这项研究得到了西班牙科学和创新部的 RTI2018-097617-J-I00 和哈恩大学的 Acción 9 资助 O.H.O. 的资助 PGC2018-096486-B-I00 来自西班牙科学和创新部的资助 -096486-B-I00 和欧盟地平线 2020 计划向 MT 提供的 H2020-MSCA-ITN-2016-722427 资助 MT JMG 得到了西班牙科学部和塞韦罗奥乔亚基金会 （PRE2022-101884） 的博士奖学金的支持。CNIC 和 CBMSO 都得到了西班牙科学部的支持，CNIC 得到了 ProCNIC 基金会的支持。

所有动物程序均已获得 CNIC 动物实验伦理委员会的批准，并符合现行法规，包括欧盟指令 2010/63EU 和建议 2007/526/EC，由西班牙法律根据 Real Decreto 53/2013 执行。对于该方案，使用了 15-21 周龄的雌性野生型 CD-1 小鼠。有关所用动物、试剂和设备的详细信息，请参阅 材料表。
1. 质粒和工具制备
<ol><li>首先，通过在冰中加入终浓度为 1 μg/μL 的所需质粒 DNA 来制备电穿孔混合物。然后，通过添加终浓度为 6% 的蔗糖（溶于 PBS）和 0.01% 的生物相容性染料来完成混合物，并加入 PBS 直至最终体积为 30 μL。 注:每个样品大约使用 1-2 μL 的电穿孔混合物。根据实验中的样品数量缩放电穿孔混合物的比例。请记住，用于电穿孔的 DNA 应从细菌培养物中纯化，以确保最佳稳定性和最少的污染物。有关本研究所用质粒的更多详细信息，请参阅代表性结果部分。</li>
	<li>通过在 Dulbecco 改良 Eagle 培养基 （DMEM） 中补充 10% 胎牛血清 （FBS）、2% Hepes 缓冲液和 1% 青霉素/链霉素来制备生长培养基。将培养基保存在 37 °C 的培养箱中直至使用。此外，在37°C下加热1x PBS和生长培养基。</li>
	<li>在以下条件下，使用移液器拉拔器拉伸直径为 20 μm 的玻璃毛细管来准备针头:热量 = 550;拉 = 100;速度 = 250;时间 = 120。拉动后的最终直径应约为 5-8 μm 之间。</li>
	<li>将电穿孔仪参数设置为 40 V 下的 5 个 50 ms 周期，间隔为 900 ms。准备电极、口移液管和钝针（图 3A）。为移液管配备针头以加载 DNA（图 3A）。</li>
</ol>2. 胚胎解剖和心脏提取
<ol><li>在野生型 CD-1 小鼠之间建立杂交。第二天早上，选择出现阴道栓的雌性，并将此时间记为胚胎日 E0.5。在胚胎日 E12-12.5 通过颈椎脱位对动物实施安乐死（遵循机构批准的方案）。</li>
	<li>小心地从老鼠身上取出子宫，并将其放入干纸巾中。然后，用细剪刀剪断子宫，将剪刀的尖端插入中侧，然后慢慢地将刀片的边缘穿过整个长度。暴露蜕膜并将它们转移到生长培养基中。有关更多详细信息，请参阅 Behringer 等人的工作20。</li>
	<li>解剖蜕膜以提取胚胎，然后将胚胎放入装有温热生长培养基的 60 毫米培养皿中。使用细镊子，小心地切开胚胎的尾巴和头部，只剩下身体的躯干。胚胎的阶段可以通过评估解剖学特征来验证，如 Wong 等人 21 所描述。</li>
	<li>然后，小心地从胚胎的躯干中取出尽可能多的组织，而不会损坏心脏或肺动脉和主动脉。一旦心脏暴露，立即将其置于新的温热生长培养基中，并将其保存在 37 °C 的培养箱中直至电穿孔。 注意:保持心脏的温度对其生存至关重要。确保培养基始终温暖，并每 5-10 分钟更换一次。另一方面，可以在不保持无菌的情况下处理样品，因为这不会影响程序的效率。请注意，生长培养基含有抗生素。</li>
</ol>3. 心脏电穿孔和 离体 培养
<ol><li>用口腔移液器小心地吸出，将大约 10 μL 质粒混合物（在步骤 1.1 中制备）装入针中。</li>
