Name: generation and long-term maintenance of nerve-free hydra
Uploaded: 2017-07-07T14:00:00.000+00:00
Duration: 6 min 33 s
Description: Watch this Scientific Journal Video about Generation and Long-term Maintenance of Nerve-free Hydra at JoVE.com

作者感谢杜克坎贝尔博士（UC Irvine）就关于产生和维持无神经动物的原始方案进行讨论，王瑞女士帮助适应注射器和针头技术，Danielle Hagstrom女士和Rob Steele博士（加州大学欧文分校）对手稿发表评论。这项工作得到RCSA和NSF授权CMMI-1463572的支持。

双秋水仙碱治疗<ol><li>在Hydra介质3,4,17中制备0.4％的秋水仙碱（重量/体积）溶液。 注意:秋水仙碱是急性毒性的，如果吞咽会致命，可能会导致遗传性缺陷，并可能导致眼睛损伤。在通风橱里处理粉末并佩戴完整的个人防护装备（PPE）。 </li><li>在室温下在黑暗中培养在培养皿中每5毫升秋水仙碱溶液在橙子每毫升秋水仙碱溶液中饥饿8小时，在0.4％秋水仙素中饥饿24小时。 注意: 5 Hydra / mL秋水仙碱是确定使用多少溶液以避免用Hydra过度拥挤的指南。使用相同体积的溶液进行后续清洗和溶液更换。 </li><li>取出秋水仙碱溶液并更换干净的Hydra培养基不含秋水仙碱。在Hydra培养基中转移至50μg/ mL利福平之前，用巴斯德玻璃杯吸管清洗Hydra培养基，将Hydra洗5次。将九头蛇保存在18°C的孵化器中。 注意:在4℃下可以储存足够的1,000X利福平（50mg / mL）的二甲基亚砜（DMSO）处理1-2周。精确量可以由每天使用的溶液的总体积确定。例如，如果有2个皿，每个包含10mL需要每天更换两次的溶液，一天中将使用总共40mL的溶液，相当于每天40×1,000个原料或560μL两周以上。然后在Hydra培养基中使用时将原料稀释1:1000。 注意: DMSO是可燃液体，易于渗透皮肤，使其他溶解的化学物质进入人体。手在通风橱中使用溶剂，并佩戴完整的PPE。值得注意的是，DMSO在4℃下冻结</li><li>每天更换含利福平Hydra培养基两次，直到Hydra停止将细胞排出到培养基中，通常在治疗后1周。此时，介质可以每天更换一次。治疗后的几天， 九头蛇将彻底失去触手。避免使九头蛇彼此接触，因为它们可能融合在一起，并导致奇怪形状的九头蛇 （ 图 2A）。 <ol><li>为了防止接触，请避免旋转盘旋转。相反，在垂直和水平方向上轻轻搅动菜肴，直到九头蛇分开。将Hydra放入18℃的培养箱中检查，然后根据需要进行调整。 注意:介质更换两次的日期，请更换介质一天早上又在下午/晚上。 </li></ol></li><li>一旦触手开始形成，每周开始强迫哺乳和打嗝九至四次（见第2和第3节）。秋水仙碱治疗约8-9天后， 九头蛇开始伸展他们的触手。 注意:触手再生根据个体而异。有些可能需要长于8 - 9天才能出现触角增长迹象。那些不会再生长它们的触手以及奇怪的形状（ 图 2B）的动物或动物太小而不能进食应该被去除。这些九头蛇可能不会在第二次治疗中生存。 九头蛇也可以芽。然而，一些芽可能仍然具有间质细胞并且能够自己进食。 <ol><li>去除任何可以在没有帮助的情况下吃的芽，并从父母中分离出来。通过添加活的卤虫来识别这些动物喂养菜，观察动物是否自行捕捉和食用卤虫 。 1或2 Hydra也可以自己吃饭。这些也应该被丢弃。 注意:无神经的九头蛇具有特征性的气球样形态，触角比正常更短和更薄（由于损失了肌细胞），因此可以容易地与正常的动物区分开（ 图 1A，1B ）。 </li></ol></li><li>在第一次秋水仙碱治疗后三周，重复秋水仙素治疗（步骤1.1-1.5）。第二次秋水仙碱治疗是消除剩余的间质细胞和神经细胞所必需的3 。 </li></ol>力量喂养<ol><li>将卤虫囊肿加入到底部变细的窄而高的玻璃容器中。