This work was supported by the University of Innsbruck, Austria, and the Austrian Academy of Sciences (&#214;AW).

1.准备显微注射和电穿孔的玻璃海鞘鸡蛋和合子<ol><li>准备10升人工海水与HEPES（ASWH）的胚胎培养。溶于420毫米氯化钠，9毫米氯化钾，10毫米氯化钙2·2H 2 O，24.5毫米氯化镁2·6H 2 O，25.5毫米硫酸镁4·7H 2 O和2.15毫米的NaHCO 3双蒸水（DDH 2 O）搅拌过夜。添加5mM的HEPES pH为8.0，并验证/调节pH至8.0。存放在4℃的ASWH。暖机至18℃，并在使用前用滤纸过滤。 </li><li>准备20X灭活dechorionation解决方案:20％的巯基乙酸钠，在ASWH 1％链霉蛋白酶。在-20℃下储存500微升等分试样，并稀释至使用前10毫升ASWH的终体积。 </li><li>准备15至20培养皿蛋/胚胎。倒入融化ASWH 1％琼脂糖到3.5，5，9或15厘米培养皿中，用1-2毫米的超薄层覆盖它们。让琼脂糖巩固和覆盖ASWH板。在4℃下最多1-2周库存它们。更换使用前ASWH。 </li><li>准备特殊的注射菜肴。 <ol><li>倒入融化的1％的5-7毫米厚的一层琼脂糖为5cm的培养皿中，并放置在琼脂糖模具（如粘到塑料盖玻片一个拉链锁关闭）在琼脂糖凝固，产生压痕可以容纳的鸡蛋。准备4〜5注射菜肴和ASWH覆盖。储存在4℃下并在使用前新鲜ASWH替换。仅使用注射新鲜平板（不超过1日龄）。 </li></ol></li><li>制备β半乳糖苷酶（LacZ基因）染色缓冲液:1mM的MgCl 2的·6H 2 O，3毫K 4的[Fe（CN）6]·3H 2 O，3毫K 3的[Fe（CN）6]在磷酸盐缓冲的盐水（PBS）吐温（PBT）（137毫摩尔NaCl，2.7毫米氯化钾，10毫的 Na 2 HPO 4，2mM的KH 2 PO 4，0.05％吐温）。我店n中的黑暗中在4℃。制备的LacZ衬底5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷（X-gal的，10单位/毫升）在40毫克/毫升在二甲基甲酰胺（DMF）和商店库存等分在-20℃直至使用。 </li><li>浸泡巴斯德吸液管中的自来水过夜，防止胚胎粘连。 </li><li>准备注射针头。 <ol><li>设置在如热425条件微量拉马，拉75，速度75和时间200（从415灯丝坡道测试）。 </li><li>拉4〜5含长丝玻璃毛细管产生8至10长，细针通常在尖端关闭。存储针在无尘针框，附着在高架的支撑双面胶带，以避免破坏的尖端和可方便地进行针填充。 注:通过注入几个鸡蛋带绿色活体染料（如第6条所述）测试获得的针和调整针头拔条件获得笔尖形状，允许微调和repetit在6.6中描述香港专业教育学院注入。 </li></ol></li><li>准备注射液。准备4微升注射液， 例如 ，1微升2毫米的MO，2微升10微克组成/微升绿活体染料和1微升的DDH 2 O.添加0.05-0.5微克/微升加帽mRNA或根据实验问题质粒DNA的20毫微克/微升的最终浓度。拌匀。置于冰上如果溶液中含有的mRNA。 </li></ol> 2.配子的收藏<ol><li>解剖玻璃海鞘成人18°C后，胚胎的房间。用小剪刀，并从该反对的呼气侧的虹吸管的末端开始（短）虹吸在其下输卵管和输精管运行。 </li><li>暴露输卵管和微妙使在使用剪刀的前端的输卵管的切口。直接丢弃流出鸡蛋打入轻轻按压与密闭剪刀剥离的如输卵管含有ASWH一个6孔板GS。用巴氏吸管预漂洗ASWH传输剩余的卵。 </li><li>从存款不同动物的卵在不同的井。根据观察解剖范围卵子的质量。 注:发育良好的蛋是圆的和粉红色至淡黄色的颜色。它们通过非蜂窝卵黄涂层，形成围绕蛋一个明确定义的卵周隙绒毛膜包围。绒毛膜与测试电池在其外侧其内部和星形毛囊细胞有关。低质量的鸡蛋往往缺乏卵周隙或毛囊细胞。他们显得不那么圆，更精细的或不同的颜色。未成熟卵母细胞体积较小，有一个银白色。 </li><li>切断与剪精子导管和收集浓缩精子成使用一个单独的巴斯德吸管1.5ml试管。凝聚的精子从不同动物（至少两个）到相同的管中。在4℃下储存精子数天。 </li></ol> 3。受精<ol><li>在步骤2中所述在18℃的收集精子和卵子。 </li><li>激活精子。 <ol><li>在1.5ml管中准备1毫升ASWH并用50μl的1M Tris pH值9.5的混合。然后加入20微升浓精，颠倒管轻轻混匀。 </li><li>检查精子的活化放在一个小培养皿的盖子或载玻片精子的下降和解剖范围下观察。该精子应该疯狂地游泳。 </li></ol></li><li>加入100-200微升活化精解的鸡蛋（100和1000之间的鸡蛋含）的每一个孔，吹打上下拌匀，使蛋在中漂浮。等待10分钟而不移动胚胎。 </li></ol> 4，卵子和胚胎的Dechorionation <ol><li>稀释20倍dechorionation溶液500微升等分10毫升ASWH。激活通过添加逐滴加入200μl的2 dechorionation溶液。5 M氢氧化钠至10毫升1X dechorionation解决方案。乳状会形成沉淀。轻轻颠倒试管混匀。 </li><li>收集卵或受精卵（在10分钟后等待在步骤3.3）转换成使用巴氏吸管的玻璃管。池鸡蛋从2至3只动物在一个管（约1,000-5,000卵/管）。沉积物与通过高速（1200 xg离心）为大约20秒纺丝手离心机，以形成在管的底部的胚胎沉淀。慢慢停止离心机并用巴氏吸管取出ASWH。 </li><li> 4毫升活化dechorionation溶液添加到各管中沉淀蛋/受精卵。轻轻吹打并上下用自来水处理巴斯德吸管用小橡皮梨荣登暂停鸡蛋/受精卵。后1-3分钟的解决方案应该变黄。 </li><li>通过除去使用巴斯德吸管dechorionating蛋/合子悬浮液的一小等分试样按照dechorionation。存款幻灯片上的下降和解剖是S观察应付。保持吹打卵/受精卵上下，检查每20-30秒。 注意:一是毛囊细胞分离，然后将蛋壳变成黄色，不透明，最后它从受精卵分离。粉dechorionated卵/合子将沉到玻璃管的底部。该dechorionation不应该超过最大值。 5分钟。 </li><li>与ASWH填充管一旦鸡蛋的50％/合子是dechorionated。 