We thank Filip Wymeersch and Anestis Tsakiridis for comments on the manuscript, staff in the SCRM animal unit for help with animal maintenance and Prof. Stuart Forbes for immunohistochemistry reagents. This work was supported by MRC grant Mr/K011200/1 and the China Scholarship Council

所有的动物实验是按照英国内政部规定如动物（科学程序）规定的法案（1986）在项目许可数量四千四百三十五分之六十〇。为了收集胚胎在特定发育阶段，定时交配共设置了O / N。中午找到一个阴道塞的一天被定胚胎一天（五）0.5。 1.剖析E7.5或E8.5着床后胚胎体外培养 <ol><li>颈椎脱位牺牲怀孕的雌性小鼠。 </li><li>用剪刀，用血管钳抱着它隔离了子宫，并放置在30毫米的菜充满了M2网上平台。 </li><li>小心地撕开两对细镊子的子宫肌层。 </li><li>撕下蜕膜，小心不要刺破胚外腔。 </li><li>用钳子捏它，慢慢从胚胎中分离取出赖克特的膜。 </li><li>检查胚胎下解剖立体显微镜，以确保卵黄囊，羊膜和ectoplacental锥体完好。 </li><li>传输用吸管和地方胚胎M2的干净的盘子上有30毫米的塑料培养皿的盖子上冰（的&#39;冰平台"），以部分寒意胚胎。 </li><li>如果需要的话，在M2的存储胚胎冰平台长达1.5小时，例如，培养基制备和操纵的小批量期间〜3-4的胚胎在RT。 注意:恢复和啮齿类动物胚胎解剖已详细描述先前7,8,16。 </li></ol> 2.准备胚胎培养液<ol><li>新鲜解冻或者市售的大鼠血清（见格兰维尔琼斯13为规格等人），或大鼠血清根据考普和克罗夫特16被热灭活30分钟，在56℃和冷冻在1ml制备在内部等分试样在-80℃ 注:市售大鼠血清是可以接受的<html> 24-36小时的培养期，虽然血清准备在公司内部，在我们的经验，卓越的文化时期长达48小时。 </li><li>新鲜制备的过量（例如，10ml）中定义的补充剂组成格拉斯哥最小的必需培养基（GMEM），1％非必需氨基酸（NEAA），2mM的L-谷氨酰胺和1mM丙酮酸钠的。 </li><li>计算是根据该将要培养的每个阶段的胚胎数所需培养基的体积（见第3节）。混合与定义的补充剂大鼠血清，以弥补50％培养基（1:1（体积/体积）的大鼠血清:定义补充剂）和/或75％的培养基（3:1（体积/体积）的大鼠血清:定义补充剂）。 </li><li>通过0.45微米的过滤器通过胚胎培养基和添加万国际单位/毫升青霉素和10微克/毫升链霉素。 注意:新鲜解冻L-谷氨酰胺和丙酮酸钠溶液是用于离体培养期间胚胎发育是至关重要的。 </li></ol>E"&gt; 3。 体外培养条件不同胚胎阶段<ol><li>为E7.5胚胎:使用4-孔板在5％的CO 2在37℃供给，在空气中24小时的孵化器静态培养。培养高达每孔两个胚胎在1毫升50％的培养基。 </li><li>为E8.5胚胎:使用一个滚子培养装置8旋转，在35转/分钟在37℃掺入连续放气，用5％ 的 CO 2在空气中放置24小时（1毫升50％，每个胚胎培养基）。 </li><li>为E9.5胚胎:使用一个滚子培养装置旋转35转速，用5％ 的 CO 2，40％O 2的55％ 的 N 2在37℃/ min供给掺入气体24小时（每毫升75％培养基胚胎）。 注:安乐死的小鼠长时间的在M2发展产生不利影响后的3小时培养的胚胎。见考普和克罗夫特16 体外胚胎培养的方法。 </li></ol> 4. Graftin摹培养细胞到E7.5或E8.5小鼠胚胎<ol><li>物理上刮去使用20-200微升枪头EpiSCs，其中遍在表达GFP，从6孔培养板，并放置在含有胚胎30mm的培养皿 注意:要插入的胚胎细胞团，细胞应该物理报废而不是胰酶消化。 </li><li>附加用手拉住嫁接毛细管的吸引管，使口吸管。 </li><li>轻轻吸口移液管吸取大小的一种或多种细胞团块&gt; 20个细胞到接枝毛细管。 </li><li>轻轻地吹出来的细胞，以部分分散成大的团块。 </li><li>选择含有一个细胞丛〜:10-20细胞并再次吸入接枝毛细管，保持它靠近毛细管的开口。要小心，不要将电池丛进出毛细血管反复，避免分裂成更小的碎片。 </li><li>松散举行的地方胚胎一双镊子和插入术毛细管到感兴趣区域来创建的开口。 </li><li>轻轻驱逐丛出接枝毛细管，留下:10-20细胞遁胚胎的短字符串。 </li><li>重复该接枝步骤胚胎的期望数量。使用3-4个胚胎批量方便。 </li><li>留在M2培养基，图像使用解剖显微镜用相机荧光化合物接枝胚胎的相同盘的胚胎，保持成像时间为最小，以避免胚胎过度光和热曝光。 注意:确定成像时间凭经验因为这些将取决于相机和显微镜，以及荧光团的性质和荧光强度的规格。 </li><li>与M2培养基的最小体积转移胚胎​​在pastette到预平衡的培养基（见第3节）成像后立即使用。 注:仔细检查胚胎的形态嫁接后。只有文化的INTAC牛逼的胚胎。 </li></ol> 5.手工电穿孔材料和设备安装（需准备以下的电穿孔实验进阶）: <ol><li>对于DNA注射移液器:使用水平微量拉马拉DNA注射移液器。注射移液管应具有精尖带有开口小于10μm，以避免DNA注射入胚胎空腔时的组织损伤。 </li><li>对于玻璃毛细管电穿孔:使用抛光仪切断DNA注射移液器到20或30微米的内径的开口。为了避免细胞损伤时，毛细管与所述胚胎电穿孔接触时，玻璃毛细管的尖端必须切割干净和不包含锐利，断裂的边缘。 </li><li>对于手工毛细管电极（正极）（ 图1）:插入直径为0.2mm的铂丝成电的玻璃毛细管20或30微米直径的固定开度集中选民RIC电流和递送质粒DNA到的在胚胎的兴趣的一个小区域。 </li><li>对于手工L形电极（阴极）（图1）:一个弯曲0.2毫米直径的铂丝创建"L"形，具有的"L"的约1毫米长的水平部分。 </li><li>将每个铂电极，以薄绝缘线，并将其插入覆盖绝缘胶带显微注射针持有人。 </li><li>安装在标准的显微操作仪​​持有人的持针器。 </li><li>连接电路，如图1:毛细管电极连接到电源的阳极;连接L形电极到万用表的阳极;所述mulimeter的阴极连接到电源的阴极。 </li></ol> 6，电穿孔E7.5或E8.5小鼠胚胎<ol><li>用PBS电玻璃毛细管填充到1-2毫米的顶部，并插入TG1的向右铂电极（阳极）放入玻璃毛细管，直到它到达毛细管的底部。 </li><li>锚30毫米培养皿中填充有PBS的表面上的L形的电极（阴极）。 </li><li>从转移M2培养胚胎成PBS充满电的菜。 </li><li>插入注射针从横向外胚层到羊水胚胎的空腔。使用气动微微泵，注入DNA溶液（pCAG-Cre重组:GFP或pCAG-GFP 1-1.5微克/毫升与0.01％的绿色食用色素染料）到空腔直到完全充满。为E7.5-E8.5胚胎，小于5微升DNA溶液需要一个胚胎。使用绿色染料作为指标，注意不要爆胚胎。 </li><li>小心地定位在电极之间的胚胎和毛细电极移动到精确的位置，其中所述DNA是待递送。 注意:胚胎的取向取决于在其中的DNA是被电穿孔的区域。 </li><li> Electropo评价使用200伏（V）的胚胎在6个脉冲，每个50毫秒持续时间与每个脉冲之间一个1秒的时间间隔。 </li><li>转移胚胎电穿孔后，立即预平衡的培养基。如果需要，重复该过程的下一个胚胎。 注:加入培养基放入无菌容器中，并把它放在培养孵化器预先平衡的媒介。 </li><li>为了检测电穿孔的细胞2小时培养后，将胚转移至用pastette M2培养基的一个干净的30个毫米的培养皿。图片使用解剖显微镜荧光化合物的胚胎如步骤5.9。 </li><li>成像后立即用pastette胚胎转移回文化。 </li><li>为了检测所造成的电死细胞，染色的胚胎用远红细胞膜​​不透核染料（1:200在胚胎培养培养基）在37℃进行10分钟2小时后电穿孔（可选） </li><li>要计算的电穿孔细胞，修复安莉芳操作系统在4％低聚甲醛（PFA）为2-4小时，在4℃下，染色用紫外线或远红荧光核染液和图像使用共聚焦显微镜（可选）胚胎的核。 注:胚胎生长，如果不太久的PBS不利影响。因此，要确保电穿孔每个胚所花费的时间最小化（&lt;5％的胚胎分钟）。 </li></ol>

