Name: viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk
Uploaded: 2022-07-27T14:10:00
Description: Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

这项工作得到了加拿大卫生研究院（CIHR）项目资助（PJT-148918和PJT-180533，WL），CIHR早期职业研究者奖（AR8-162705，WL），加拿大心脏和中风基金会（HSFC）麦当劳奖学金和新研究者奖（S-17-LI-0866，WL），学生奖学金（JW和YX）和渥太华大学心脏研究所心脏捐赠基金的博士后奖学金（AL）的支持。作者感谢理查德·西摩先生的技术支持。 图 2 是使用 Biorender.com 批准的许可证创建的。

所描述的所有方法和程序都得到了渥太华大学动物研究伦理审查委员会和渥太华大学心脏研究所生物安全审查委员会的批准。制定的安全规程包括，处理重组腺病毒或腺相关病毒（AAV）的所有程序均在II级生物安全柜中进行。实验结束后，所有与病毒接触的物品都进行了彻底净化。Ctnnb1 flox / flox和αMHC-MerCreMer小鼠（8-12周龄，任何性别）和Sprague-Dawley大鼠（200-250g，雄性）用于本研究?...

<ol>
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有几个步骤对于朗根多夫灌注的分离心脏准备的成功至关重要。首先，重要的是要避免在心脏采集过程中对心脏造成任何损害（例如，由于意外挤压或剪刀切割）。其次，尽快将收集的心脏放入冷的Tyrode溶液中至关重要，因为这会停止心跳并减少心脏的耗氧量。第三，针插入主动脉不能太深——理想情况下，针尖靠近主动脉瓣水平，以便心脏通过冠状动脉充分灌注。最后，灌注针中的气泡需要在?...

心血管疾病是全球主要死因，仅在2017年就夺走了1800万人的生命1。啮齿动物，尤其是小鼠和大鼠，由于易于处理和各种转基因过表达或敲除系的可用性，已成为心血管研究中最常用的模型。啮齿动物模型对于理解疾病机制和确定心肌梗塞2，高血压3，心力衰竭4和动脉粥样硬化5的潜在新治疗靶点至关重要。然而，与人类或大型动物模型相比，啮齿动物在心律失常研究中的使用受到其小心脏尺寸和更快心率的限制。因此，心肌梗死后小鼠或大鼠的自发致死性心律失常很少见2。研究人员被迫关注可能反映促心律失常底物的间接继发性变化，例如纤维化或基因表达，而没有显示出心律失常负担或促心律失常倾向的有意义的变化。为了克服这一限制，本协议描述了一种允许可靠评估小鼠和大鼠心脏在基因修饰6，7 或心肌梗死2 后对室性快速性心律失常的易感性的方法。该方法将肾上腺素能受体刺激与程序性电刺激相结合，以在孤立的，Langendorff灌注的8 个小鼠和大鼠心脏中诱导室性快速性心律失常。
啮齿动物心肌组织中病毒基因转移的标准方法通常涉及通过开胸术9，10，11暴露心脏，这是一种侵入性手术，与手术后动物恢复延迟有关。本文介绍了一种在超声成像引导下直接心肌注射病毒以治疗转基因过表达的方法。与开胸术相比，这种侵入性较小的手术允许动物在病毒注射后更快地恢复和更少的组织损伤，减少动物的术后疼痛和炎症，从而可以更好地评估转基因基因对心脏功能的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G 1/2 PrecisionGlide Needle</td>
			<td>Becton Dickinson (BD)</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>adeno-associated virus (AAV9-GFP)</td>
			<td>Vector Biolabs</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-GFP)</td>
			<td>Vector Biolabs</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-Wnt3a)</td>
			<td>Vector Biolabs</td>
			<td>ADV-276318</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet (Level II)</td>
			<td>Microzone Corporation</td>
			<td>N/A</td>
			<td>Model #: BK-2-4</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Vetergesic</td>
			<td>DIN 02342510</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>102378</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL Clipper Corporation</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Sigma-Aldrich</td>
			<td>20701</td>
			<td>705 LT, volume 50 &#956;L</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Life Brand</td>
			<td>E12107</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresenius Kabi</td>
			<td>DIN 02264315</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi Ltd.</td>
			<td>M60303</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1351005</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart 8 software</td>
			<td>ADInstruments Inc.</td>
			<td>Version 8.1.5</td>
			<td>for ECG recording</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (Ctnnb1flox/flox)</td>
			<td>Jackson Labs</td>
			<td>4152</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (&#945;MHC-MerCreMer)</td>
			<td>Jackson Labs</td>
			<td>5650</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>MS400 transducer</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Systane</td>
			<td>DIN 02444062</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure meter</td>
			<td>NETECH</td>
			<td>DigiMano 1000</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Cole-Parmer</td>
			<td>UZ-77924-65</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Rat (Sprague-Dawley, male)</td>
			<td>Charles River</td>
			<td>400</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10007-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Down Inc.</td>
			<td>Sylgard 184</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Small animal ECG system</td>
			<td>ADInstruments Inc.</td>
			<td>N/A</td>
			<td>Powerlab 8/35 and Animal Bio Amp</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>567530</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>IonOptix</td>
			<td>MyoPacer EP</td>
			<td></td>
		</tr>
		<tr>
			<td>VEVO3100 Preclinical Imaging System</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk

心脏病是全世界发病率和死亡率的主要原因。由于易于处理和丰富的转基因品系，啮齿动物已成为心血管研究的重要模型。然而，经常导致心脏病患者死亡的自发致死性心律失常在心脏病啮齿动物模型中很少见。这主要是由于人类和啮齿动物之间心脏电特性的物种差异，并对使用啮齿动物研究心律失常提出了挑战。该协议描述了一种使用超声心动图引导的重组病毒（腺病毒和腺相关病毒）肌内注射在小鼠和大鼠心室心肌中实现有效转基因表达的方法。这项工作还概述了一种方法，该方法能够使用孤立的，Langendorff灌注的小鼠和大鼠心脏以及肾上腺素能和程序性电刺激来可靠地评估心律失常的心脏易感性。这些技术对于研究与损伤后不良心脏重塑（如心肌梗塞）相关的心律失常至关重要。

当按照此处描述的方案（图1）灌注时，分离的大鼠或小鼠心脏有节奏且稳定地跳动至少4小时。如果实验设计需要较长时间的心脏灌注，则在灌注液中加入白蛋白有助于减少长时间灌注后心肌水肿的发生14。在灌注溶液中加入异丙肾上腺素模仿交感神经系统的激活，这发生在许多致心律失常疾病，如心肌梗塞和心力衰竭中。在程序化的电刺激期间，通过（1?...

Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

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

本协议描述了在超声心动图引导下通过直接心肌内注射病毒在大鼠和小鼠心脏中表达转基因的方法。本文还解释了通过对孤立的朗根多夫灌注的心脏进行程序性电刺激来评估心脏对室性心律失常易感性的方法。

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 ...

该协议允许用户在心室心肌中实现有效的转基因表达，促进基因表达研究以检查心律失常和心室组织中的功能。病毒转基因表达技术对动物来说是微创的，与涉及开胸术的传统方法相比，造成的损害更小，恢复时间更短。诱导小啮齿动物心脏心律失常的技术有助于评估心脏病小动物模型中心律失常的易感性。演示该程序的将是来自实验室的博士后研究员Alice Lu博士。首先，用基于碘或基于氯己定的磨砂膏交替进行消毒，在酒精中以圆周运动三次。在超声成像引导下，将含有病毒的注射器的针头插入动物的胸部。将针尖接近左心室前游离壁，缓慢注射 10 至 15 微升病毒。通过针尖附近的增强亮度验证超声图像中的成功注射。从心脏中取出针头并插入左心室的其他区域，进行第二次和第三次注射相同数量的病毒。要对小鼠心脏进行朗根多夫灌注，请将空心放入涂有硅胶弹性体的 10 厘米塑料培养皿中，左心室朝上。在恒定流速模式下，用连接到改良的朗根多夫灌注系统的钝针将心脏的主动脉插管。在 37 摄氏度下用氧气气泡的 Tyrode 溶液灌注心脏。在灌注开始时，通过观察前两到三次心跳期间血液从心脏排出并将心脏颜色从红色变为苍白来验证正确的主动脉插管。调整流速，将灌注压力保持在70至80毫米汞柱。确认后，将小动物心电图系统的电极插入培养皿中的有机硅弹性体涂层中，将其放置在心脏周围。然后，使用兼容软件记录心电图。接下来，进行肾上腺素能受体刺激，并用异丙肾上腺素和Tyrode溶液灌注心脏。10分钟后，通过用连接到电刺激器的两个铂电极刺激心尖处的心脏，进行程序性电刺激以诱导室性快速性心律失常。从最初的 10 个连续刺激 S1 开始刺激程序，然后是额外的刺激 S2，初始间隔为 80 毫秒。然后，每次重复将 S2 间隔减少两毫秒，直到无法再捕获心跳或达到心脏的有效不应期或 ERP。通过心电图监测任何诱发的室性快速性心律失常，包括室性心动过速和颤动。如果没有诱发心律失常。在 S2 之后添加另一个额外的刺激 S3，使用相同的参数，直到达到 ERP。如果室性快速性心律失常仍未诱发，应停止电刺激并考虑心脏非诱发。在程序化的电刺激期间，通过连续 S1 刺激期间的心跳一对一捕获和 S1 起搏期间延长的 QRS 波群来验证心脏的成功起搏。这些肾上腺素能和电刺激的结合在健康的假手术野生型小鼠心脏或对照腺病毒注射GFP的大鼠心脏中没有引起室性快速性心律失常。相比之下，相同的方案在心肌梗死后77%的野生型小鼠心脏中诱导室性快速性心律失常，在心肌内注射Ad-Wnt3a后，在四分之三的大鼠中诱导室性快速性心律失常。插入针头时，确保针尖嵌入游离壁内，而不是心室内。此外，固定和插管心脏主动脉是最关键的一步。遵循这些程序后，心脏可用于标准的组织学研究或分子生物学测定。或者，可以分离活细胞以进行单细胞电生理学研究。

Research

JoVE Journal

Biology

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

科研

生物学

Optical Mapping of Langendorff-perfused Rat Hearts

光学测绘的Langendorff灌注大鼠心脏

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...