注:非常轻轻离心（8 XG），用于相应于一个非常低的速度刚好够以沉淀dechorionated卵/胚胎约10-15秒。慢慢停止离心机以不是在管的底部扰动卵/受精卵的沉淀。 </li><li>从玻璃管中取出，几乎所有的液体包括不完全dechorionated卵/受精卵的漂浮物和ASWH替换。慢慢吸管上下洗并轻轻如在步骤4.2再次离心。或者，等待受精卵通过重力定居下来。洗一次，直到没有绒毛膜碎片为lEFT。 </li><li>转印的蛋/合子到新鲜漂洗使用巴斯德吸管培养皿。保持在低浓度的受精卵（不蛋）（约每10 cm培养皿200胚胎），以避免它们粘在一起。培养胚胎到所需阶段在13和20℃之间的温度。可替代地，电穿孔合子（步骤5）或注入未受精的卵子（步骤6）。 </li></ol> 5.电穿孔<ol><li>受精的卵和dechorionate在18℃的受精卵中第3和第4，提供每电穿孔样品鸡蛋50和400之间。 </li><li>在1.5ml管（最大在50μlDDH 2 O的100微克的质粒DNA）制备50微升质粒DNA，并添加200μl的0.95摩尔的甘露醇。涡旋拌匀。 </li><li>调度和重力沉淀在硅化1.5毫升管的dechorionated受精卵。除去ASWH下降到管中的100μl的标记。 </li><li>继续，连续，每个受精卵管如下:add使用巴氏吸管的DNA /甘露醇溶液。与受精卵轻轻混匀，并立即采取了DNA /甘露醇/受精卵悬挂，并转移到4毫米的电比色皿。 </li><li>立即将试管放入电穿孔保持器，并在50五得到的16毫秒的单脉冲</li><li>使用相同的巴斯德吸管取下试管受精卵并传播出来的含有新鲜过滤ASWH培养皿。移液器受精卵他们开始在18℃裂解约1小时受精后（HPF）之前。 </li><li>冲洗样品之间的吸移管在ASWH的烧杯中。 </li><li>在文化以每10cm皿约200胚胎足够低的密度15-20℃的受精卵，以避免其粘在一起。 </li></ol> 6.显微注射<ol><li>准备Dechorionated鸡蛋注射。 <ol><li>分别Dechorionate鸡蛋，2-4的动物，如第4节所述保持受精和dechorionatedËGGS在独立的小琼脂糖涂层的培养皿。将鸡蛋洗净后多次在菜取出碎片。 </li><li>测试从每个受精鸡蛋dechorionated小批量（大约50）。使用一个个单独的5 cm的天线每批并申请2微升激活精子（步骤3.2）。纷飞的菜拌匀即可通过侧向运动的菜摊开鸡蛋的菜。保持精子和卵子一起约10分钟不动。 </li><li>消除一个最大精子如下:受精卵用新鲜ASWH转移到新鲜培养皿或冲洗两次。离开切割胚胎未扰动直到32细胞阶段（在18℃约3小时后受精）。选择最好的发展批次（施肥的最佳比例，最常规的切割方式）的显微注射鸡蛋。 </li></ol></li><li>建立了注射针。 <ol><li>回填在垂直位置4注射针头，以允许重力FL沿着长丝流。包含在该被定位针尖端向下针的后端绿色活体染料存款0.5微升注射溶液（步骤1.8）。等待液体底部填充针尖。 </li><li>水平重新定位针，并用细而长的金属或塑料吸管尖端与矿物油回填他们。慢慢地覆盖以矿物油排出所有气泡却使针的注射液。 </li></ol></li><li>设置在18℃的显微操作冷却的房间。 <ol><li>针保持器的塑料管连接到填充有矿物油10毫升玻璃注射器。回填管和油针保持器和排出任何气泡。 </li><li>插入针座的针，并在显微保持器的位置。 </li><li>调整在相对于表面45°的角度沿直线针托移动。 </li></ol></li><li>定向dechoriona泰德鸡蛋沿着喷射盘，使得蛋可以由一个在解剖显微镜下被注入一个的琼脂糖缩进。 </li><li>打破针尖，如果需要，通过轻轻推针对琼脂糖或针对放置在琼脂糖一块玻璃盖玻片。小幅承压注射器旋钮验证针尖是开放的（绿色针的内容将驱逐）。 </li><li>通过首先将针引入到蛋和略微吸打破蛋膜，随后通过注射注入由一个未受精的卵之一。注入绿色注射溶液进入蛋的中间到最大的泡孔直径的1/3。 （这对应于大约为30pl为140微米的卵直径）。 </li><li>转移注入未受精卵到一个新的培养皿中，并在15-18℃下孵育，直到施肥。 </li><li>施肥注入鸡蛋新鲜精子激活在测试施肥（步骤6.1.2）。 EliminatEA最大精子由受精卵转移到新鲜培养皿中。摊开胚胎，并在切割阶段不扰乱它们（最多32细胞阶段）。 </li><li>培养胚胎到所需阶段在由13和20℃之间的温度。 </li><li>由漩他们到板的中部，并将它们转移到硅化1.5毫升管收集胚。修复和染色，或装入（步骤7,8）。 </li><li>比较所得到的表型来控制注射的胚胎。 注:注入的第二，非重叠的MO靶向同一转录物以获得相同的表型。具有修饰的mRNA编码的感兴趣的蛋白质，但并非由该MOS识别营救特定的MO表型（S）。注射对照MO，让没有表型。 </li></ol> 7.染色LacZ的<ol><li>收集胚胎在硅化1.5毫升管所需的阶段，并修复它们在0.2％的戊二醛在1毫升ASWH 15至30分钟的最大值。 </li><li>洗2×10分钟用1ml 1X PBT。 </li><li>在500微升的LacZ染色缓冲液冲洗一次。用X-gal底物代替补充1毫升染色缓冲液（用10微升/毫升的40毫克/毫升X-gal的股票）。胚胎转移到一个12孔板为更好观察染色的。 </li><li>孵育在黑暗中。从0.5小时变化温育过夜，在4℃下，在室温或在37℃下根据表达LacZ的驾驶员的强度。 </li><li>洗净的胚胎达4倍在1毫升1X PBT，除去染色缓冲液。 </li><li>后修复程序，在室温下，用2％多聚甲醛（PFA）在1ml 1X PBT或在1毫升1×PBT 0.2％戊二醛染色的胚胎的解释和可视化10分钟。 </li></ol> 8.安装将荧光标记的Wmbryos的<ol><li>修复所需的发育阶段的胚胎在2％PFA中于1ml ASWH 20分钟。 </li><li>洗净的胚胎在1毫升1X PBT 2倍。避免任何光线博览会通过保持胚胎在黑暗条件下URE。 </li><li>传输高达50胚胎一张幻灯片。用纸巾先用吸管和小心地取出最长的PBT。 </li><li>添加20-25微升的安装介质。 </li><li>由（首先从一个侧面）的角度定位它，然后慢慢用尖头物体（针，镊子，枪头，），避免气泡，直到样本覆盖降低它添加盖玻片。修正了使用透明的指甲油点在其边缘盖玻片。 </li><li>用密封干燥后的指甲油整个盖玻片。储存在-20°C黑暗。 </li></ol>