	<li>将单个心脏置于干净的 60 mm 培养皿中，用加热至 37 °C 的无菌 1x PBS 制备用于电穿孔。 将培养皿置于解剖显微镜下（图 3A）。</li>
	<li>通过将正极和负极之间的距离调整到大约 4 mm 来准备电极（图 3B）。 注意:4 mm 是 E12.5 鼠标心脏与本文中使用的电极相配的孔径距离。请考虑根据不同的样品量或电极规格更改此距离。</li>
	<li>用口腔移液管中的针头轻轻穿刺心脏的最浅层。小心吸取 1-2 μL 质粒混合物并取出针头。可以进行多次注射以确保最佳渗透，以及扩大电穿孔细胞的数量。</li>
	<li>将电极固定到位，使心脏位于两极之间（图 3C）。然后，使用上述条件对心脏进行电穿孔。 注:为确保最大的电穿孔效率，可以切换电极的极性并发出另一个脉冲。</li>
	<li>将电穿孔的心脏转移到含有 1 mL 生长培养基的新 12 孔板中，并将其置于 37 °C、5% CO2 下。</li>
	<li>重复步骤 3.1 到 3.6，直到实验中的所有样品都电穿孔。最后，将所有心脏在 37 °C、5% CO2 的培养箱中孵育 24 小时或直到分析。 注意:在我们手中，心脏可以在培养物中放置长达 48 小时，而没有明显的恶化迹象（补充电影 1;补充电影 2;补充图 1）。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66754/66754fig03.jpg"> 图 3:电穿孔设置。 （A） 用于电穿孔的设置。红色箭头表示电穿孔仪、电极和培养皿，其中心脏被电穿孔。（一'）电穿孔针的近距离细节，如 （A） 中白色矩形突出显示的。（B） E12.5 心脏中使用的电极调整距离的详细信息。（C） 电穿孔的示意图。 <a href="https://www.jove.com/files/ftp_upload/66754/66754fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
4. 细胞分选和免疫组化分析
<ol><li>对于样品的细胞分选分析，通过将 400 μL liberase （5 mg/ml）、300 μL 分散酶 （0.1 g/mL）、4 μL CaCl2（0.4 μM）、50 μL MgCl2 （5 μM） 混合在一起，制备消化培养基以解离组织，并完成直至 10 mL DMEM。放在冰上直到使用。每个心脏大约需要 1 mL 的消化培养基。根据样品数量调整体积。 注:对于免疫组织化学，直接在 1.5 mL 的 4% PFA 中固定心脏在 RT 下 2 小时或在 4 °C 下过夜。 第二天，用 PBS 洗涤心脏 3 次，然后进行下游组织学和免疫染色程序。有关详细的免疫组织化学方案，请参阅 Morris 等人 22。</li>
	<li>将培养物中的每个心脏转移到含有 1 mL 冰冷消化培养基的新试管中。</li>
	<li>为了促进解离，用注射器轻轻涂抹 pleasure 3-4 次，将组织切碎，并立即在 37 °C、600 rpm 的热板中孵育 45 分钟。</li>
	<li>通过 70 μm 细胞过滤器将消化混合物过滤到新管中，然后通过重力流将其排空。然后，挽救通过体积，并使用 40 μm 细胞过滤器将其再次过滤到新的 50 mL 试管中。</li>
	<li>加入 500 μL 胎牛血清 （FBS），并使用冷 DMEM 完成至 30 mL。</li>
	<li>在室温下以 240 x g 离心混合物 10 分钟。</li>
	<li>弃去上清液，将 50 mL 试管倒置在纸巾上完全干燥。 注:这是通过冷冻细胞沉淀来暂停实验的便捷点。如果是这样，用 FBS 中的 10% DMSO 重悬细胞并储存在 -80 °C。</li>
	<li>对于细胞分选，将细胞重悬于 300 μL 分选缓冲液（1% FBS、1% 青霉素/链霉素的 PBS 溶液）中，并添加 0.3 μL DAPI （1000x）。执行单元格分选。</li>
</ol>