用容器装满卤水 （6.72M NaCl）。避免擅长每1升水加1克囊肿以获得更高的孵化率。 <ol><li>用石蜡膜覆盖容器的顶部，并通过石蜡膜将10mL血清移液管插入容器中。通过将管道连接到水族箱气泵并将管道安装在移液管周围来进行曝气。确保移液器尖端到达容器的底部，并且没有囊肿在底部沉降。 卤虫将在48小时后孵化。 注意:有许多不同的方法来孵化卤虫 ，这也可能会起作用。 </li></ol></li><li>在卤虫网（可从水族馆供应公司获取）中将卤虫从卤水中应变，并将其在去离子水中洗涤约20秒，然后将其置于含有Hydra培养基的盘中。 卤水的盐度对于九头蛇来说太高了，所以卤虫在使用前必须洗净。 </li><li>在解剖显微镜下，通过用一对精细镊子轻轻地挤压它们来安乐死卤虫 。避免挤压足够的力量排出胆量，因为这会粘在镊子上，使喂养困难。将卤虫轻轻压榨，然后将其送入Hydra将有助于消化过程14 。将新鲜杀死的卤虫附近的九头蛇放在盘子里，以方便快速喂养。 注意:所有后续步骤都在解剖显微镜下进行。 </li><li>使用一对镊子将花to握住九头蛇 。在喂养九头蛇的过程中继续握住花序梗。使用第二对钳子捏住身体柱，以使Hydra合同。 </li><li>在将第二对镊子的尖端保持在一起的同时，点击下丘的中心（动物口端的圆顶结构）。这有时会导致嘴巴打开。一世通过将镊子插入臀部的中心，然后轻轻地释放镊子的尖端上的压力，嘴巴不会通过开口打开，刺穿口腔。这应该伸出由穿刺造成的开口。 注意:嘴巴打开时， 九头蛇可能会放气和塌缩（ 图2 C ）。如果发生这种情况， 卤虫仍然可以插入九头蛇 。如果这样做太难了，请转到下一个九头蛇，然后再回到原来的水Hyd ，再试一次。 Hydra倾向于在随后的口腔中不会泄漏一样多。 </li><li>用镊子快速拿起卤虫 ，将其插入九头蛇的胃腔。插入尽可能多的卤虫 ，直到胃腔充满，更多的卤虫不能插入而不损坏九头蛇 ，事故盟友拉扯任何卤虫 ，或阻止嘴巴闭合。平均来说，这是5 - 6 卤虫 ，然而最多12种已经喂给较大的动物。 <ol><li>如果嘴巴开始闭合，如上所述用镊子再次拉伸;但是，当口腔重新开放时，动物内已经有的卤虫可能开始出现，应该小心。 </li><li>如果Hydra对于全卤虫来说太小，用手术刀切割卤虫 ，并将Hydra较小的部分喂食。如果卤虫没有留在九头蛇眼中 ，可能需要将镊子压在卤虫上 ，使其保持在九头蛇之内，直到嘴巴闭合。 </li></ol></li><li>使用玻璃巴斯德移液管转运Hydra ，仔细避免意外打嗝，在Hydra培养基中加入新鲜的50μg/ mL利福平的菜肴。如果卤虫被驱逐f在转移的同时，在新的菜中重新插入Hydra 。 注意:使用玻璃移液器来转移Hydra ，因为与塑料相比， Hydra比玻璃更少。移液器的长度并不重要，但是使用5英寸移液器可能会更容易一些。 </li></ol>打嗝<ol><li>在进食后8至20小时之间任何时候，打开九头蛇去除未消化的物质。 </li><li>用P320或类似的砂砾砂纸将30G皮下注射针点降下，直到尖端平坦。 注意: 27G皮下注射针也可以工作，但较高规格的较小尖端是优选的。 </li><li>将针头连接到一次性1mL塑料注射器，并在Hydra培养基中用新鲜的50μg/ mL利福平填充注射器。 注意:打破单个Hydra需要约0.05 mL至0.1 mL溶液。 1 mL syringe是优选的，因为用更大体积的注射器控制出来的溶液的力和体积更加困难。 </li><li>在解剖显微镜下，用精细镊子握住九头蛇的花序梗，并用针头敲击hypostome中心。如果嘴巴没有打开，请将镊子插入腰包，然后按照上述方式打开嘴巴进行喂食。 注意:所有后续步骤都在解剖显微镜下进行。 </li><li>使用注射器非常轻轻地冲洗，以避免用50μg/ mL利福平溶液吹走整个动物，胃腔，直到所有碎片都被排出。如果Hydra的尺寸不允许，则针不一定需要插入Hydra 。针可以保持靠近开口并且直接指向嘴。 </li><li>使用玻璃巴斯德移液器，将九头蛇转移到一个新鲜的50碟81; g / mL利福平在Hydra培养基中。 </li></ol> 4. 