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The authors declare that they have no competing financial interests.

有两种主要的技术来产生转基因玻璃海鞘胚胎:RNA或DNA和DNA的电穿孔的显微注射。这些技术在玻璃海鞘胚胎基因摄于90年代7,8首次使用，并从那时起陆续改善，现在在玻璃海鞘 （和其他海鞘属）20世界范围内使用。 在协议中的关键步骤成功的转基因成玻璃海鞘配子，很容易从成年动物收集取决于许多的关键因素。配子的质量取决于产卵季节（一般在5〜12月在北大西洋，水温和充氧变化），在水族箱海水质量（在正确的盐度，成年人保持健康的2-4周） ，最后在正确处理来自成人经萃取配子，如下详述在各个步骤协议。 在准备步骤，用自来水孵化巴斯德吸液管将防止从坚持和琼脂糖涂层培养皿海水预孵化的胚胎将删除琼脂糖释放有毒物质。通常，餐具可制备最多使用前2周，保存在4℃，以避免细菌生长并在使用前新鲜漂洗。有一天老了，小菜似乎最适合注射。 dechorionation效率和长度在协议作为长期治疗的蛋/胚胎会损害发展的关键一步。精心的准备（无晃动），以及正确贮藏（4℃长达一周）保持dechorionation解决方案的质量。在溶液中的蛋白酶失去效率如果无论是在制备或随时间混合过于剧烈，特别是如果在室温下保持更长的小时/天。我们通过优化使用便利，新鲜dechorionation解决这个关键的一步从20倍原液冷冻等分准备。 而移液将不利于脆弱dechorionated蛋/胚胎的正确处理是必要的，剪切或胚胎捅死。当移液，将胚胎保持尽可能在悬浮液（轻轻&#39;吹&#39;液体漩涡它们随后光滑但快速移液），同时保持玻璃吸管在垂直而非水平位置。这样的关心内容适用于洗涤，再悬浮和输送蛋的所有移液步骤/胚胎进出管，试管或板，前，后固定。一旦固定，特别小心也应采取独立的移液设备的用于实时与固定的胚胎，以避免固定剂的遗体任何毒性。 显微注射是玻璃海鞘蛋/胚胎相当棘手，由于其体积小，卵黄膜的弹性和关键取决于优化针尖大小注射针，注射的一个完美的角度（在一行针运动，沿着蛋半径）和一个微调，手动卵黄膜的（抽吸之前通过抽吸注射）断裂。后者是唯一可能的，如果气泡完全从包含矿物油用于平滑吸入/注射压力注射管线消除。此外，灰尘或蛋碎片的最小件将堵塞针，并很容易被清洁的工作条件和用于向在注射培养皿转移之前鸡蛋漂洗步骤避免。注射后，蛋转移到新鲜的菜肴会避免死胚不具有存活的注射过程毒性作用。 故障排除在程序中的潜在问题可通过以下解决方案来解决。低受精率可能与精子的活化不足，低蛋清洁或大体积施肥出现。使用freshlŸ汇集，少精子稀释。验证在活化的精子的运动。如步骤4.2中所述洗净与ASWH卵受精之前。减少水量和降低每受精菜蛋量/孔。在准备步骤失去胚胎可以通过添加的dechorionation溶液滴下来的undechorionated卵/胚胎更有效地离心来避免。 Dechorionation时间应优化为部分dechorionated鸡蛋不会沉积，延长dechorionation是有毒的，导致胚胎爆炸。 异步的发展可以从解剖过程中释放残留的精子产生。远离不同动物中分离鸡蛋批，以避免不受控制的相互交流。发育异常可能有多种原因。在开始和在赛季结束，保持分开不同个体的胚胎后，最好的批次可能被选中。避免扰动的胚胎下面受精10分钟，以避免与它们的卵胞质偏析干扰。确保在被转让的移液中分离胚胎部分的姐妹卵裂球和结果的胚胎没有开始分裂。使用较少的精子卵子dechorionated避免多精入卵。使用新鲜配制，但冲洗琼脂糖菜肴。确保移液设备固定液免费。记住，没有dechorionated，而不是在电哪一步发展被打乱检测控制胚胎。用抗生素补充的胚胎，如果污染和发生分解。 如果该注射装置已经&#39;堵塞"的注射针开口可以通过驱逐一些其染色内容的验证。堵塞材料可以通过琼脂糖小心拖动针尖被擦去。气泡在注入管道和/或针应该被删除。