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The authors have no conflicts of financial or other interest to declare.

嫁接 用于细胞移植实验的关键步骤是细胞的相干串的插入理想地在一个单一的动作，避免了丛的解体。这种技术需要在控制口吸管一些练习。如果供体细胞掺入以及在宿主，其衍生物将分散在胚胎。为了进一步确定分散供体衍生的细胞是否分化适当的宿主，免疫染色可以在胚胎切片来执行。如果供体细胞是不与主机环境相兼容，它们或者不能被检测（因为它们是从胚胎排出）或形成培养后的胚胎未掺入团块。如果这两个分散的细胞和细胞块进行了观察，这可能表明过多的细胞移植和过度供体细胞不能与周围的宿主细胞相互作用导致丛形成。在这种情况下，包含其他移植物可以进行细胞的数量较少。 细胞移植技术的主要限制是它不可能确定随时间超过48小时一直没有实现，因为鼠离体培养的细胞的全部体内潜力。然而，如果结合超声引导细胞注射，它可能会向在子宫内培养的细胞转移到胚胎。总之，细胞移植实验已经广泛应用在我们的组和给予我们关于各种细胞类型4,21,22 的体内潜在有价值的线索。它是一种通用的技术来评估在早期胚胎着床后在体外培养的细胞的体内潜在。 电穿孔 虽然在本研究中，我们只表明，它是有效的是使用毛细管电穿孔技术目标外胚层，我吨，还可以有意地靶向其它胚层如内胚层细胞。用于毛细管电穿孔技术的关键步骤是尽量减少电穿孔每个胚（&lt;5％的胚胎分钟）所花费的时间以来的PBS是非常不理想的早期小鼠胚胎。我们的数据上面已经表明，在大部分地区，在胚胎中，电穿孔不影响胚胎的生长。然而，电的节点造成畸形发展，导致胚胎的过早死亡。这可能是由于形成重要的信号的中心23上的细胞的损伤或死亡。因此，这个区域就必须避免与这种技术。另一个需要注意的是，正如结果部分，而外胚层或原条细胞进行有针对性的，有内胚层细胞也被电。这可能是因为DNA通过下外胚层上皮间隙到达内胚层。内胚层是由上皮细胞，并在我们的经验日ESE细胞具有更高的倾向占用的DNA。因此，应用这种技术的命运映射时，必须评估哪些细胞最初占用的DNA是非常重要的。 还应当指出的是，虽然pCAG-GFP和pCAG-Cre重组:GFP的质粒可以有效地使用在本研究中所示的电穿孔参数递送，其他DNA构建的效率可能会发生变化，并且需要单独优化。在DNA浓度，电穿孔电压或脉冲数改变可以如果质粒被发现是困难的转染来制备。 总之，我们已优化的毛细管电系统能够高效，重复地提供绿色荧光蛋白或酶Cre:GFP质粒到极少数的细胞在有限的细胞死亡的胚胎。由于该方法不需要昂贵的或高度专业化的设备，它可以是对细胞跟踪研究或在测试中异位表达或条件缺失邻的效果大用处在早期胚胎˚F基因，如果电在胚胎携带两侧装接loxP条件突变等位基因进行。因此，该电穿孔技术为理解上的细胞通过细胞基础的细胞内在因素的作用在局部野生型胚胎的环境的上下文中有用的官能工具。

