这项工作是由德国研究基金会（德意志研究联合会）资助。

1。制备DNA和玻璃注射微量移液器<ol><li>质量好的玻璃的微量是必不可少的，降低了初始的高流产率，由于羊水流失。已经有据可查的程序拉玻璃微13,18,25。使用直径1.2mm的毛细管拉在常规萨特设定P = 500;热= 300 P-97的移动设备，上拉= 40，速度= 50;时间= 50。适合拉马用3毫米的"药谷"丝（萨特仪器FT330B）。 2毫米大小的细丝已经取得了对我们不太满意的结果。另一方面，坡口的微量提示似乎并没有改善我们的结果。 </li><li>溶解纯化的无内毒素的质粒DNA在PBS中（细胞培养级），含0.1％固绿，以使最终浓度为1〜2微克/微升。快速的绿色会让可见胚胎脑室注射的溶液。 </li><li>装入到玻璃微10μl的DNA溶液。 </lI&gt; <li>玻璃微喷射系统（碧泵）或口吸管连接。 </li></ol> 2。麻醉准备一个加热垫和手术器械的手术表。打开的冷光源，方便的可视化的胚胎。例如使用玻璃珠灭菌消毒外科手术工具。 三种不同的麻醉程序，此协议（见讨论）。在这里，我们将介绍一个我们认为是最好的，使用异氟醚吸入麻醉诱导（流量为0.5升/分钟）以及维护（流量1升/分钟）。 <ol><li>介绍孕鼠在一个透明的小（使鼠标仍然可见）的容器，连接管长度短的科米萨拉夫马克5麻醉设备。 </li><li>填写异氟醚蒸发器，然后打开氧气瓶连接到设备。吹塑一个或两个脉冲isoflurane /按住鼠标和手表昏迷设置英寸到容器中的氧气（0.5升/分钟） </li><li>拿鼠标，它的眼睛用膏干燥，以防止他们在它的头立刻适应流量麻醉面罩。 </li></ol> 3。剖腹探查<ol><li>将鼠标肚皮朝上加热垫（否则体温会下降速度非常快，复苏的机会减少），其四条腿的两侧用胶带固定到位并确保其身体。评估麻醉深度通过检查反射性刺激的反应（ 如脚趾或尾巴捏用力压）损失。 </li><li>剃腹部表面消毒的iodophorpovidone碘（Braunoderm）。 </li><li>腹部皮肤上作一纵切口（1〜1.5厘米长）。然后切开腹膜。将纱布切口周围。一个子宫角可见拉出，到PBS漂洗的gauz的钝钳Ë。用PBS冲洗子宫，很多时候，维持常湿（ 图2A和2B）。在整个过程中，避免的系膜及或子宫拉紧，由于在子宫内的高压力将发送到注射后增加流体损失的机会导致流产胚胎。 </li></ol> 4。 DNA注射到脑室<ol><li>保持子宫在这样一种方式，可以可视化脑室。不要广泛胚胎的位置，是一个不利的位置 - 这样只会增加流产的机会。 </li><li>在胚胎的头部从顶部看，视觉上的间隙或裂缝之间的左，右皮质半球本地化。半球很容易区分，颜色稍深的形状通常可以被视为侧脑室内（不是针对）。如果胚胎被发现是完全面向DNA注射（FIGUR上课2C和2D），可以刺穿子宫壁，并进入第三脑室一次。保持在45°到子宫壁穿刺喙端皮质半球之间的差距，玻璃微穿透约1毫米。在这种方式中的玻璃微吸管的前端进入大脑第三脑室（侧脑室）（ 图2C和2D）。在胚胎逊色导向的，它是有用的第一个捅破子宫壁（总是在45°）在胚胎头附近，将皮质半球之间在正确的位置，然后才枪头刺穿大脑。约1μlDNA溶液注入到第三脑室（一个很好的注射填充绿色液体的脑室）。重复同样的过程，有一个角子宫的胚胎。这使得一段时间的DNA溶液混合均匀，脑室液，达到entirê神经上皮。 </li></ol> 5。针对下丘脑进行电穿孔<ol><li>电穿孔的切换和调整设置，根据胚龄（E12.5我们使用5方波脉冲，50 V，"50毫秒ON/950毫秒关）。使用正极不锈钢针电极（CUY550-10），并作为负极圆扁电极（CUY700P4L）。 （重要的是，电极是小于胚胎，因为否则电流流动的，但不是通过它的胚胎。） </li><li>那些胚胎背侧（ 即转向实验者），因为他们大多是选择电，并丢弃那些方向是不是有利的。现在是安全的乙醇消毒手指或戴手套触摸到子宫。然而，用钳子持有子宫会干扰在电穿孔过程中电流的流动。 </li><li>皮尔斯胚胎的头部子宫壁之间的安姆的iotic囊和胎盘通过它向下推压，与针状电极的尖端。用食指，另一方面作为推力块。现在必须是约5毫米的电极尖端之间的羊膜囊和子宫壁，在大约水平的中脑（ 图2E和2F）。请记住，这是"针对电极"，所以它的位置将决定哪些是最有可能转下丘脑神经上皮的一部分。