换针更好地控制注射量为东东DLE提示可能会破坏或沿注射堵塞。冲洗蛋并将其放置在干净的菜注如果碎片在盘中已积累。离心之前，以沉淀任何小颗粒或晶体填充新鲜针注射溶液。也有利于在练习注射只活体染料。通过注射施肥和非注射的卵验证注射技术。最后，拥有一支经验丰富的同事咨询可以帮助改善注射技术。 为了控制脱靶效应的第二非重叠MO和救援与没有由该MOS识别能够忠实排除在玻璃海鞘的非特异性的表型的影响的改性的mRNA。 显微注射:意义和局限在玻璃海鞘显微注射被利用在一个脊索动物11产生用于全胚胎基因调控网络（GRN）第一蓝图。然而，尽管这样的REMArkable成就，显微注射在玻璃海鞘的胚胎具有局限性，由于玻璃海鞘卵的相对小的尺寸（0.14毫米直径）。相比那些外源DNA / mRNA通过显微注射引入其他脊索动物物种的， 玻璃海鞘卵是比较难以处理和每个实验注入玻璃海鞘胚胎率仍然很低（每个实验平均30至50的胚胎），阻碍定量分析。然而，在玻璃海鞘扰动母体因素，MOS管显微注射是首选的也是研究从16到32个细胞及其在发展合子发病参与15级的方法。此外，有可能虽然更加难以m​​icroinject个体的卵裂球到16-32细胞阶段。最后，显微注射仍然是最有效的技术，在比玻璃海鞘其它海鞘物种产生转基因胚胎（通过mRNA或DNA注射），其充分优化解不存在ZED电协议。 电:意义和局限为了克服低的数字和显微注射的其他方面的限制，大部分的玻璃海鞘社区已经用质粒DNA的电，因为它是开发和泽勒等人发表的优化和标准化的协议，于2004年9。事实上，成千上万的转基因玻璃海鞘胚胎可以在一小时内多达500胚在一个实验皿同时电穿孔用的16毫秒的单个50 V脉冲而产生。产生转基因胚胎的庞大的数字的方法仍然是脊索动物之间的模式生物是独一无二的。这已经导致玻璃海鞘被迅速确认为进行体内强大的模型系统中潜在的顺 -regulatory地区和蜂窝/亚细胞定位的分析，如下例证。 虽然electroporati在玻璃海鞘受精卵从根本上彻底改变了脊索动物GRN研究领域，该技术仍然面临一些限制。首先，质粒瞬时引入到玻璃海鞘胚胎和最有可能继承作为染色体外的阵列，使得电穿孔质粒DNA投机的稳定性。此外，它是非常困难的，甚至是不可能的，以控制将要占用每合子电穿孔质粒的拷贝，从而导致表型的波动。进一步的问题，使得它难以解释电穿孔的结果是，我们称之为嵌合的现象。电穿孔质粒DNA可以被吸收在单细胞阶段的胚胎的不同位置;这决定了如何受精卵的许多方面将获得质粒由卵裂球后代继承。这种变化显然使定量效应的解释更加困难。然而，大多数的问题随之而来electropora灰变性是由生物样品适量解决则计数和LacZ基因（或GFP）阳性细胞，这是在一个单一的批次容易获得的统计信息。 多功能性和电基因工程的潜力最终电穿孔允许一次克隆到记者还是表达质粒潜在顺 -regulatory区和基因功能的定量分析的中期通量筛选。由于紧凑的玻璃海鞘的基因组，识别和潜在的监管区的克隆是相当简单的3。一种使用社区的大量数据的策略是表现在图2A。记者基因编码质粒的克隆是由玻璃海鞘的产生进一步促进适应，GATEWAY相容载体套房19和兼容全长ORF库18。这两个工具都提供给社会和允许调整驱动器和编码区的高效率，限制无酶的重组，如在图2B中描绘。 近年来，电穿孔被成功适于实现用表达基因编辑工具，例如组织特异性的驱动程序21,22的控制下CRISPR / Cas9部件质粒组织靶向基因操作。这些方法将迅速推进我们GRNS的动态和潜在保守的信号机制，包括参与组织形成转录因子代码和多能性的逐步退出的理解。此外，使用电穿孔的组织特异性损失-或获得性功能的方法（由CRISPR / Cas9或过度表达）将工具来研究人类疾病相关的基因的玻璃海鞘直向同源物。事实上，人类疾病基因的同源玻璃海鞘和他们的全长ORF编码克隆目录很容易availab乐在玻璃海鞘1 8分析。由于脊椎动物的全基因组重复，大多数人类基因拥有该基因的功能1的分析复杂功能冗余的旁系。 玻璃海鞘是在幼虫典型的脊索动物组织安排应揭露这种重要的是，往往在功能上保守的基因的基本角色，一个简单的系统。