细胞培养已被广泛应用在发育生物学研究中。小鼠胚胎干细胞（ESC）和外胚层干细胞（EpiSCs）可以分化成在体外所有三种胚层，并且是细胞分化早期哺乳动物胚胎发育的有用模型。这些细胞系的衍生开辟了体外操纵和本地化信号事件和转录网络在早期胚胎图案形成操作的详细调查的机会。然而，它确定在培养完成的任何操作的体内相关性仍然是重要的。对早期胚胎来源的小鼠胚胎干细胞在体内的潜力进行了评估通过引入它们放回植入前胚胎（桑椹胚或囊胚）1。然而，这代表外胚层细胞在胚胎着床后EpiSCs不能有效地在植入前胚胎2,3集成。我们previo我们的研究结果表明，EpiSCs可以有效地生成嵌合体，促进所有胚层，当移植到着床后的胚胎4。因此，为了评价在体外培养的细胞的最好方法是将它们引入到它们相应的体内环境。 电穿孔是一种广泛使用的方法，提供外源分子导入靶细胞在体内和体外实验 。电能可以生成大量的细胞膜，允许外源脱氧核糖核酸（DNA）或核糖核酸（RNA），以进入细胞孔。其中一个这种技术的最大挑战是最佳的细胞活力结合高electrotransfection效率5,6。为在胚胎组织核酸电穿孔，镀金电极已最常被使用，允许细胞在一个宽广的空间范围7-9定位。为了获得更多的LOCAlized基因转移，一个针状电极已被用来实现一个焦点的电场10,11。使用这种方法，作者表明，电穿孔后，约为30-60细胞已经占用的DNA构建11。不过，似乎能准确调节电穿孔细胞的数量保持很难与一个固定宽度电极。毛细管电穿孔技术已被用于递送质粒到单个细胞12-14中 。然而，这种技术还没有得到应用电穿孔质粒胚胎体外。最近，一个微型装置已报道在早期着床后小鼠胚胎15局部地电穿孔几远侧内脏内胚层细胞（小于4细胞）。然而，它仍然是未知的，这设备是否能够有效地靶向外胚层和中胚层离体进行。 在这项研究中，我们描述了两种新的方法来评估在后早期细胞和基因功能-implantation胚胎。我们首先演示了如何移植体外培养的细胞变成早期小鼠胚胎中定义的网站，以评估它们在体内的潜力。在移植细胞及其后代的融合，所有标记的由例如遗传标记（绿色荧光蛋白（GFP），可以通过组织特异性蛋白的免疫染色来进一步检查4;其次，我们描述一种改进的方法，以精确地将DNA传递在通过电穿孔胚胎定位的位点。而不是使用一个针状电极，我们插入一个细金属丝的细尖玻璃毛细管内，并表明，该变形例可以提供的DNA，以少量的细胞具有较高的效率和有限的细胞死亡。此外，我们还表明，采用玻璃毛细管不同的开口尺寸，我们可以控制电穿孔细胞的数量。因此，我们认为这种方法可以有很大的应用研究早期胚胎图案涉及少数ØF细胞。

