土压痕与它的内表面朝上，并放置在盖的卵母细胞;保护与粘土的小扩展重组。 </li><li>文化在20°C，直到控制胚胎达到所需要的阶段检测。 </li><li>分离重组去除盖玻片/粘土和外胚层分离钳和吹风循环。 </li><li>修复外胚层片段1小时的MEMFA（3.8％甲醛在MEM [0.1M的MOPS pH为7.4，2mM的EGTA，和1mM MgSO 4干燥]）。 </li><li>转移片段（和控制胚胎）从MEMFA成乙醇和储存在-20℃。 </li></ol>反应在外胚层原位杂交4.分析（ISH） <ol><li>准备ISH的解决方案。 <ol><li>制备的1×PBS:0.01M磷酸盐缓冲盐水，氯化钠0.138男，pH 7.4的</li><li>制备PBS-吐温（PTW）:1×PBS，0.1％Tween-20的</li><li>准备100X Denhart的解决方案:2％BSA，2％的聚乙烯吡咯烷酮，2％聚蔗糖</li><li>制备杂交缓冲液:50％甲酰胺，5×SSC，1毫克/毫升圆酵母RNA，1微克/毫升肝素，1×Denhart溶液，0.1％吐温-20，0.1％CHAPS，10毫摩尔EDTA，DEPC-H 2 O的</li><li>准备马来酸缓冲器（MAB）:100毫米马来酸，150毫米氯化钠，pH值7.5 </li><li>准备MAB +块:MAB，2％封闭试剂（加热至60℃溶解） </li><li>准备碱性磷酸酶（AP）缓冲液:100毫米的TrispH值9.5，50毫米氯化镁2，100毫米氯化钠，0.1％吐温，卫生署2 O </li></ol></li><li>准备RNA探针。 <ol><li>用市售RNA聚合酶试剂盒，挖-NTP混合物，添加到1.5ml试管（50微升反应）:25.5微升DEPC-H 2 O，10微升5X转录缓冲液，2.5微升10X掏-NTP混合物，5微升100毫DTT，2微升的RNasin，2微升线性DNA模板（约1微克/微升），3μl的核糖核酸聚合酶，孵育37℃90分钟。 </li><li>添加2微升的RNA聚合酶，孵育37℃60分钟。 </li><li>检查2微升反应的1％琼脂糖凝胶。 </li><li>加入1μlRQ1无RNA的DNA酶孵育37℃20分钟。 </li><li>通过加入50μlDEPC-H 2 O，25微升10μM的乙酸铵和313微升乙醇沉淀探头。储存在-20℃过夜（O / N），然后通过离心分离回收的RNA在13800×g离心20分钟。用500微升75％乙醇洗涤，短暂离心，去掉乙醇并允许沉淀风干。加入50微升DEPC-H 2 O </li><li>添加杂交缓冲液至0.5微克/微升〜的终浓度。 </li></ol></li><li>准备组织进行杂交。除非另有说明，填充小瓶与每个解变化描述（约4毫升）中的顶部。 <ol><li>除去从小瓶和移植胚乙醇到75％乙醇/ PTW，然后用50％的乙醇/ PTW，每次10分钟，水平方向上的摇杆。 </li><li>在PTW每个摇杆上的5分钟洗三次。 </li><li>转移到10微克/微升蛋白酶K处理的PTW;岩管垂直15分钟。 </li><li>岩管垂直 - 在0.1M三乙醇胺pH值7.8冲洗两次，每次10分钟。 </li><li>添加12.5微升乙酸酐到管和岩石竖直5分钟。重复用另外的12.5微升乙酸酐5分钟。 </li><li>在PTW 5分钟垂直于摇杆清洗。 </li><li> Refix在4％多聚甲醛20分钟的摇杆。热杂交缓冲液到60℃。 </li><li>在PTW每个摇杆上的5分钟洗三次。 </li><li>从各管中取出PTW的所有，但约1毫升加250微升HYB缓冲;轻轻摇动管混匀。岩管竖直5分钟。 </li><li>替换60℃HYB缓冲液（0.5毫升）和轻轻搅动在60℃10分钟。更换新鲜HYB缓冲器和搅在60℃两至4小时。 </li><li>热探针（1混合物在0.5微克/微升在HYB缓冲液）到60℃（3分钟）。删除HYB缓冲区，并添加探头管。轻轻搅动O / N在60℃。 </li></ol></li><li>准备组织的抗体。 <ol><li>温暖HYB缓冲器和2x SSC + 0.1％Tween-20的溶液至60℃。 </li><li>更换HYB缓冲探针溶液（保存探头在-20℃下2 - 3倍重用）。在60℃10分钟洗净。 </li><li>在60℃在2×SSC-吐温（每个搅拌20分钟）洗涤三次。 </li><li>在60℃在0.2×SSC-吐温（每个搅拌20分钟）洗涤三次。 </li><li>在生物圈（RT）搅拌15分钟，各水平上摇杆洗两次。 </li><li>加入1毫升MAB +块。在摇杆垂直洗2小时。 </li><li>转移至1ml MAB +模块包含一个1 / 2,000抗洋地黄毒苷-AP。垂直岩石在4 CO / N。 </li></ol></li><li>准备组织的显色剂。 <ol><li>更换单克隆抗体的解​​决方案;在人与生物圈洗三次，每次5分钟水平上的摇杆。 </li><li>人与生物圈1小时，各水平上的摇杆洗三次。 </li><li>在AP缓冲液10分钟，各水平上摇臂洗两次。 </li><li>转移组织到多个孔塑料托盘，取出AP缓冲液，并添加BM紫试剂（〜1毫升）中。允许反应进行在黑暗5分钟，O / N。 </li><li>在适当的时候染色来实现，组织转移到PTW的小瓶。在摇杆的PTW洗两次，每次10分钟。 </li><li>修正了布安的4 CO / N在摇杆的解决方案。 </li><li>洗出布安的三个70％乙醇/ 30％PTW洗涤在室温。继续牛逼Ø图像采集使用的外部照明，并在显微镜安装摄像头，漂白如有必要，18。 </li></ol></li></ol> 5.同胞选择和克隆<ol><li>最高的活动选择池（外胚层组织中最大的响应）。 </li><li>滴定（的LB稀释至合适的密度）的甘油原液用于与活性最高的池和积垢10的新板用大约十分之一从（使用协议部2的上方）上一步骤的菌落。 </li><li>减少池大小，直到活动被追踪到单一的殖民地。 </li><li>使用标准的引物中的载体得到的DNA序列。 </li></ol>

<ol>
	<li>Yergeau, D., Kelley, C. ., Zhu, H., Kuliyev, E., Mead, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+Genetic+Screens+in+Xenopus.+Using+Transposon-Mediated+Insertional+Mutagenesis.">Forward Genetic Screens in Xenopus. Using Transposon-Mediated Insertional Mutagenesis.</a> Methods in Molecular Biology. 917, 111-127 (2012).</li><li>Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.+as+a+Model+Organism+for+Genetics+and+Genomics:+Past,+Present+and+Future.">Xenopus tropicalis. as a Model Organism for Genetics and Genomics: Past, Present and Future.</a> Methods in Molecular Biology. 917, 3-15 (2012).</li><li>Nakayama, T., Fish, M., Fisher, M., Oomen-Hajagos, J., Thomsen, G., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+Efficient+CRISPR/Cas9-Mediated+Targeted+Mutagenesis+in+Xenopus+tropicalis.">Simple and Efficient CRISPR/Cas9-Mediated Targeted Mutagenesis in Xenopus tropicalis.</a> Genesis. 51, 835-843 (2013).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis.">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.</a> Biology Open. 1 (12), 1273-1276 (2012).</li><li>Zhao, Y., Ishibashi, S., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetic+Studies+Using+Antisense+Morpholino+Oligonucleotides.">Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides.</a> Methods in Molecular Biology. 917, 143-154 (2012).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injected+Xwnt-8.+RNA+acts+early+in+Xenopus.+embryos+to+promote+formation+of+a+vegetal+dorsalizing+center.">Injected Xwnt-8. RNA acts early in Xenopus. embryos to promote formation of a vegetal dorsalizing center.</a> Cell. 67, 753-765 (1991).