小鼠心脏中基于电穿孔的心肌细胞转基因

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A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ex+vivo+culture+method+for+the+embryonic+mouse+heart.">A novel ex vivo culture method for the embryonic mouse heart.</a> J Vis Exp. (75), e50359 (2013).</li></ol>

作者没有需要披露的利益冲突。

总体而言，此处描述的方法为在发育中的心外膜中表达转基因构建体提供了一个强大的框架（图 4B），如 Meis1 过表达所示（图 4C）。通过适当的构建体，该方案可用于瞬时评估特定基因的功能获得 （GOF） 或功能丧失 （LOF） 的影响。LOF可以通过 RNA 干扰转染靶向候选基因的质粒来实施到该技术中 26。基因 GOF 或 LOF 的快速评估特别有?...

心脏是胚胎发育过程中形成的第一个器官。这个过程涉及来自胚胎不同区域的各种祖细胞群的时空协调。所有这一切都是在发育中的心脏继续跳动和功能的同时发生的，强调了其形成所需的非凡协调性 1,2,3。鉴于心脏的关键作用，细胞和分子水平的严格调节对于心脏的正常形成至关重要 4,5。确定控制心脏发育的机制一直非常有趣，因为它们对于解开先天性心脏病至关重要，先天性心脏病影响着全世界的大量患者6。此外，理解心脏发育对于破译心脏再生至关重要，因为出生后哺乳动物的心脏保留了在成年后丢失或受阻的再生能力 7,8。因此，剖析心脏发育的分子调节因子对于推进先天性心脏病和心脏再生的研究工作至关重要。
为了实现这一目标，人们越来越关注研究心外膜在心脏发育和再生中的作用9。心外膜是一层薄薄的间皮组织，构成哺乳动物心脏的最外层（图 1）。最近的研究表明心外膜在心脏损伤期间的重要性，表明该组织能够向受影响区域的心肌细胞发送增殖信号以减轻损伤10,11。尽管心外膜很重要，但进行进一步的分子研究一直受到其巨大异质性的挑战。单细胞 RNAseq 实验揭示了心外膜的异质性，包含具有不同转录组特征的多个细胞亚群12、13、14、15、16。因此，筛选心脏发育潜在调节因子的策略应适应心外膜祖细胞的多样性。
从这个意义上说，小鼠模型对基因改造的适应性促进了对心脏发育至关重要的许多基因的鉴定，从而可以产生具有特定基因功能获得 （GOF） 或功能丧失 （LOF） 的突变系。然而，这些方法意味着大量的时间和实验资源的投入;因此，在评估大量候选基因的作用时，它们是不切实际的。此外，发育基因通常在不同的组织中发挥多效性功能，或者是早期胚胎发育所必需的，从而阻碍了对它们在特定过程中对发育的贡献的解释。虽然可以在特定结构或发育时间点靶向基因功能，但这通常需要使用更复杂的遗传结构，这可能难以生成或通常不可用。
为了克服这些限制，我们提出了一种对小鼠胚胎心脏进行电穿孔以进行瞬时转基因的方法（图 2）。与离体培养和荧光激活细胞分选 （FACS） 配合使用，该策略通过 Meis1 的瞬时 GOF 展示其能力，Meis1 是一种与心脏发育和再生有关的特征明确的基因 17,18,19。在本文中，还探讨了该方法的其他潜在应用，并讨论了其优点和局限性，以及与瞬时调节基因表达的现有方案的比较。我们相信所提出的框架和视觉示例将增强对发育和疾病过程中心外膜生物学的理解。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66754/66754fig01v2.jpg"> 图 1:小鼠胚胎心脏层。 E13-14 小鼠胚胎心脏的冠状图示意图。心脏的三个主要细胞层以黄色（心内膜）、红色（心肌）和蓝色（心外膜）表示。心包用棕色线表示。心脏的四个腔室缩写为 LV， 左心室; RV， 右心室; LA， 左心房; RA， 右心房。 <a href="https://www.jove.com/files/ftp_upload/66754/66754fig01largev2.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66754/66754fig02.jpg"> 图 2:心脏电穿孔方案的示意图概述。<a href="https://www.jove.com/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#55 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Clear Flat Bottom Multiwell Cell Culture Plate</td>
			<td>BD Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm vise table&#160;</td>
			<td>Grandado</td>
			<td>SKU 8798771617573</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Fischer Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP Policlonal Antibody</td>
			<td>Invitrogen</td>
			<td>A10262</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Anti-Myosin 4 (MF20) Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>14-6503-82</td>
			<td>1:500 dilution used</td>
		</tr>
		<tr>
			<td>CD1 Wild Type mice</td>
			<td></td>
			<td></td>
			<td>Provided by Animalary Unit (CNIC)</td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Cell Signalling Technologies</td>
			<td>9661</td>
			<td>1:400 dilution used</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signalling Technologies</td>
			<td>4083</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Dispase/collagenase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>10438-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51032720</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Stereoscopic Microscope S8AP0</td>
			<td>Leica</td>
			<td>11524102</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller Model P-97</td>
			<td>Sutter Instrument</td>
			<td>SU-P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG expression plasmid</td>
			<td>Addgene</td>
			<td>#89689</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 &#215; 10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 60 &#215; 15 mm</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Merck</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td></td>
			<td></td>
			<td>Reused from old laboratory equipment</td>
		</tr>
		<tr>
			<td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td>
			<td>Janvier Labs</td>
			<td>9979</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant anti-Wilms Tumor Protein 1 (WT1) Antibody</td>
			<td>Abcam</td>
			<td>ab89901</td>
			<td>1:300 dilution used</td>
		</tr>
		<tr>
			<td>Square Wave Electroporator CUY21SC</td>
			<td>Nepa Gene</td>
			<td>CUY664-10X15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td></td>
			<td></td>
			<td>Provided and autoclaved by technical unit</td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Millipore</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer electrodes with variable gap</td>
			<td>Nepa Gene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient transgenesis approach for gene delivery in the mouse embryonic heart