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methods for precisely localized transfer of cells or dna into early postimplantation mouse embryos

操纵小鼠早期胚胎和文化是一个功能强大且主要是利用不足的技术增强这个模型系统的价值。相反地​​，细胞培养物已被广泛用于发育生物学的研究。然而，它确定在体外培养的细胞是否真正代表在体内的细胞类型是重要的。移植细胞成胚胎，随后开发期间的贡献的评估是一种有用的方法，以确定在体外培养的细胞的潜力。在这项研究中，我们描述了用于移植的细胞置于定义早期植入后的小鼠胚胎，随后离体培养的部位的方法。我们还介绍了一个使用已知直径的玻璃毛细管，允许接收与两个高转染效率和低的细胞死亡的外源DNA的细胞数的精确定位和调整优化的电穿孔法。这些技术，它不需要任何属ecialized设备，使实验操作的原肠胚形成和早期器官形成阶段的小鼠胚胎成为可能，从而在培养的细胞亚群的承诺和遗传操作原位对细胞分化的影响进行分析。

嫁接 EpiSCs该无所不在表达EGFP（R04-GFP，从E6.5外胚层衍生，和C2，从mESCs衍生体外）4是手动从培养皿刮出并移植到不同的网站E7.5胚胎（图2A）。将胚胎体外培养和24小时后进行分析。供体细胞的分布通过荧光显微镜进行评估。如果供体细胞掺入，它们增殖和主机胚胎（图2B）内其衍生物分散。它已经观察到含有在宿主胚（图2A， 和图2B）有效地结合10月16日的细胞移植物，但是，接枝更多的细胞不会导致更好chimaerism。相反，移植细胞产生的未掺入的团块（图2C和2D）。 电穿孔 要在ssess我们的电系统的效率，我们提供绿色荧光蛋白表达质粒（pCAG-GFP和pCAG-Cre重组:GFP）在胚胎特定的网站。符合先前的研究图11，在胚胎中检测到GFP +细胞后电穿孔（ 图2E与2G）1-2小时。当远端外胚层细胞在原始社会后期连胜阶段的胚胎进行电，标记细胞24小时培养（图2E和2F）后促成了神经外胚层。这一结果对应很好地外胚层细胞在原肠胚期胚胎17知天命的地图。类似地，当GFP表达质粒在E8.5（2-5个体节）电穿孔在原条，GFP +细胞促进了近轴中胚层（图2G和2H），与已故原条18的公知的命运地图一致。此外，我们观察到的贡献从电穿孔的细胞的所有三种胚层（图2I-K）这表明电穿孔过程不会损害体内细胞的行为。然而，我们也注意到，虽然外胚层（E7.5）或原条细胞（E8.5）进行了针对性，某些内胚层细胞还电穿孔（图3C和表1）。 一种利用毛细管电极的主要优点是，电穿孔的细胞的数量可以被控制，简单地通过改变其开口的直径。为了确定电穿孔的细胞的数量，胚胎固定电穿孔后2小时，在成像wholemount上的共焦显微镜。 GFP +细胞的数量是手动计算在共焦的z栈。表1表明，对于一个给定的阶段中，由多个小区增加从20至30微米的结果中的DNA摄取的玻璃毛细管的开口尺寸。当单个开口大小级之间比较（E7.5与E8.5），发现更多的细胞在后期被电。这种效果可能是由于存在于羊膜腔在E8.5的DNA的浓度较高。因为DNA溶液与绿色食用色素混合，我们可以使用绿色，以评估在羊膜腔DNA浓度。在显微镜下，很明显的是，当与E8.5胚相比，DNA注射后的绿色的颜色是在E7.5胚胎的空腔轻得多。虽然DNA溶液相同浓度注入E7.5和E8.5的胚胎，多个DNA溶液在E8.5胚胎为了羊膜腔以完全填充它，因为它们在大小。抽出注射针后，总有一定程度的从羊膜腔DNA溶液的泄漏，并且由于穿孔是在比较早期胚胎羊膜腔的大小大，则很可能是有比例更泄漏从E7.5 E8.5比胚胎，从而导致较低的DNA浓度。的不同数量的染细胞还可能是由于在不同阶段的不同直径的细胞或诱导的跨膜电压（ITV）阈值。 电穿孔的一个缺点是相关联的细胞死亡。类似于传统的镀金或针状电极，使用毛细管电极电穿孔也导致细胞死亡。电穿孔后的目标区域中的颜色出现较暗相比邻近区域（图3A和3B），表明某种程度的细胞死亡的必须发生在这一区域中。