作者想感谢约瑟夫LoTurco和丹尼斯Selkoe这种技术的有益的讨论。作者感谢美国健康援助基金会的捐助者，为支持这项研究。

在子宫内电的基本协议已经详细描述了在另一个1，2 Kriegstein实验室的朱庇特的文章。这种技术最初是在大隅实验室3和描述我们的协议是基于后在LoTurco 实验室 4发达的地区之一。我们将为您提供概述大鼠胚胎在子宫内电“我们的协议，专注于最重要的细节，说明了我们的协议， 在子宫内电适用于研究基因功能的神经过程的产物。 在子宫内电穿孔1。 <ol><li> 准备DNA和装载针 在子宫内 electroporations的第一步是设计实验，以确定要注入的DNA的结构。这种方法是有用的misexpressing或撞倒基因。如果您在规划misexpressing或过度表达的基因，一定要使用的发起人，是活跃在神经前体细胞。我们建议CAGGS发起人，其中包括鸡β-肌动蛋白启动子和CMV增强5。由于只有一个细胞的一小部分是在子宫内电转，关键是要包括如GFP荧光蛋白的质粒编码，因此，您可以按照那些成功的电穿孔细胞。对于质粒GFP，我们建议准备DNA的浓度在0.5微克每微升。对于shRNA的结构，我们发现，你感兴趣的基因敲0.5-1.0微克每μL，结果在有效。对于过度表达或错误表达，我们用每微升1.0和3.0微克之间，取决于大小的基因表达水平的实验要求。国家主管部门编制使用Qiagen公司的内毒素的制备试剂盒，并在1 × PBS稀释。我们注入胚胎脑约0.5-1.0％微升，因此，对于我们准备注射液10μLDNA混合物的动物垃圾。我们添加1μL快绿的DNA，所以，我们可以按照注入的DNA。 拉针正确的形状是关键的一步。 Walantus 等人使用不同的系统提供的DNA，所以他们的针准备性能稍微不同的，那么我们的1,2。的设置，你来拉你的针，将取决于针车夫，你的品牌。我们从大卫Kopf使用型号750。我们使用的设置是：烧热1：9.0，2：0热，Soleniod：0，灯丝尺寸3.0毫米，感应加热器：3毫米，时间：10秒。一旦拔出，我们将用刀片在一个45度角，从开放给小费的最大距离是11毫米的针头。然后，我们从针后端负载的DNA。然后填写在玉米油针的剩余空间。 DNA注射，我们使用了Picospritzer三。根据确切的斜面，每针切不同，我们设置Picospritzer从4.0至6.0。我们用一个脚踏板提供增压空气，排出针的DNA。 </li><li> 准备手术的动物 我们使用无病原体的SD大鼠，专​​门为这些手术。其他几个实验室的使用以及不同基因型的小鼠。在这里，我们描述了我们的协议显示E15大鼠胚胎电，但在子宫内的电是经常的E13和E18之间的年龄大鼠进行。虽然初期电目标的皮质深层，后一阶段electroporations的目标更多的表面层。 动物都获得了手术前剂量丁丙诺啡（0.05-0.1毫克/公斤），手术开始前。对麻醉动物有多个选项。 Walantus 等。利用isofuorane吸入，而我们经常使用腹腔注射氯胺酮（40-80毫克/公斤）和甲苯噻嗪（5-10毫克/公斤）1,2。一个脚趾头掐应始终执行，以确保动物完全麻醉，反应迟钝。动物保持在整个手术过程中的加热垫。 动物的毛皮剃光在切口的地区，并与乙醇用碘酒三次洗三次。皮肤外侧向中线切口，切口在肌肉。子宫角接触非常谨慎。他们轻轻地梳理出人体腔，用你的指尖。请用无菌PBS胚胎水合，而他们之外的体腔。 </li><li> 注射的DNA和电 当你第一次开始执行这些手术时，最难的是熟悉，你需要注入的DNA，让你填写的侧脑室，并有多深，你注入你的针，为了达到正确的区域。胚胎轻轻用指尖操纵，让你可以IDENTIFY的头部在哪里，如果你仔细观察，你将能够看到中线缝合。这作为一个一般的具有里程碑意义的，你可以用它来确定是位于侧脑室。我们通过子宫壁，进入侧脑室注入的DNA。我们用一个脚踏板来控制注射的DNA - DNA的多个脉冲进行，直到侧脑室与DNA /染料混合物填充。我们然后将胚胎的头两侧桨电极和使用另一脚踏板，提供脉​​冲，整个胚胎的头部。电极的位置是在确定皮层区域是电穿孔的关键。由于DNA是带负电荷的DNA将旅行时负责整个桨消除朝正电极。根据电极的确切位置，将针对不同的细胞亚群。我们经常把整个大脑的背内侧位置附近的正极。然而，LoTurco实验室精美表明，如果你把更多的大脑腹外侧地区的电极，可以针对 6外侧骨皮质流的皮质纹状体边界和命中细胞的细胞。每个胚胎可以被电，并可以在每个胚胎的DNA结构的不同组合。 </li><li> 缝合和术后护理 电所有胚胎，子宫角仔细返回到体腔，肌肉层和皮肤缝合。这种技术是在Walantus 等 1,2概述。动物连续监测，直到他们从麻醉中恢复，管理和镇痛丁丙诺啡（0.05-0.1毫克/公斤），每8-12小时。 </li></ol> 2。培养电穿孔皮层神经元<ol><li> 收获电穿孔的大脑解剖电穿孔地区 在子宫内电以下的体内分析，动物可以被收获从出生到成年后早期电后24小时在任何时间点。然而，当培养初级神经元，我们收获了24小时后在E16电。此时，电穿孔胚胎表达GFP的检测水平。 动物安乐死使用吸入二氧化碳和快速断头。胚胎的子宫解剖和二价阳离子的HBSS，保持跟踪胚胎的DNA质粒电穿孔。使用过滤的HBSS，无菌管和板，蒸压清扫工具，这一点至关重要。皮层是解剖和脑膜中删除使用显微镜在引擎盖。这些皮层，然后与绿色荧光蛋白的能力，可视化的解剖显微镜下观察。 GFP阳性的皮层区域确定，我们UE一双一万纳剪刀剪出从皮质的GFP阳性的地区。