通过免疫组织化学的质量控制注意:以下协议由Shenk，M.A 等人的协议改编18 ，Böttger，A 19 。所有步骤均在室温（RT）下进行，除非另有说明。培养步骤可以使用养分器，并且可以改善染色质量。然而，如果九头蛇相互纠缠，所有的步骤也可以不执行。 <ol><li>准备封闭溶液:10％胎牛血清（FBS）和1％DMSO在1×磷酸盐缓冲盐水（PBS）中。将此溶液储存在4°C直至使用（步骤4.6和4.11）。溶液可以储存1 - 2天。 注意:本协议中使用的所有解决方案必须作为危险废物进行适当的收集。 </li><li>在Hydra m的200μL2 ％氨基甲酸酯中放置Hydra在1.7 mL微量离心管中放置1 min。在4种情况下，每个管使用5只Hydra ，无神经，未经治疗，未经第一抗体治疗，未经二抗治疗。 注意:不要超过1分钟。这里的时间是至关重要的，因为在氨基甲酸乙酯花费太多时间之后，动物会变得不好。 </li><li>取出氨基甲酸酯并将Hydra在Hydra培养基中的200μL4 ％多聚甲醛（PFA）中固定15分钟。 </li><li>用1x PBS洗涤样品3次，每次10分钟。 注意:用PBS和PBSTx洗涤全部用500μL溶液完成。 </li><li>用500μL的0.5％Triton X-100在PBS中渗透15分钟。 </li><li>取出0.5％Triton X-100并加入500μL封闭溶液。阻止样品至少1小时。 </li><li>在阻滞溶液中稀释抗酪氨酸 - 微管蛋白抗体1:200。 注意:这个浓度是de实验终止。如果发现控制信号太弱，可能需要增加浓度。如果存在非特异性结合，则可能需要降低浓度。抗体的预孵育也可以有助于减少非特异性结合。 </li><li>从样品中取出封闭溶液，并向无神经的Hydra ，未处理的对照组和未处理的对照组中加入200μL的无抗体，无次要的。对于未经初步处理的未处理对照，仅添加200μL无抗体的封闭溶液。 </li><li>在4℃下孵育样品过夜（&gt; 12小时）。 注意:或者，在室温孵育5 - 6 h。 </li><li>取出第一抗体，并用1×PBS（PBSTx）中的0.3％Triton X-100大量洗涤样品。 注意:保存一次抗体并保存在4°C，因为它可以重复使用至少2-3次。 </li><li>稀释山羊抗小鼠hP二抗ody 1:500阻塞溶液。向无神经的Hydra ，未处理的对照组和未处理的对照组中加入200μL，无原代培养。对于没有继发的未处理对照，只加入200μL无抗体的封闭溶液。 注意:或者，可以使用荧光二抗，不需要步骤4.13-4.17。使用荧光辅助器节省时间并且通常是足够的，但hP次级提供增加的灵敏度。实验确定次级浓度。如果控制中的信号太弱，则可能需要增加浓度。如果存在非特异性结合，则可能需要降低浓度。 </li><li>在4℃下孵育样品过夜。 </li><li>取出第二抗体并用PBSTx广泛洗涤样品。 </li><li>在1×PBS中制备1×PBT:0.2％牛血清白蛋白（重量/体积）和0.05％Tween20。孵出来在1×PBT中的30分钟。 </li><li>取出1×PBT并在1x PBT中1:1,000 NHS-荧光素和1:10,000H 2 O 2的样品在黑暗中孵育15分钟。从这一步开始，尽可能保持样品在黑暗中，因为荧光信号会在暴露于光下时减少。 注意: NHS-荧光素是按照King，RS和Newmark，PA 20的详细FISH方案制备的</li><li>用PBSTx快速洗涤样品3次，然后2次30分钟。 </li><li>将样品放在PBSTx中4℃过夜，继续洗涤。 </li><li>再用1x PBSTx洗几次。为了可视化细胞核，使用4&#39;，6-二氨基-2-苯基吲哚，二盐酸盐（DAPI）进行DNA复染的以下任选步骤。否则，现在可以对样品进行成像。 </li><li>在PBSTx中稀释5 mg / mL DAPI原液至1:500的工作浓度，并向每个管中加入200μL。 INCU将样品叮咬30分钟。 注意: DAPI是一种已知的致癌物质。穿全PPE。 </li><li>在用荧光显微镜成像之前，用PBSTx去除DAPI并洗涤样品2-3次。 </li></ol>