最近，基因组序列和转录基因库已经成为了许多模式生物的访问。确定基因功能链接，但是，这些连接如何在整个胚胎时空执行转录活性的DNA的硬连线，仍然是一个重大的挑战，特别是在脊椎动物。监管相互作用的复杂性和基因组1,2的复杂性，尤其是在脊椎动物中，放慢有效功能分析，特别是在体内 DNA水平。事实上，没有GRN目前从受精分析，发育中的生物体的最后，终末分化的状态。 该海鞘C.代表肠等在那里完成GRN分析是可能的模式生物。它具有不重复的基因组典型的少基因冗余脊椎动物。脊椎动物，在对比已经历了两轮的甲肝病毒基因组复制的Ë产生较大的基因家族和功能的多样化，也旁系间的冗余。 C的紧凑顺 -regulatory序列（GRNS的控制单元） 肠 ，和几个细胞的胚胎不变的血统都让人联想到的C.线虫 。此外，其独特的系统发育地位作为一个无脊椎动物姐妹群脊椎动物脊索动物内允许发展的观察，在细胞的分辨率，形成脊索动物体计划3-6。 确保功能基因组学的模式生物的未来成功的关键问题是大量的胚胎进行定量体内扰动的产生。在玻璃海鞘鸡蛋7的显微注射的技术，并迅速通过体内电穿孔在数百玻璃海鞘的获得许多瞬时转基因动物的可能性发展受精卵8,9-已突破<em&gt;玻璃海鞘功能基因组学。在这里，我们首先提出了针对单个基因的比较费力显微注射技术剩下首选吗啉（MO）介导的基因敲除的方法，特别是产妇的成绩单。莫寡聚通常靶向起始子ATG或RNA的剪接位点，并与蛋白质的翻译干扰。然后，我们表明电穿孔在玻璃海鞘的受精卵如何允许对大量的胚胎以定量的方式将待分析，从而打开增强剂的有效分析的途径在体内和组织特异性GAIN-或损失的功能的方法，用可能性同时操作和分析。我们进一步证明玻璃海鞘社区工具如何在调节区和在表达载体中充分开放读框（ORF）的有效克隆的硅片预测用于。最后，我们表现出的不同亚细胞定位的荧光标记或者通过电穿孔后，标记的蛋白质过度表达。

<table><tbody><tr><td>Lab with constant temp. (~18 &amp;deg;C)&amp;nbsp;</td><td></td><td></td><td>For embryo work</td></tr><tr><td>&lt;em&gt;Ciona intestinalis&lt;/em&gt; adults</td><td>UPMC - Station Biologique,&lt;br/&gt; Roscoff, France</td><td></td><td>For collecting gametes</td></tr><tr><td>NaCl</td><td>Roth</td><td>3957.1</td><td>For ASWH</td></tr><tr><td>KCl</td><td>Roth</td><td>6781.1</td><td>For ASWH</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;x2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth</td><td>A119.1</td><td>For ASWH</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;x6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth</td><td>2189.1</td><td>For ASWH</td></tr><tr><td>MgS0&lt;sub&gt;4&lt;/sub&gt;x7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth</td><td>P027.2</td><td>For ASWH</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Roth</td><td>6885.1</td><td>For ASWH</td></tr><tr><td>Hepes&amp;nbsp;</td><td>Roth</td><td>HN78.3</td><td>For ASWH</td></tr><tr><td>Sodium thioglycolate&amp;nbsp;</td><td>Sigma</td><td>MZ-6</td><td>For Dechorionation</td></tr><tr><td>Pronase&amp;nbsp;</td><td>Sigma</td><td>T0632-100G</td><td>For Dechorionation</td></tr><tr><td>D-Mannitol&amp;nbsp;</td><td>Sigma Aldrich</td><td>M9546-250G</td><td>For electroporation</td></tr><tr><td>Plasmid DNA (prep &gt;1&amp;micro;g/&amp;micro;l)</td><td>Macherey-Nagel</td><td>740410.100</td><td>For electroporation or microinjection</td></tr><tr><td>Agarose</td><td>any supplier</td><td></td><td>For coating embryo culture dishes</td></tr><tr><td>Plastic Petri dishes</td><td>VWR</td><td>612-2079</td><td>Coated with 1% Agarose in ASWH, covered with ASWH</td></tr><tr><td>Small scissors</td><td>any supplier</td><td></td><td>For dissecting gametes</td></tr><tr><td>NaOH&amp;nbsp;</td><td>Roth</td><td>6771.1</td><td>For activating dechorionation solution</td></tr><tr><td>Tris&amp;nbsp;</td><td>Roth</td><td>5429.3</td><td>1 M pH 9.5, for sperm activation</td></tr><tr><td>Pasteur pipettes&amp;nbsp;</td><td>VWR</td><td>612-1701</td><td>Treated by immersion in tap water for at least 12 hr</td></tr><tr><td>Hand centrifuge</td><td>Hettich Zentrifugen</td><td>1011</td><td>Use glass centrifuge tubes</td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Sigma</td><td>T3406-250EA</td><td></td></tr><tr><td>2 ml Eppendorf tubes</td><td>Sigma</td><td>T3531-200EA</td><td></td></tr><tr><td>Square electroporator</td><td>Bio Rad Gene Pulser Xcell</td><td></td><td></td></tr><tr><td>Electroporation cuvettes 4 mm</td><td>Eurogentec</td><td>CE-0004-50</td><td></td></tr><tr><td>Coverslips</td><td>Sto Premium</td><td>PR248212600</td><td></td></tr><tr><td>Glass slides</td><td>VWR</td><td>ECN 613-1551</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257-10ML</td><td>For fixation in LacZ staining</td></tr><tr><td>Paraformaldehyde</td><td>Sigma&amp;nbsp;</td><td>P6148-500G</td><td>For fixation of fluorescent samples</td></tr><tr><td>Vectashield</td><td>Vector</td><td>H-1000</td><td>For mounting of fluorescent samples</td></tr><tr><td>X-Gal (5-Bromo-4-chloro-3-indolyl &amp;szlig;-D-Galactopyranoside)</td><td>Roth</td><td>2315.2</td><td>LacZ substrate; stock in 40 mg/ml DMF (Dimethylformamide)</td></tr><tr><td>K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;&amp;#8226;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>4984</td><td>For LacZ staining buffer</td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Merck</td><td>4973</td><td>For LacZ staining buffer</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;2HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>4984.2</td><td>For PBT</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>3904.2</td><td>For PBT</td></tr><tr><td>Tween&amp;nbsp;</td><td>Sigma</td><td>SLBJ5103V</td><td>For PBT</td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410</td><td>Embryo culture tested</td></tr><tr><td>Green Vital Dye</td><td>Fast Green Sigma&amp;nbsp;</td><td>F7251</td><td>for microinjection; 10&amp;micro;g/&amp;micro;l</td></tr><tr><td>Micropipette Puller (Flaming/Brown)</td><td>Sutter Instruments</td><td>Model P-97</td><td>For pulling injection needles</td></tr><tr><td>Borosilicate glass capillaries GC100TF-10</td><td>Harvard Apparatus Ltd. UK</td><td>30-0038</td><td>1.0 mm O.D. x 0.78 mm I.D., containing filament, for microinjection</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MWS-2</td><td>Three-axis Oil Hydraulic System; for 3D fine movement during injection</td></tr><tr><td>Injection holder</td><td>Narishige</td><td>1M-H1</td><td>For holding injection needles, entire system filled with mineral oil</td></tr><tr><td>All Glass Syringe</td><td>Walter Graf &amp;amp; Co GmbH &amp;amp; Co. KG&amp;nbsp;</td><td>No. 95199.10427A4</td><td>10 ml FortunaOptima; filled with mineral oil, for fine tuning of microinjection pressure&amp;nbsp;</td></tr><tr><td>Morpholinos&amp;nbsp;</td><td>GeneTools LLC, Pilomath, OR, USA</td><td></td><td>Injection at 0.25-0.5 mM</td></tr><tr><td>Capped mRNA</td><td>Ambion mMessage mMashine kit</td><td></td><td>Injection at 0.05-0.5&amp;micro;g/&amp;micro;l&amp;nbsp;</td></tr><tr><td>Plasmid DNA</td><td></td><td></td><td>Injection at 20 ng/&amp;micro;l</td></tr><tr><td>Recombination cloning kit</td><td>Gateway BP Clonase Enzyme Mix Invitrogen</td><td>11789-013</td><td></td></tr><tr><td>Recombination cloning kit</td><td>Gateway LR Clonase Enzyme mix Invitrogen</td><td>11791-019</td><td></td></tr><tr><td>anti-HA tag antibody</td><td>abcam</td><td>ab9110</td><td>for Immunofluorescence, use 1:400 in 3% BSA/PBT</td></tr><tr><td>TRITC goat anti-rabbit antibody</td><td>Jackson</td><td>111-025-144</td><td>for Immunofluorescence, use 1:300 in 3% BSA/PBT</td></tr></tbody></table>