胚胎有非常温和地电极之间的"挤压"。 </li><li>用另一只手的位置以外的子宫壁胚胎的头（ 图2E与2G）的相对侧上的圆形平板电极。 </li><li>使用脚踏开关施加电压（50 V，50毫秒，950毫秒关，5个方波脉冲）。 </li><li>慢慢拔出针头电极的子宫，而子宫用食指另一方面 - 如果羊膜抽动液体丢失通过刺破壁，通常胚胎发生流产。重复上述步骤，所有的胚胎。 </li><li>返回到腹部和子宫角，残角重复注射和电。 </li></ol> 6。完成手术<ol><li>所有胚胎注射和电后，将子宫放回腹部非常小心，定位，正是因为他们之前和潮湿关闭前用生理盐水腹腔。 </li><li>外科肠线（5-0薇乔的polyglactin 910 V4914），爱惜康缝合腹膜。对于封闭的皮肤使用"打断十字绣"methodwith更耐缝合（6-0 Supramid尼龙，，Serag维斯内尔07171L）。 </li><li>消毒腹部表面用碘伏（Braunoderm）中和注入的非类固醇的抗发炎的皮下（ 例如 Rimadyl 1:10的溶液中加入100μl0.9％NaCl溶液中），或者甚至更好，阿片样物质像蒲式耳prenorphine（Temgesic，0.05〜0.1毫克/公斤体重0.9％氯化钠），以减轻疼痛。 </li><li>母亲从麻醉机移除，并将其放置在一个笼子里这是通过加热垫加热。不断监视鼠标，直到它完全从麻醉中恢复。后来，每天检查的动物，以确保他们正在恢复的过程没有感染或痛苦的任何迹象。 </li></ol> 7。分析结果<ol><li>根据所需的分析当天收获胚胎（或postnatals），。出生后的小鼠（1〜2日龄）断头处死。 </li><li>根据以前的电穿孔注释分隔每个胚胎。大脑解剖立体显微镜下检查，在显微镜下绿色荧光信号（来自GFP记者）在适当的地区。 </li><li> "新鲜"的，可以分析所选择的脑组织固定在4％多聚甲醛和很短的时间在4％琼​​脂糖或明胶一个嵌入lbumin，则第一个vibratome的类型的移动设备上（ 图3）。如需更详细的免疫组化分析（ 图4），低温保护脑嵌入华侨城安装介质（组织TEK）在30％的蔗糖溶液，然后切成20微米的部分在低温恒温器。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammillary+bodies:+two+memory+systems+in+one.">The mammillary bodies: two memory systems in one.</a> Nat. Rev. Neurosci. 5, 35-44 (2004).</li><li>Gratsch, T. E., De Boer, L. S., O'Shea, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+inhibition+of+BMP-4+gene+expression+in+postimplantation+mouse+embryos.">RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.</a> Genesis. 37, 12-17 (2003).</li><li>Wu, N., Yu, A. B., Zhu, H. B., Lin, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., 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=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, H., 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=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. L., 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=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