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin.,+a+new+dorsalizing+factor+localized+in+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin., a new dorsalizing factor localized in the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Plautz, C., Zirkle, B., Deshote, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+induction+of+anterior+head+ectodermal+properties+in+Xenopus.+embryos+are+mediated+by+transcriptional+cofactor+ldb1.">Early stages of induction of anterior head ectodermal properties in Xenopus. embryos are mediated by transcriptional cofactor ldb1.</a> Developmental Dynamics. 243 (12), 1606-1618 (2014).</li><li>Servetnick, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+neural+and+lens+competence+in+Xenopus.+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus. ectoderm: evidence for an autonomous developmental timer.</a> Development. 112, 177-188 (1991).</li><li>Nieuwkoop, P., 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). , (1994).</li><li>Lemaire, P., Garrett, N., Gurdon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+Siamois.,+a+Xenopus.+homeobox+gene+expressed+in+dorsal-vegetal+cells+of+blastulae+and+able+to+induce+a+complete+secondary+axis.">Expression cloning of Siamois., a Xenopus. homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.</a> Cell. 81, 85-94 (1995).</li><li>Lustig, K., Kroll, K., Sun, E., 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=Expression+cloning+of+a+Xenopus+T.-related+gene+(Xombi.)+involved+in+mesodermal+patterning+and+blastopore+lip+formation.">Expression cloning of a Xenopus T.-related gene (Xombi.) involved in mesodermal patterning and blastopore lip formation.</a> Development. 122, 4001-4012 (1996).</li><li>Hsu, D., Economides, A., Wang, X., Eimon, P., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus.+dorsalizing+factor+Gremlin+identifies+a+novel+family+of+secreted+proteins+that+antagonize+BMP+activities.">The Xenopus. dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.</a> Molecular Cell. 1 (5), 673-683 (1998).</li><li>Lustig, K., 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=Use+of+an+oocyte+expression+assay+to+reconstitute+inductive+signaling.">Use of an oocyte expression assay to reconstitute inductive signaling.</a> Proc. Natl. Acad. Sci. USA. 92, 6234-6238 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE Science Education Database. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests.">Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> JoVE Science Education Database. , (2015).</li><li>Viczian, A., Zuber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Determination+Using+the+Animal+Cap+Transplant+(ACT)+Assay+in+Xenopus+laevis.">Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis.</a> J. Vis. Exp. (39), e1932 (2010).</li><li>Sive, H., Grainger, R., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of.Xenopus laevis.: A laboratory manual. , (2000).</li><li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activation+during+early+stages+of+lens+induction+in+Xenopus.">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li><li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxe+view+of+lens+development+and+disease.">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li><li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism.">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li><li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project.">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li><li>Karpinka, J., 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=Xenbase,+the+Xenopus.+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li><li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis.">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469 (2013).</li></ol>

The authors declare that they have no competing financial interests.

此处描述的基因能够诱导在主管外胚层的响应的功能性克隆的方法可以用于鉴定范围广泛的基因产物。该方法通过将组织诱导试验用表达克隆技术扩展了过去的工作。我们利用爪蟾卵母细胞的代谢途径如生产诱发因素，直接或间接地，以下的RNA注射的来源。此，在与使用已建立的方法进行克隆目的6,7-使用从cDNA文库或其他集合克隆的产生转录物表达的基因组合，提供了一个有价值的...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">12/101 Antibody</td>
			<td style="width:191px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:135px;">12/101</td>
			<td style="width:233px;">Monoclonal antibody for detection of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">20X SSC Buffer</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">S6639</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Acetic anhydride</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A6404</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Anti-Dig-AP</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11093274910</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Aurum Plasmid Mini Kit</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:135px;">732-6400</td>