这些作品被放置在一个15毫升的锥形管中的二价阳离子的HBSS没有。 </li><li> 游离和电镀神经元 HBSS中，一旦所有的GFP阳性地区都解剖，是用0.25％胰酶替代，孵育5分钟，在37度。胰蛋白酶被删除，取而代之的是与电镀媒体（DMEM培养基+ 5％胎牛血清+佩恩/链球菌+谷氨酰胺），和磨碎的5-7倍，游离细胞。卷使用后，取决于组织目前的金额。游离细胞，然后镀上CC2涂层玻片。对于两个玻片，我们板体积1.5毫升电镀媒体200,000-350,000细胞每室。 4小时后，电镀媒体吸气和1.5毫升每室（神经元培养基Neurobasal媒体+ B27的补充+ glutamax +庆大霉素）所取代。 </li></ol> 3。神经过程的产物分析<ol><li> 固定和免疫文化 为了测量这些细胞的遗传操作的短期内effecs，我们收获后三天在体外的主要神经元。如果是要培养更多的分析不再的初级神经元，一半的媒体应更换一次3天。固定的文化，我们从分庭吸媒体，并在15分钟的4％paraformaldhyde的神经元修复。录制后，细胞用PBS洗涤两次，然后在封闭液（2％驴血清，用0.1％Triton X - 100的PBS）一小时的。细胞，然后在第一抗体孵育1小时。分析神经过程的产物，我们使用抗βtubululin抗体，以确定神经元 - 测试版II微管蛋白的免疫标签neruonal细胞体，树突和轴突。细胞，然后洗五分钟，在PBS的3倍，然后孵育1小时，由三个PBS洗涤counterstaining细胞核用DAPI和安装，Cy3标记的抗小鼠。 </li><li> 测量NEurite长度 蔡司Axioskop GFP阳性，β-Ⅲ微管蛋白阳性神经元的图像采集与MC100摄像机系统。这些包括所有突起的长度，最长突起长度的GFP阳性细胞，可以检查几个变量，分支的突起，细胞胞体的大小等，我们已经使用这种方法来分析后神经元过程中击倒或基因的表达产物有关的神经发育和神经退行性疾病，神经过程的长短上的焦点。为了测量神经元突起的生长，我们使用Axiovision LE 4.4软件（蔡司）。在这个软件中，有一个选项，选择“纲要”的工具。使用此工具，您可以使用您的计算机的鼠标跟踪的每个神经元的过程的长度。确定的目标，您使用的是为了得到准确测量您的突起，这是关键。对于这些分析，我们通常使用20倍的目标。 </li></ol> 4。代表性的成果我们发现，只Dawley产仔数范围在6日和14胚胎。我们通常electroporate所有的胚胎。每个胚胎可以被电具有不同的DNA结合。然而，我们通常electroporate至少有四个相同的条件下，游离和电镀前池这些大脑大脑。 我们发现，使用这种技术，大约75％的电穿孔的大脑有针对性地所需的皮质区域，不管是背内侧或腹外侧皮层（图1）。此外，我们已经发现E13 - 14的目标，如Tbr1积极第六层神经元的深层神经元的早期electroporations，同时目标CTIP2积极，TBR1负V层细胞，再后来electroporations目标Brn2积极的II / III层细胞​​以后electroporations。一个优秀的描述不同的标记和皮层神经元亚型规范的解释的发现 Moleneaux等7条。图2显示了在任胚胎每天15.5或17.5电穿孔和大脑在出生后第5天收获的冠状切片。图中红色是免疫的Oct6。您可以免疫染色标记在文化，以确认有针对性的细胞层的人群。我们发现，你可以期望目标在同一窝的每一个胚胎细胞相同的细胞层人口（换言之，目标取决于胚胎的时间点而不是在其他技术变化）。 ％GFP阳性细胞，可以在文化上，范围广泛，取决于你如何保守解剖GFP阳性区域（图3）时，。然而，即使我们是非常保守的，并剖析GFP阳性细胞修补，我们观察到的是5-10％的比例最高 - 虽然你解剖电穿孔皮质区域，在只有一层的细胞会做到有的放矢。这种低转effciency有助于确定哪些进程属于你分析electoporated细胞。然而，在这个高密度电镀细胞有助于heathier文化，它是很难辨别哪些进程属于细胞体中GFP阴性细胞（图3）。 如果你看到的所有的电穿孔细胞的精细的工序有麻烦，你可以增加你是电穿孔增加GFP表达GFP的DNA浓度，你可以使用抗GFP抗体免疫染色游离细胞（自Invitrogen ）沿与CY2二级抗体。 <img src="/files/ftp_upload/2103/2103fig1.jpg" alt="图1" /> GFP质粒电穿孔与图1。E15.5 Sprague - Dawley大鼠，3天后收获。根据电极的位置，皮层的不同区域将有针对性。在整个大脑的自动对焦显示绿色荧光。 <img src="/files/ftp_upload/2103/2103fig2.jpg" alt="图2" /> 图2。E15.5（A）或E17.5（二）Sprague - Dawley大鼠电穿孔与GFP质粒，并在出生后第5天收获。大脑进行固定，切片冠使用vibratome（100微米节），并Oct6（红色）的免疫染色。 A和B的免疫染色部分的共聚焦图像。 <img src="/files/ftp_upload/2103/2103fig3.jpg" alt="图3" /> 图3。E15.5 Sprague - Dawley大鼠与GFP质粒电穿孔。 24小时电后，大脑收获GFP阳性，电穿孔地区进行解剖和分离，如在视频中所述。经过3天的体外，细胞固定和免疫染色BIII -微管蛋白（红色）和染色用DAPI（蓝色）（A，C）的原子核。最长突起长度测量AxioVision软件（B，D）。