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Biol. 172 (1), 170-181 (1995).</li><li>Miljkovic-Licina, M., Chera, S., Ghila, L., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+regeneration+in+wild-type+hydra+requires+de+novo+neurogenesis.">Head regeneration in wild-type hydra requires de novo neurogenesis.</a> Development. 134 (6), 1191-1201 (2007).</li><li>Wenger, Y., Buzgariu, W., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+neurogenesis+in+Hydra+leads+to+compensatory+regulation+of+neurogenic+and+neurotransmission+genes+in+epithelial+cells.">Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells.</a> Phil. Trans. R. Soc. B. 371 (20150040), (2015).</li><li>Campbell, R. D., Josephson, R. K., Schwab, W. E., Rushforth, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+nerve-free+Hydra.">Excitability of nerve-free Hydra.</a> Nature. 262, 388-390 (1976).</li><li>Takaku, Y., Hwang, J. S., 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=Innexin+gap+junctions+in+nerve+cells+coordinate+spontaneous+contractile+behavior+in+Hydra+polyps.">Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.</a> Sci. Rep. 4 (3573), (2014).</li><li>Marcum, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Epithelial+Hydra.">Culturing Epithelial Hydra.</a> Hydra: Research Methods. , 287-290 (1983).</li><li>David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+method+for+maceration+of+Hydra+tissue.">A quantitative method for maceration of Hydra tissue.</a> Wilhelm Roux Arch. Entwickl. Mech. Org. 171, 259-268 (1973).</li><li>Gr&#246;ger, H., Schmid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+development+in+Cnidaria:+A+connection+to+Bilateria.">Larval development in Cnidaria: A connection to Bilateria.</a> Genesis. 29 (3), 110-114 (2001).</li><li>Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water,+Culture+Solutions,+and+Buffers.">Water, Culture Solutions, and Buffers.</a> Hydra: Research Methods. , 29-34 (1983).</li><li>Shenk, M. A., Bode, H. R., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cnox-2,+a+HOM/HOX+homeobox+gene+in+hydra,+is+correlated+with+axial+pattern+formation.">Expression of Cnox-2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation.</a> Development. 117, 657-667 (1993).</li><li>B&#246;ttger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+Gene+Transfer+Contributed+to+the+Evolution+of+Extracellular+Surface+Structures:+The+Freshwater+Polyp+Hydra+Is+Covered+by+a+Complex+Fibrous+Cuticle+Containing+Glycosaminoglycans+and+Proteins+of+the+PPOD+and+SWT+(Sweet+Tooth)+Families.">Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families.</a> PLoS One. 7 (12), (2012).</li><li>King, R. S., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+protocol+for+enhanced+detection+of+gene+expression+in+the+planarian+Schmidtea+mediterranea.">In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.</a> BMC Dev. Biol. 13 (8), (2013).</li></ol>