embryo microinjection and electroporation in the chordate ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

显微注射对基因调控网络的研究的海鞘的胚胎玻璃海鞘非常适合于在细胞谱系水平基因功能或基因调控的研究，并与蜂窝分辨率。的mRNA，MOS管或标签可显微注射到未受精的卵子或进入受精个人卵裂球。使用显微注射技术拦截MO介导的基因在步骤中描述6.的MO特异性靶向选择的基因的mRNA及防止其翻译成蛋白质。莫介导的失功能（LOF）发育基因的改变下游基因的浩如烟海的表达，整体透出一窥胚胎GRN 10。在玻璃海鞘这种分析在后生动物11提供的第一个完整胚胎的监管蓝图。 图1显示了如何microinjecti的例子在被利用在玻璃海鞘胚胎的原肠期研究保守慈 -Myelin转录因子（CI -MYT）表达的上游调控。通过原位杂交（ISH），内源表达（ 图1a， 图1a&#39;系统化）监测在6行神经板前体观察特别是脑，（行Ⅲ/Ⅳ，在红色）和脊髓（我排在绿色）。此外， 慈 -MYT上游调控是通过MO注射分析瞄准了在表达或邻近之前慈 -MYT表达发病（ 图1b-G）神经前体几个因素。的早期胚胎的因素，如叉头框了Aa MO击倒（FOXA-a）的转录因子和成纤维细胞生长因子20年9月16日（FGF9 / 16/20）（ 图1b或1c，分别地）消除整体词 -MYT表达。其它转录因子仅部分影响词 -MYT表达导致MO介导的下调在脑前体（蓝色箭头头部在图1d 中，f和g）的子集。相反， 慈 -MYT表达异位节点时（ 图1E，红色箭头头）的下调激活，这表明节点通常压制在这些侧神经索前体慈 -MYT表达。 电穿孔高效的基因功能和增强研究质粒DNA电穿孔到玻璃海鞘合子是用于瞬时转基因和在体内的表型改变随后观测的有效方法。不像mRNA的显微注射，电穿孔允许组织特异性表达，其中基因编码区（个ORF，开放读框）的控制下表达组织特定的驱动程序。这些通常构成的公知的基因（如在脊索前体8的短尾增强剂表达） 顺式 -regulatory区域。相反地，电穿孔是工具为新颖调控区的有效分析，其中报告基因（LacZ的或绿色荧光蛋白，GFP）揭示其在体内的活性。用于鉴定和这样一种新颖的调节区的后续电穿孔介导的分析（对词 -MYT）工作流程示于图2。 玻璃海鞘社区数据，在海鞘特定基因组浏览器12,13，如海鞘网络易于接近原位表达与胚胎学资料（大料）23的一个巨大的剧目，被检查监管区域（ 图2A）的，在硅片鉴定。随后聚合酶链反应（PCR）的这些区域的基于克隆到表达载体允许在体内电穿孔介导的检测（ 图2B）。基因组浏览器屏幕截图在图2A中示出了用于词 -MYT对KH转录模型的上游区域（前三个外显子，橙色，和两个内含子是可见的）。不同的基因组浏览器轨道注释这个地区的功能基因组数据的预测，如染色质免疫沉淀（ChIP）数据14（这里示出为ZicL，小脑升词 -锌指），两种相关的玻璃海鞘物种15,24或核小体之间的序列保守占用16,17（从顶部至图2A中底部）。一般地，外显子蛋白编码区是高度保守的（ 图2A中对准轨道）。额外的高保护峰出现在上游非编码区，并在第一个内含子。其中两个被预测为核一自由ccording到暗示无障碍转录因子基于序列算法16,17（ 图2A中的红色帧）。事实上， 慈 -ZicL转录因子结合芯片片上信号14（ 图2A绿线）富集在这两个保守区域。此外，使用该搜索潜在的转录因子结合位点的接口25，我们确定了位于被C -ZicL的ChIP簇（橙色箭头和序列与下划线ZIC核， 图2A）标记的基因组序列内ZIC位点。在保守的C -ZicL转录因子的词 -MYT的自由调节区的结合和可预测的核小体与在莫注射介导的敲除实验靶向ZicL（ 图1d）观察到的C -MYT表达的下调是一致的。 威慑后在挖掘潜在的硅片 顺 -regulatory地区的慈 -MYT表达，这些都是PCR方法从玻璃海鞘基因组DNA扩增和重组克隆到LacZ报告质粒19插入。然后将构建体电穿孔入受精玻璃海鞘卵（ 图2B）以及它们的活性通过的LacZ染色（ 图3）进行分析。 图3示出了由这种在硅片的方法是可能的，以确定概括词 -MYT mRNA表达域（对比图1a）两个单独的顺 -regulatory序列。一般情况下，瞬时转基因胚胎电在只有已经纳入了DNA的那些细胞质粒DNA显示马赛克的表达。马赛克的LacZ表达因此被pmyt-R3驱动，可以在所有也表达慈前体发现</em&gt; -MYT表达即行后我的神经板前体和前行III / IV在原肠胚和早期神经胚阶段（分别为图3a，B，紫色和红色箭头头）。相比之下，pmyt-R5是活动仅在后（行I）的神经板前体，并在后来的神经胚期（ 图3c，红色箭头头）开始。这两个地区由此编码的C MYT基因调控的两个独立的空间和时间方面的问题。有趣的是，这两个调节区还染色在ISH没有看到额外的前体，尤其是在前方和横向神经板和肌肉（ 图3a，B，粉红色，白色和黄色的箭头头部，分别地）。这样更宽表达可能指向压制性未包含在分离的调控片段元件（如用于交点该压制横向神经命运， 见图1e）的或与累积检测的低表达水平LacZ的酶信号。 除了增强的研究，电穿孔玻璃海鞘也被他们的表达促进了玻璃海鞘编码基因的功能分析。大集玻璃海鞘全长的ORF是提供给社会，并进一步注明为人类同源基因，包括疾病相关的基因18。单个基因或从该重组克隆兼容完整的ORF cDNA文库（ 图2B）的基因的基团很容易转移到含有适当的驱动程序在胚胎中表达目的载体。所得表达克隆可以进一步与顺 -regulatory记者共电穿孔的构造，以测试在增强激活他们的推定作用。 图2B示出了用于转录充分的ORF克隆的分离因子词 -ZicL和CI- GATAa及其recombination为进可能包含翻译标签19（ 如绿色荧光Venus-或病毒血凝素（HA） -标记）和组织特定的驱动程序，如早期的泛外胚层表达15朋友加塔调控区域（pfog驱动程序）改编目的载体在本研究中使用。通过pmyt-R3&gt;的LacZ和pmyt-R5&gt; LacZ的记者的共电穿孔（ 图3b&#39;，B&#39;和c&#39;，c&#39;的&#39;，分别地）分析词 -ZicL过度表达在外胚层和神经外胚层组织的效果。我们的电分析表明， 词 -ZicL可以激活词 -MYT表达通过pmyt-R3和pmyt-R5增强子区既报道构建表明在外胚层区域（异位的LacZ表达在图3b中的绿色箭头的头&#39;，B&#39;和c＆＃39;，C&#39;&#39;）。 