			<td style="width:233px;">Plasmid DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Blocking Reagent</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11096176001</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">BM Purple</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11442074001</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Boekel Hybridization Oven</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">13-245-121</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Bouin&#39;s Solution</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">HT10132</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">BSA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A9647</td>
			<td style="width:233px;">for OCM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">CHAPS</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C3023</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Collagenase A</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">10103578001</td>
			<td style="width:233px;">Defolliculation of oocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cysteine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">C121800</td>
			<td style="width:233px;">Dejelly embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">DEPC-H2O</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">BP5611</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Dig-RNA Labeling Mix</td>
			<td style="width:191px;">Roche</td>
			<td style="width:135px;">11277073910</td>
			<td style="width:233px;">for ISH probes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Dumont #5 forceps</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">500233</td>
			<td style="width:233px;">for Vitelline envelope removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ethyl 3-aminobenzoate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">A5040</td>
			<td style="width:233px;">MS222 anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Ficoll PM 400</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F4375</td>
			<td style="width:233px;">for Injection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Formamide</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">F9037</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Gentamicin sulfate</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G1914</td>
			<td style="width:233px;">for OCM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Glass capillaries</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">4878</td>
			<td style="width:233px;">3.5&#34; long, I,D, 0.530mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glass sample vials</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">06-408B</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Hair loop</td>
			<td style="width:191px;"></td>
			<td style="width:135px;"></td>
			<td style="width:233px;">Hair affixed in pasteur pipette for tissue manipulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium salt</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">H4784</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Injector Nanoliter 2010</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">Nanoliter 2010</td>
			<td style="width:233px;">Microprocessor-controlled microinjector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Instant Ocean</td>
			<td style="width:191px;">Carolina</td>
			<td style="width:135px;">972433</td>
			<td style="width:233px;">Aquarium Salt for frog recovery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">IRBG XGC Xenopus verified full-length cam cDNA</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">989_IRBG</td>
			<td style="width:233px;">cDNA library&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">LB Agar plates with 100 &#181;g/mL Ampicillin</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L5004</td>
			<td style="width:233px;">150mm pre-poured LB-Amp plates for sib selection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">LB Luria Broth</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:135px;">L8650</td>