<ol>
	<li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, 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+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, 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+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J Vis Exp. , (2007).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70 (4-5), 155-1562 (2002).</li><li>Bai, J., Ramos, R. L., Ackman, J. B., Thomas, A. M., Lee, R. V., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+doublecortin+is+required+for+radial+migration+in+rat+neocortex.">RNAi reveals doublecortin is required for radial migration in rat neocortex.</a> Nat Neurosci. , 1277-1283 (2003).</li><li>Okada, A., Lansford, R., Weimann, J. M., Fraser, S. E., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+the+developing+nervous+system+with+retrovirus+expressing+modified+green+fluorescent+protein.">Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.</a> Exp Neurol. 156 (2), 394-406 (1999).</li><li>Bai, J., Ramos, R. L., Paramasivam, M., Siddiqi, F., Ackman, J. B., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+DCX+and+LIS1+in+migration+through+the+lateral+cortical+stream+of+developing+forebrain.">The role of DCX and LIS1 in migration through the lateral cortical stream of developing forebrain.</a> Dev Neurosci. , 144-156 (2008).</li><li>Molyneaux, B. J., Arlotta, P., Menezes, J. R., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat Rev Neurosci. , 427-437 (2007).</li><li>Young-Pearse, T. L., Bai, J., Chang, R., Zheng, J. B., Loturco, J. J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Function+for+-Amyloid+Precursor+Protein+in+Neuronal+Migration+Revealed+by+In+Utero+RNA+Interference.">A Critical Function for -Amyloid Precursor Protein in Neuronal Migration Revealed by In Utero RNA Interference.</a> J Neurosci. 27, 14459-14469 (2007).</li><li>Young-Pearse, T. L., Chen, A. C., Chang, R., Marquez, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+APP+regulates+the+function+of+full-length+APP+in+neurite+outgrowth+through+interaction+with+integrin+beta1.">Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1.</a> Neural Develop. 3, 15-15 (2008).</li></ol>

