Name: an efficient transgenesis approach for gene delivery in the mouse embryonic heart
Uploaded: 2024-05-24T14:25:00.000+00:00
Duration: 5 min 6 s
Description: The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

<table><tbody><tr><td>#55 Forceps</td><td>Dumont&amp;nbsp;</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&amp;nbsp;</td><td>Grandado</td><td>SKU 8798771617573</td><td></td></tr><tr><td>40 &amp;micro;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 &amp;micro;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&#039;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 &amp;times; 10 mm</td><td>BD Falcon</td><td>351008</td><td></td></tr><tr><td>Petri dishes 60 &amp;times; 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&amp;nbsp;</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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

570 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

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

科研

遗传学

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

生物工程

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 ...

神经科学

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

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 ...

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 ...

发育生物学

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 ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

生物学

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

激光辅助慢病毒基因传递到小鼠受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

啮齿动物心脏中的病毒转基因表达和心律失常风险的评估

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

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