			<td style="width:233px;">LB for collecting colonies in sib selection from plates and dilution of cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Magnetic mRNA Isolation Kit</td>
			<td style="width:191px;">New England BioLabs</td>
			<td style="width:135px;">S1550S</td>
			<td style="width:233px;">for isolation of poly(A)-enriched RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Maleic Acid</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">M0375</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Manual Microfil Micromanipulator</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">M3310R</td>
			<td style="width:233px;">Manual micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Nutating Mixer</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">22-363-152</td>
			<td style="width:233px;">Rocker for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Permoplast</td>
			<td style="width:191px;">Nasco</td>
			<td style="width:135px;">SB33495M</td>
			<td style="width:233px;">Clay for injection and dissection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate Buffered Saline</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P5368</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PMSG</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">G4877</td>
			<td style="width:233px;">to stimulate oocyte development</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Polyvinylpyrrolidone</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">PVP40</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Programmable Puller</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:135px;">PUL-1000</td>
			<td style="width:233px;">Micropipette needle puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Proteinase K</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P6556</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">pTnT Vector</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">L5610</td>
			<td style="width:233px;">cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Riboprobe Combination System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">P1450</td>
			<td style="width:233px;">in vitro transcription</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Superscript Full Length cDNA Library Construction Kit</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:135px;">18248013</td>
			<td style="width:233px;">kit for cDNA library construction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Sutures, 3-0 silk</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:135px;">19-037-516</td>
			<td style="width:233px;">Suture thread and needle for post-oocyte removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Torula RNA</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">R3629</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Triethanolamine</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">T1502</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween 20</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:135px;">P9416</td>
			<td style="width:233px;">for ISH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Universal RiboClone cDNA Synthesis System</td>
			<td style="width:191px;">Promega</td>
			<td style="width:135px;">C4360</td>
			<td style="width:233px;">alternative kit for cDNA library construction</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:207px;">Xenopus Full ORF Entry Clones - ORFeome Collaboration</td>
			<td style="width:191px;">Source Bioscience</td>
			<td style="width:135px;">5055_XenORFeome</td>
			<td style="width:233px;">ORFeome Clones</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">XL2-Blue Ultracompetent Cells</td>
			<td style="width:191px;">Agilent Technologies</td>
			<td style="width:135px;">200150</td>
			<td style="width:233px;">cells for transformation of cDNA library</td>
		</tr>
	</tbody>
</table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

响应于mRNA的注入卵母细胞，响应动物帽组织表达测定为OTX2的表达通过原位杂交（ 图2和表1）; OTX2通过透镜placode表达在推定透镜外胚层（PLE）从神经管闭合增厚19。然而，由于OTX2也表达于前神经外胚层以及PLE外非神经外胚层头，它与这两个神经和placodal应答有关。使用foxe3筛选文库为能够产生在透镜感受态动物帽外胚?...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K 次观看.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

视频: Functional Cloning Using a Xenopus Oocyte Expression System

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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 ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...