作者没有什么可以披露的。

Hydra间质细胞可以通过双秋水仙碱治疗3,4消除。在第一次处理之后的几天内，防止个人Hydra之间的接触，以避免Hydra碎片融入变形Hydra中至关重要。此外，第一次治疗后可以不助食的动物必须去除，因为第二次秋水仙碱治疗可能不足以从这些动物中消除剩余的间质细胞。为了使第二次秋水仙碱治疗后无神经性九头蛇的存活率最大化，从第一次治疗?...

九头蛇神经系统由神经网组成，神经元与上皮组织层1相关联。神经网在体积较小的密集体和花序梗中较密集，体积不足2 。神经细胞来自间质干细胞，这是造成分泌细胞，神经细胞，生殖细胞和神经元的多能干细胞。可以通过用秋水仙素3,4 （一种杀死分裂细胞的植物来源的毒素）处理来消除寻常性黑草的间质细胞。尽管秋水仙碱已被发现抑制其他生物体中的微管聚合，但以前的研究表明，在整个治疗过程中， Hydra存在微管，这表明秋水仙素在Hydra 3中不起作用。另一个udy表示秋水仙素在一些生物体中不能有效地结合微管蛋白，包括四膜虫，三角豆，衣藻和粟酒裂殖酵母，这可能解释了这种差异。秋水仙碱治疗通过内胚层上皮细胞3诱导间质细胞的吞噬，从而允许产生缺乏神经细胞，腺体细胞和神经细胞的动物。不清楚为什么间质细胞对秋水仙碱治疗特别敏感。鉴于有丝分裂后间质细胞和间质干细胞谱系都受损和吞噬，Campbell认为秋水仙素不直接影响有丝分裂活性3 。值得注意的是，秋水仙碱治疗在寻常 藿香中效果良好，但在其他物种如Hydra oligactis 6中也 表现不佳 。 </用秋水仙碱和羟基脲进行改良处理可用于产生无神经的水ra草 7 。因此，无神经的Hydra （有时也被称为"上皮Hydra " 8 ）是研究来自间质细胞系的这些特殊细胞类型在组织稳态和再生中的作用的有用工具。 九头蛇可能是没有神经系统能够生活的动物的唯一已知的例子。无神经的九头蛇作为一种特别有用的模型，用于解剖神经网在调节Hydra再生，体内平衡和行为中的作用。例如，通过移植将间质细胞引入无神经的Hydra ，允许将神经细胞分化表征为高度区域特异性9 。此外，因为无神经的Hydra可以再生，它们可以调查替代的，神经系统独立的再生途径。一个这样的例子是顶端神经发生和头部形成，其已显示取决于野生型Hydra中神经系统中的cnox-2功能，但似乎在无神经的Hydra中是不必要的 ，这表明可能存在替代的头部再生过程10 。 神经发生丧失后，神经元和神经传递基因也被用于研究上皮细胞的表达和调节。无神经的九头蛇不会发生自发的收缩爆发12 ，表明这些爆发是由神经系统调节的。然而，无神经的水ra ra ，，in to to the the the force force u u u u u u u u u u u u u u u u u u ugh间隙连接在上皮细胞，而自发收缩行为介导通过间隙连接在神经细胞13 。 无神经的九头蛇在呈现食物或还原型谷胱甘肽3时不要张开嘴巴，这表明感觉神经元是检测食物存在所必需的，并且指示口腔开放。此外，神经网似乎在感测渗透压方面发挥作用，因为无神经动物不能通过开口自主调节其内部静水压力，导致其特征性的气球样外观3,4 （ 图1B ）。通过频繁的手动通气调节无神经的Hydra的静水压，导致hypostome和身体柱中的一些异常形态的损失。然而，长期通缩导致了对经济增长的干扰发病，萌芽和组织组织8 。 虽然无神经的九头蛇不能自己饲养和自发，但可以通过手动强制喂养和打击每只动物在实验室中无限期地保持它们。以前的出版物已经描述了强制喂养和打嗝无神经Hydra的方法 ，然而这些方案涉及使用微量移液器吸头，其必须被小心地手动地拉到适当的尺寸，以及使用通过管道14连接到移液管的接口管。这里描述了一种更简单，更安全，更节省时间的喂食和打嗝方法。 此外，以前的研究涉及通过将固定动物解离成单个细胞来检查神经细胞的缺失和细胞形态的检查3,4,15。 H使用抗α微管蛋白的酪氨酸羧基末端的单克隆抗体的免疫组织化学作为一种补充方法，用于检查hypostome 13,16中神经元的消耗。以前的研究已经表明，也可以使用这种抗体13可视化花序梗中的神经元，然而这些神经元以及体列中的这些神经元更难以形成。虽然免疫组织化学足以证实在hypostome中不存在神经细胞，并且不需要关于细胞类型形态的专门知识，但是它不能用于检查间质干细胞和这些细胞的其它衍生物的缺失。解离和细胞形态学研究更加严格，并且可以给出每个治疗阶段后剩余的每种细胞类型的数量的定量分析。