哺乳动物心脏是一个复杂的器官，在发育过程中 通过 高度多样化的祖细胞群形成。这些祖先的起源、招募时间和命运对于该器官的正常发育至关重要。控制心脏形态发生的分子机制对于理解先天性心脏病和胚胎心脏再生的发病机制至关重要。研究这些机制的经典方法采用转基因小鼠的产生来评估心脏发育过程中特定基因的功能。然而，小鼠转基因是一个复杂、耗时的过程，通常无法评估特定基因在心脏发育过程中的作用。为了解决这个问题，我们开发了一种小鼠胚胎心脏高效电穿孔和培养的方案，使瞬时转基因能够快速评估参与心脏发育的基因功能获得或丧失的影响。使用这种方法，我们成功地在胚胎心脏中过表达 Meis1，并优先进行心外膜细胞转染，证明了该技术的能力。

为了证明该技术在对相关心脏发育调节因子进行功能获得 （GOF） 实验中的有效性，对过表达 Meis1 转录因子的构建体进行了电穿孔。为此，从 E9.5 胚胎中提取 RNA，并进行逆转录以获得互补 DNA （cDNA）。使用 cDNA 作为模板，将 Meis1 编码序列（补充表 1）克隆到 pCAG 表达质粒（以下简称 pCAG::Meis1）中，同时使用组成型 GFP 表达质粒 （pCAG::GFP） 作为对照。然后仅使用 pCAG::GFP ?...

作者聚焦:用于快速基因筛选的心肌细胞转基因

该协议为发育中的小鼠心脏中基于电穿孔的心肌细胞转基因提供了详细的方法框架。此处提供的视频资源将有助于学习这种通用技术。

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

an-efficient-transgenesis-approach-for-gene-delivery-mouse-embryonic

Research

JoVE Journal

Developmental Biology

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

527 次观看.  Severo Ochoa Center for Molecular Biology (CBMSO). 我们的目标是建立一种新的方法，促进某个基因的过表达或下调，以了解心脏发育。小鼠转基因是一项巨大的时间和精力投资。因此，一个一个地评估基因是不现实的，也因为基因组的高度复杂性。因此，需要一种快速的筛查方法。我们向任何研究人员提供了一种工具，用于分析候选基因在心脏发育过程中的功能获得或丧失。我们方案的主要优...

视频: 作者聚焦:用于快速基因筛选的心肌细胞转基因

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

在小鸡视网膜有效的基因转移的原代细胞培养研究:<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...