如图3d中所示，在数百个玻璃海鞘卵质粒DNA的电穿孔允许的表情变化，在浓度依赖性，泛外胚层慈 -ZicL表达异位pmyt-R3&gt; LacZ的表达出统计学相关的量化。 电穿孔用于体内亚细胞定位 图4描绘了标记的转录因子的亚细胞定位使用适于表达构建体（在图2B中图式）的外胚层卵裂球在电穿孔时表达。通过C-末端标记的转录因子（例如C -GATAa）配有一个金星标签（ 图4b）或HA-标签（ 图4c）和外胚层组织过表达它们（pfog）我们可以看到， 在体内，&#39;慈 -GATAa大多定位于细胞核，如预期的转录因子，而金星表达无处不在，包括细胞质中。类似的实验可进行研究的亚细胞定位的动态随时间的个人或转录因子的组合，有可能揭示它们的共定位与不同标签。 <img alt="图1" src="/files/ftp_upload/54313/54313fig1.jpg" /> 图1:在玻璃海鞘卵评估慈 -MYT调节通过显微注射 慈 -MYT表达WT和吗啉（MO）注射胚胎。在神经板词 -MYT的（ 一 ）WT，表达模式。 （a&#39;）的在神经板（NP）染色前体的示意图。行I / II:后神经命运;排III / IV:前神经的命运;排V / VI:触须的命运。 （ <stron。摹&gt; B - G）对时撞倒参与早期玻璃海鞘 GRN（改编自今井等人的照片显示的基因各种MOS管注射慈 -MYT表达，2006年）11 慈 -MYT， 慈 -myelin转录因子; WT，野生型;蓝色箭头: 慈 -MYT信号丢失;红色箭头:异位慈 -MYT信号。比例尺= 100微米是指所有图像。 <a href="http://ecsource.jove.com/files/ftp_upload/54313/54313fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54313/54313fig2.jpg" /> 图2:工作流在硅片搜索增强地区和重组克隆通过在玻璃海鞘胚胎电瞬时转基因 （A）从大料23 ASCI的快照。滇基因组浏览器的标识词的上游调控序列- MYT。红色标记的帧两个潜在的顺 -regulatory地区，pmyt-R3（scaffold_196:76883..77079）和pmyt-R5（scaffold_196:75856..76041）通过电被选作分析的。曲目的名称:ZicL探头:芯片级芯片数据14; KH2012成绩单: 玻璃海鞘成绩单模型;比对得分铯/ CI LastZ: 玻璃海鞘savignyi（CS）/ 玻璃海鞘 （CI）15,24之间的序列保守性; 。2006年西格尔预测核小体占据第1版:小鸡由西格尔等人训练的计算算法，2006年16适用于慈在Khoueiry 等 ，2010青17条:。 慈 -ZicL沉淀峰14;橙色箭头:ZIC预测网站; GCTG:芯ZIC结合位点。 （B）方案从接穗产生过度的结构全长ORF cDNA文库，重组到含有组织特定驱动程序（pfog）和蛋白质标记随后共电穿孔与报道构建（如pmyt-R3&gt;的LacZ或pmyt-R5&gt; LacZ基因） 在体内再生词目的载体。 - MYT， 慈 -myelin转录因子; CI -ZicL，小脑大号转录因子慈 -锌手指。pfog，GATA的慈 -Friend的调控区域<a href="http://ecsource.jove.com/files/ftp_upload/54313/54313fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54313/54313fig3.jpg" /> 图3:电穿孔玻璃海鞘胚胎显示在中间gastrul 慈 -MYT的LacZ染色胚胎慈 -ZicL诱导时空顺 -regulation。一个（MG）/晚原肠（LG）或早期神经胚（EN）阶段被证明是共电有两种pmyt-R3&gt;的LacZ或pmyt-R5&gt;的LacZ和控制结构（pfog&gt; mCherry）或pfog&gt; ZicL。该pfog司机19介导外胚层神经外胚层和异位（泛动物）ZicL表达，导致这些细胞中的LacZ异位污点。 （ 一 ）pmyt-R3 LacZ的活性毫克/ LG阶段的胚胎（小箭头，左图），或在相应颜色的原理领地（蓝色圆圈，右图）;植物视图（VG）。行I / II:后神经命运;行III / IV:前神经的命运;行V / VI:触须的命运。 （b）于组织中EN阶段，因为在一个pmyt-R3活性。 （B&#39;）异位时的C -ZicL共电穿孔pmyt-R3活性被绿色箭头表示。 （B&#39;）胚胎同在B&#39;，面向动物极了（一个）。 （ <str翁&gt; c）在EN阶段（C&#39;）在慈 -ZicL共同电异位pmyt-R5活动pmyt-R5 LacZ的活动是由绿色箭头指示。 （C&#39;&#39;）胚胎同在C&#39;，但面向动物极了（一个）。 （ 四 ）对慈 -ZicL代表性实验量化在低浓度过度激活pmyt-R3。一个生物重复表示慈 -MYT， 慈 -myelin转录因子; 慈 -ZicL，小脑大号转录因子慈 -锌手指; CI -FOG，GATA的慈 -Friend; WT，野生型; MG，中期原肠胚; LG，后期原肠胚;恩，早期神经胚;一，动物图。 VG，植物图。 NLS的LacZ，对于LacZ的核定位信号。比例尺= 100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54313/54313fig3large.jpg" target="_blank">请点击此处查看该图的放大版本</a>。 <img alt="图4" src="/files/ftp_upload/54313/54313fig4.jpg" /> 图4:蛋白质在金星标记或Hemagglutinin-（HA）的电本地化差亚细胞定位 -tagged在使用pfog司机19外胚层细胞中过度表达的蛋白质。 （A - C）DIC图像。 （A&#39;，B&#39;）相应的荧光图像。 （C&#39;）在与TRITC免疫组织学标记对应的荧光图像。比例尺= 100微米是指所有图像。 DIC，微分干涉对比。 <a href="http://ecsource.jove.com/files/ftp_upload/54313/54313fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis at JoVE.com