<table><tbody><tr><td>Colchicine</td><td>Acros Organics</td><td>227120010</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD</td><td>301025</td><td></td></tr><tr><td>Brine Shrimp Eggs</td><td>Brine Shrimp Direct</td><td>N/A</td><td>Can be purchased locally</td></tr><tr><td>Brine Shrimp Hatchery Dish</td><td>Brine Shrimp Direct</td><td>N/A</td><td></td></tr><tr><td>60 mm x 15 mm Petri Dish</td><td>Celltreat</td><td>229663</td><td></td></tr><tr><td>30 G x 3/4&quot; Hypodermic Needle</td><td>Covidien</td><td>1188830340</td><td>A 27G needle may also be used</td></tr><tr><td>2 x Fine-tip Tweezers</td><td>Dumont</td><td>0109-5-PO</td><td></td></tr><tr><td>Rifampicin</td><td>EMD Millipore</td><td>557303</td><td></td></tr><tr><td>Goat anti-mouse lgG, Pab (HRP Conjugate)</td><td>Enzo</td><td>ADI-SAB-100-J</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher</td><td>BP9703-100</td><td></td></tr><tr><td>Scalpel</td><td>Fisher</td><td>08-920A</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>PBS Tablets</td><td>MP Bio</td><td>2810305</td><td></td></tr><tr><td>Monoclonal Anti-Tubulin, Tyrosine</td><td>Sigma</td><td>T9028</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma</td><td>216763</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Triton X - 100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Urethane</td><td>Sigma</td><td>U2500</td><td></td></tr><tr><td>Air Pump</td><td>Tetra</td><td>77846-00</td><td></td></tr><tr><td>Glass Pasteur Pipette</td><td>VWR</td><td>53283-916</td><td>Length of the pipet does not matter</td></tr><tr><td>15 mL Tube</td><td>VWR</td><td>89039-670</td><td></td></tr><tr><td>P320 Sandpaper</td><td>3M</td><td>IBGABBV00397</td><td>Can be purchased at local home improvement store</td></tr></tbody></table>