为了进一步确定所造成的电穿孔程序死细胞的数量，胚胎染色用荧光细胞膜不透核染料。死细胞的细胞核进行标记，用膜不透远红光荧光染料。染色确认这毛细管电穿孔技术只导致小的数目邻近电穿孔部位死细胞（图3D和Ta的BLE 1）。 我们注意到，虽然死细胞出现在电现场，GFP +细胞和死细胞也最独特的彼此（图3E和3F）。此外，当在E8.5尾侧外胚层电穿孔用pCAG-GFP和20微米的玻璃毛细管开口，大量的GFP +细胞的48小时培养中（图3G和3H）后进行检测。两者合计，这些结果表明大多数的 GFP +细胞中检测到2小时电穿孔后仍存活在进一步培养。 我们后24小时体外培养打进的 GFP +细胞的数量。六个胚胎电穿孔pCAG-GFP在E7.5，使用20微米直径的毛细管开口。 107±31（平均值±标准差） 的 GFP +细胞/胚胎进行检测。因为在培养物（2小时）的开始，9细胞进行电穿孔，平均每个胚胎（ <s<html>仲&gt;表1），这表明，电穿孔的细胞进行3-4司内2小时。平均气泡从E7.5倍增时间至E8.5胚胎是6-7小时内所有细胞除了那些在腹侧节点19,20。这表明，电穿孔过程不妨碍正常的细胞生长。 <img alt="图1" src="/files/ftp_upload/53295/53295fig1.jpg" /> 图1.电路图表示电穿孔设置。在其羊膜腔含胚胎DNA溶液置于两个电极之间。电流在所选定的参数是由方波脉冲发生器（电源）提供的。万用表串联连接，检测电流通过胚胎。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53295/53295fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> jove_content"FO:保持together.within页="总是"&gt; <img alt="图2" src="/files/ftp_upload/53295/53295fig2.jpg" /> 图2.接枝或电穿孔的细胞在宿主胚胎的分布。（AH）GFP荧光重叠（绿色）上wholemount胚胎（灰度）（A）的明视野图像10月16日的 GFP + EpiSCs移植到较晚的远端区域-streak阶段的胚胎。（℃） 的 GFP + EpiSCs的较大丛被嫁接到中间条纹阶段的胚胎的远端区域。（B和D）在宿主胚胎EpiSCs衍生的细胞（绿色）的分布（在所示A和C）24小时培养后，分散在宿主胚（B）中 的 GFP +细胞，提示供体细胞的正确整合。（D）的接枝较大的细胞团块导致非法人丛形成的宿主胚。 （EK）pCAG-Cre重组:GFP质粒ELE<html> ctroporated成野生型胚胎的特定区域。电穿孔的早期芽阶段胚胎（E）或一个2-5体节阶段胚胎（G）中的原条的远端区域造成的GFP +细胞在这些区域中2小时后的程序。 （F和H）的GFP +细胞在宿主中的胚胎24小时培养后的分布，显示出电穿孔细胞向神经外胚层（黑色箭头）（F）和近轴中胚层（白色箭头）（H）（IK） DAB免疫染色将GFP +细胞显示出电穿孔的细胞可产生神经外胚层（I）中，中胚层（J）和内胚层（J和K）24小时培养后。比例尺（AH）= 250微米;比例尺（IK）= 100微米。注意:图1A和1B是从我们的先前出版物4重印。HREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53295/53295fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53295/53295fig3.jpg" /> 图3.分销GFP + 细胞和死细胞的电穿孔后的胚胎 （AC）pCAG-Cre重组酶:GFP质粒电穿孔到E8.5的尾部横向外胚层细胞（2-5体节期）胚胎（毛细管开孔尺寸:20微米）（A）的 2小时后的程序，该目标区域呈暗颜色（白色箭头）相比，胚胎的其他部分。插图示出电穿孔区域的放大图。（B）的明场图像（灰度）覆盖有绿色荧光通道表示电穿孔的细胞（绿色）。