作者宣称，他们有没有竞争经济利益。

关于麻醉:由于在子宫内的电穿孔进入下丘脑可以在技术上是艰巨而需要较长的昏迷时间，我们优选通过氧和异氟醚的混合物给药麻醉诱导和维持。根据我们的经验，动物可以保持适当的麻醉，在这样的时间长达一个小时，至少，母亲的恢复是非常快的，提高胚胎存活率。其他麻醉方法也可提供。最简单的程序由腹腔注射诱导和维持麻醉状态2.5 0.9毫升/千克体重的氯胺酮（25毫克/毫升），赛?...

遗传操纵胚胎小鼠大脑是首选的方法，了解发育调控。然而，突变小鼠线的产生是缓慢和昂贵。一个强大的方法来引入特定的基因哺乳动物的大脑发育中神经元的变化是在子宫内的电。本质上，该技术包括DNA转染到胚胎脑的神经上皮通过电脉冲，然后使胚胎存活了一段时间，收集大脑，并分析它的可能的新颖，翔实的表型。以这种方式，实验者可以几乎立即测试假设没有小鼠突变体的生产所需的等待时间长。 到胚胎发育开始的DNA转染在OVO电鸡胚1。基本概念证明型的鼠标进行文化<s高达2。这很快就被随后该技术在子宫内 3,4鼠标的第一个描述。 主要的问题是转染而不杀死它们，或者母亲的大脑在子宫内的胚胎发展。学习进行必要的手术（开腹手术，注射，电穿孔），需要一个漫长的训练期。一旦手术已经掌握的胚胎存活率比是可以接受的地步，接下来的关键问题是:大脑结构进行访问？这并不奇怪，首次发表的论文，得到的结果与在子宫内的电主要集中在皮质发育5-9。这是使用这种技术刊物的大部分仍然是正确的，因为该区域是在显影最访问外科手术的小鼠大脑皮质（ 图1）。已经描述了该程序为在子宫内的电穿孔到皮质在打印10 11-14视频。可以使用的技术的修改目标端 ​​脑腹侧部，在基底神经节15。 除了端脑，间脑（传统分为丘脑和下丘脑）是前脑更难以达到的区域。少数的论文报告，针对其背鳍和最容易的部分，丘脑16-19。 下丘脑是最腹侧的前脑的一部分，因此，一个本地化的最深刻的背侧表面（皮层）（ 图1）。 在子宫内基因操作的小鼠大脑的研究人员致力于该地区仍然是一个艰巨的挑战。据我们所知，只有极少数文章报道的在宫内转染到小鼠下丘脑20,21的结果。然而，canno下丘脑功能的重要性被夸大了，因为它规定的行为，如吃喝，交配，繁殖和养育子女的22。此外，改变下丘脑发展有助于以后的生活条件，如肥胖，高血压，糖尿病，性早熟的23起源。基因能够改变下丘脑在开发过程中会提供一个非常强大的工具来了解它。 基本手术开腹孕鼠，我们这里使用的是类似的协议，用于其他协议11,13,14,24。我们将在这里简要地描述他们的完整性。我们的程序的关键，在另一方面，是不同的麻醉，注射的地方，不同的电极的正极相对于胚胎的头部插入和位置。我们优选通过气体吸入超过简单的腹腔麻醉诱导和维持麻醉，因为前者允许有些较长的麻醉需要一个艰难的手术。吸入异氟醚麻醉恢复快，因为通常是母亲表现出正常的行为已经分钟后手术。最简单的点注入的DNA溶液的玻璃微侧脑室内，但它是完全不适合丘脑电穿孔。直接在第三脑​​室注射液确实是针对深间脑结构的关键。这是可能的转染与标准的，现成的电极从E12.0或E12.5下丘脑。我们已经发现了一些特别适合来此的目的NEPA基因（日本千叶）制造的电极。 随着我们的过程中，我们得到整个下丘脑神经上皮或部分区域转取决于电极方向转。在这里，我们展示了乳头体转染技术，可以说是最深和最凹进的所有丘脑核团。此外，我们详细的组织学分析转染细胞的细胞层次的解析。 在子宫内的电转鼠标在子宫内发育中的大脑与其他方法的比较，可以发现在讨论部分。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

基因改造发展哺乳动物大脑的特定区域，是一个非常强大的实验方法。但是，生成新的小鼠突变体往往是令人沮丧的缓慢。它已被证明， 在子宫内的合理的操作室后的生存期的小鼠脑开发的访问是可能的。不过，此过程的结果与已报道几乎完全发育中的大脑最肤浅和容易获得的部分， 即皮层。丘脑，更窄，更内侧的区域，已被证明更加困难的目标。转染到更深的原子核，特别是下丘脑，可能是最具挑战性的，因此很少的结果已被报道。在这里，我们展示了一个过程为目标的整个下丘脑神经上皮或它的一部分（下丘脑地区）通过电穿孔转染。我们的方法的关键是昏迷时间较长，注射在T希尔德心室，以及适当的种类和电极的定位。此外，我们将展示最凹陷的下丘脑核，乳头体定位和随后的病理分析结果。

大多数丘脑神经元之间E11.5到E15.2出生，根据在大鼠有点短的鼠标发展27,28翻译成26的出生约会分析。下丘脑神经发生的高峰期达到在E12.5 29-31。因此，在本研究转染选择的年龄（E12.5），相当大的比例的下丘脑神经元可以在任何给定的尾鳍rostro的水平标记。 分析E18.5厚vibratome的类型的段（ 图3A和3B）表明，下丘脑神经上皮的整个...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

尽管下丘脑的功能和医学重要性，<em&gt;在子宫内</em&gt;遗传操纵其发展已经很少有人尝试。我们显示的详细步骤<em&gt;在子宫内</em&gt;电到老鼠的下丘脑和展示具有代表性的结果整体和局部（区域）下丘脑转。

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.8K 次观看.  University of Heidelberg. 尽管下丘脑的功能和医学重要性，在子宫内遗传操纵其发展已经很少有人尝试。我们显示的详细步骤在子宫内电到老鼠的下丘脑和展示具有代表性的结果整体和局部（区域）下丘脑转。

视频: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...