generation and long-term maintenance of nerve-free hydra

Hydra的间质细胞谱系包括多能干细胞及其衍生物:腺体细胞，线粒体细胞，生殖细胞和神经细胞。间歇细胞可以通过两次连续的处理来消除，秋水仙素是一种植物来源的毒素，其杀死分裂细胞，从而消除了从间质干细胞衍生的分化细胞的更新潜力。这样可以产生缺乏神经细胞的九头蛇 。无神经的息肉不能打开嘴巴进食，最小化或调节渗透压。然而，如果经常强制喂养和褥疮，这样的动物可以在实验室中无限期地存活和培养。神经细胞的缺乏允许研究神经系统在调节动物行为和再生中的作用。以前发布的无神经性Hydra维持方案涉及过时的技术，例如用手拉式微量移液管吸嘴ips喂养和清洁九头蛇 。在这里，介绍了一种用于维持无神经的Hydra的改进方案。精细的镊子用于强制开口并插入新鲜杀死的卤虫 。在强制喂养之后，使用注射器和皮下注射针将动物的体腔用新鲜培养基冲洗以除去未被消化的材料，这里称为"打嗝"。通过使用镊子和注射器，这种通过使用镊子和注射器的强制喂养和打嗝无神经的九头蛇的新方法消除了使用手拉式微量吸头的吸嘴的需要。因此使得该过程更加安全和显着地更加高效。为了确保hypostome中的神经细胞已被消除，使用抗酪氨酸 - 微管蛋白进行免疫组织化学。

在最初的8小时秋水仙碱治疗之后，所有Hydra都存活下来。其中一些Hydra只会有触角残留物（ 图3A ），而其他的则会完全失去触手（ 图 3B ）。在接下来的1或2天之内，触手将继续缩小，直到所有的九头蛇都已经失去触手。治疗后约1周， Hydra将在触角充分再生之前显示触角再生的迹象，如触...

Watch this Scientific Journal Video about Generation and Long-term Maintenance of Nerve-free Hydra at JoVE.com

Generation and Long-term Maintenance of Nerve-free Hydra

通过秋水仙碱的双重治疗，可以产生一种杀死分裂细胞的植物来源的毒素，可以产生无神经的九头蛇 。这些九头蛇不能自己饲养或觅食。本文介绍了实验室中长期维持无神经寻常性黑 。 病的改进方法。

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

generation-and-long-term-maintenance-of-nerve-free-hydra

Research

JoVE Journal

Neuroscience

Generation and Long-term Maintenance of Nerve-free Hydra

12.4K 次观看.  UC San Diego. 通过秋水仙碱的双重治疗，可以产生一种杀死分裂细胞的植物来源的毒素，可以产生无神经的九头蛇 。这些九头蛇不能自己饲养或觅食。本文介绍了实验室中长期维持无神经寻常性黑 。 病的改进方法。 

视频: Generation and Long-term Maintenance of Nerve-free Hydra

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

科研

发育生物学

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

神经科学

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and free-swimming medusae. As exemplified in established models such as Hydra and Nematostella, stem cells and/or proliferative cells contribute to the development and regeneration of cnidarian polyps. However, the underlying cellular mechanisms in most jellyfish, particularly at the medusa stage, are largely unclear, and, thus, developing a robust method for identifying specific cell types is critical. This paper describes a protocol for visualizing stem-like proliferating cells in the hydrozoan jellyfish Cladonema pacificum. Cladonema medusae possess branched tentacles that continuously grow and maintain regenerative capacity throughout their adult stage, providing a unique platform with which to study the cellular mechanisms orchestrated by proliferating and/or stem-like cells. Whole-mount fluorescent in situ hybridization (FISH) using a stem cell marker allows for the detection of stem-like cells, while pulse labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), an S phase marker, enables the identification of proliferating cells. Combining both FISH and EdU labeling, we can detect actively proliferating stem-like cells on fixed animals, and this technique can be broadly applied to other animals, including non-model jellyfish species.

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2&#39;-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected.&#160;

太平洋水母干细胞样细胞的荧光原位杂交和5-乙炔基-2'-脱氧尿苷标记

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

重建神经元电路:快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

转基因的产生<em&gt;水润</em&gt;通过胚胎显微注射

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

生物学

<meta charset="utf8"></head><body>对从小鼠脑室下区分离的 NSC 进行高效成核

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

无神经的一代和长期维持

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