（C）的共焦的z切片显示出2内胚层细胞（绿色）也采取了质粒时，尾部的横向外胚层细胞进行有针对性的。细胞核示于红色（DF）pCAG-Cre重组:GFP的质粒电穿孔在E8.5胚胎的节点的尾部方面（毛细管开口尺寸:30微米）。胚胎中培养2小时。电穿孔的细胞中显示绿色和死细胞为红色。（D）中的电穿孔的区域包含的 GFP +细胞以及死细胞。在白盒的区域中一个共焦显微镜进一步分析。的z叠层的人工计数表明，有33的GFP +细胞和 23的死细胞在这个区域。只有两个细胞均为阳性两个荧光团。（E和F）共焦的z切片从白盒装区域D中的XYZ视图，示出的 GFP +细胞是分离的死细胞。细胞核以蓝色显示（G和H）pCAG-Cre重组:GFP的质粒电穿孔到几大公在E8.5胚胎的尾侧外胚层的ls（毛细管开口尺寸:20微米）和成像后两（G）和 48（H）的小时体外培养注:（H）的胚胎进行培养后切断为两半。除去头部和心脏地区。比例尺（A，B，D，G和H）= 250微米;比例尺（C，E和F）= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53295/53295fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td>开口毛细管的直径</td><td>胚胎阶段</td><td>电穿孔效率:没有。胚胎含有绿色荧光蛋白+细胞 2H /总无后。中电穿孔胚胎（无。GFP +胚胎在24或48小时培养后发育正常） </td><td> GFP平均数+细胞 ，每个胚胎±标准差（n = NO。检验胚胎） </td><td>数按每个胚胎GFP +内胚层细胞±标准差（n = NO。检验胚胎） </td></tr><tr><td> 20微米</td><td> E7.5（LS-LB） </td><td> 7/9（7） </td><td> 9±3（n = 4时） </td><td> 4±2（n = 4时） </td></tr><tr><td>为30μm </td><td> E7.5（LS-LB） </td><td>十五分之十三（12） </td><td> 17±2（n = 4时） </td><td> 6±1（n = 4时） </td></tr><tr><td> 20微米</td><td> E8.5（2-5体节） </td><td> 12个/ 13（10） </td><td> 21±4（n = 4时） </td><td> 11±4（n = 4时） </td></tr><tr><td>为30μm </td><td> E8.5（2-5体节） </td><td> 2/2（2） </td><td> 33和26（N = 2） </td><td> 14和16（N = 2） </td></tr></tbody></table> pCAG-Cre重组酶的表1电穿孔效率:在小鼠胚胎GFP质粒。 缩写:LS，已故原条的阶段; LB:后期萌芽阶段。胚根据上演Downs和戴维斯12

Watch this Scientific Journal Video about Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos at JoVE.com

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

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JoVE Journal

Developmental Biology

10.3K 次观看.  School of Biological Sciences, University of Edinburgh. We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

视频: Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

科研

发育生物学

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...