允许初级神经元文化的体外研究的突起生长的定量分析。为了研究遗传变异如何影响神经过程的产物，通过化学转染或病毒转导的主要神经元的shRNA或错误表达结构可以引入。然而，初级皮层细胞的细胞类型（从不同层次，抑制性神经元，神经胶质细胞的谷氨酸能神经元）的异构池是转使用这些方法。引进胚胎鼠皮质DNA的结构，允许使用在子宫内电靶细胞的某些子集，而早期胚胎皮?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Cortical Neuron Preparation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dissection Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10X Hanks' Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14185-052</td>
<td>&nbsp;</td>
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<td>10X Hanks' Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 )</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14065-056</td>
<td>&nbsp;</td>
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<td>1M HEPES pH 7.4</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630-080</td>
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<td>Dishes and Vials:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>100 x 15 mm Petri Dishes</td>
<td>
</td>
<td>Fisherbrand</td>
<td>08-757-12</td>
<td>&nbsp;</td>
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<td>60 x 15 mm Petri Dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>351007</td>
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<td>15 mL conical vial</td>
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<td>Sarstedt</td>
<td>62-547-205</td>
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<td>50 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-554-205</td>
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<td>Dissection Tools:</td>
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<td>&nbsp;</td>
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<td>Scissors </td>
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<td>Fine Science Tools</td>
<td>91402-12</td>
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<td>Standard Forceps</td>
<td>
</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
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<td>Curved Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11273-20</td>
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<td>Fine Forceps</td>
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<td>Fine Science Tools</td>
<td>11255-20</td>
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<td>Vannas spring scissors </td>
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<td>Fine Science Tools</td>
<td>15000-00</td>
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<td>Miscellaneous:</td>
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<td>.25% Trypsin-EDTA</td>
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<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Reichert Bright-Line Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td>&nbsp;</td>
<tr>
<td>Hand-Held Tally Counter</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Z169021</td>
<td>&nbsp;</td>
<tr>
<td>Plating Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (D-MEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Growth Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEUROBASAL Medium </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX -I Supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin Reagent Solution </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>Immunostaining:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fixative, Washes, and Blocking Buffer:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>017-000-121 </td>
<td>&nbsp;</td>
<tr>
<td>Antibodies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>beta-III tubulin antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB1637</td>
<td>&nbsp;</td>
<tr>
<td>MAP2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>AB15452</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy3 anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>715-166-151</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy2 anti-chicken</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>703-226-155 </td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>D3571</td>
<td>&nbsp;</td>
<tr>
<td>Slide Preparation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC2 Coated Two-Chamber Slides </td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>154852</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>24 x 60 mm Micro Cover Glasses</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48393-106 </td>
<td>&nbsp;</td>
<tr>
<td>Clear nail polish</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72180</td>
<td>&nbsp;</td>
<tr>
<td>Electroporation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>995-2949</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>4015809TV</td>
<td>&nbsp;</td>
<tr>
<td>buprenorphine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>1118217</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III</td>
<td>&nbsp;</td>
<td>Parker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTX square wave electroporator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BTXECM830</td>
<td>&nbsp;</td>
<tr>
<td>Tweezertrodes, 7 mm, platinum</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450488</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

在初级神经元文化的体外研究允许突起生长的定量分析。为了研究遗传变异如何影响神经过程的产物，或shRNA的基因结构，可以通过化学转染或病毒转导的主要神经元介绍。然而，初级皮层细胞的细胞类型（从不同层次，抑制性神经元，神经胶质细胞的谷氨酸能神经元）的异构池是转使用这些方法。引进胚胎鼠皮质DNA的结构，允许使用在子宫内电靶细胞的某些子集，而早期胚胎皮层目标的皮质深层电，在后期的胚胎时间点电的目标更多的表面层。此外，差分跨越个别胚胎在背内侧与腹外侧皮层区域的定位结果头上的电极位置。电，转染的细胞可以被解剖，分离，镀在突起生长的体外定量分析。在这里，我们提供了一步一步的方法，定量测量皮层细胞亚群的神经过程的产物。 电在子宫内的基本协议已经Kriegstein 实验室1，2两个其他的Jove文章中详细介绍了。我们将提供我们的协议概述在子宫内电，专注于最重要的细节，说明我们的协议，适用于研究基因功能的神经过程的产物，在子宫内电。

Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内电转染神经祖细胞在体内的一个有价值的方法。根据电极和电发育时间点的位置，才能有的放矢。皮层细胞的某些子集靶细胞，然后可以在体内或体外基因改变的影响进行分析。

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

in-utero-electroporation-followed-primary-neuronal-culture-for

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

17.6K 次观看.  Brigham and Woman's Hospital and Harvard Medical School. 在子宫内电转染神经祖细胞在体内的一个有价值的方法。根据电极和电发育时间点的位置，才能有的放矢。皮层细胞的某些子集靶细胞，然后可以在体内或体外基因改变的影响进行分析。

视频: In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

科研

神经科学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...