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

我们目前的转基因瞬时和基因敲除在玻璃海鞘 ，脊索动物姐妹群脊椎动物，利用显微注射和电技术。这种方法有利于在这个简单的无脊椎动物，具有脊椎动物中最起码的特征，包括脊索和头部感觉上皮细胞，与人类疾病相关的基因的同源基因的许多功能基因组学。

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

embryo-microinjection-electroporation-chordate-ciona

Research

JoVE Journal

Developmental Biology

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

15.8K 次观看.  University Innsbruck. 我们目前的转基因瞬时和基因敲除在玻璃海鞘 ，脊索动物姐妹群脊椎动物，利用显微注射和电技术。这种方法有利于在这个简单的无脊椎动物，具有脊椎动物中最起码的特征，包括脊索和头部感觉上皮细胞，与人类疾病相关的基因的同源基因的许多功能基因组学。 

视频: Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

科研

遗传学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

神经科学

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

果蝇中 phiC31 整合酶介导的胚胎转基因

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

作者聚焦:优化胚胎显微注射以促进果蝇的转基因

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

胚胎显微注射用于果蝇转基因

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

生物学

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

发育生物学

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

胚胎显微注射和电的脊索动物

摘要

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此视频中的章节

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

在蛋和卵外研究中研究鸟类内耳发育的方法

胚胎显微注射和电的脊索动物

摘要

探索更多视频

此视频中的章节

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

胚胎显微注射用于果蝇转基因

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

在蛋和卵外研究中研究鸟类内耳发育的方法