作者要感谢国家实验动物中心的Minna Törrönen，Riikka Venäläinen，Heikki Karhunen和Inkeri Niemi在动物工作方面的帮助。这项研究得到了芬兰科学院、ERC和CardioReGenix EU Horizon资助的支持。

所有实验均得到东芬兰大学动物实验委员会的批准。该协议描述了一种临床相关的猪跑步机运动测试，以评估心脏病新疗法的安全性和有效性。本研究使用体重25-80公斤的雌性家猪。这些动物是从商业来源获得的（见 材料表）。
1. 设置跑道
<ol><li>设置跑道，使动物只能向一个方向移动。使用门和舱口防止动物后退。跑道的平面图如图 1 所示，跑道示例如图 2 所示。</li>
	<li>确保跑步机（见 材料表）有足够的空间来改变倾斜度。</li>
	<li>确保跑步机的宽度可调，以防止动物在跑步过程中转动。</li>
	<li>使用透明塑料制作跑步机的前壁。这可以防止动物从跑步机上跑开，但仍然允许动物透过墙壁看到。 注意:动物必须能够透过前墙看到，因为我们的经验表明，如果猪看到墙的另一侧的猪同伴，它们就会更有动力奔跑。</li>
	<li>将心电监护仪和除颤器（见 材料表）放在跑步机旁边。 注意:在压力测试期间可能发生致命的心律失常，特别是如果猪有心肌缺血14,15,16。</li>
	<li>确保跑道包括一个饮水点，动物可以在跑步后喝水和降温。</li>
</ol>2.猪在试验前的适应期
<ol><li>在开始实验之前，将动物安置2周。</li>
	<li>在适应环境的第一周，确保动物习惯他们的处理者和新的住房环境，不包括跑道。</li>
	<li>在适应期的第二周，确保动物习惯跑道。</li>
	<li>开始习惯，让动物熟悉跑道。首先，保持所有大门打开，以便动物可以在赛道上自由行走并探索环境。</li>
	<li>当动物对赛道比较熟悉时，打开跑步机，让动物一次跑一小段时间，比如7分钟。运行时间的长度必须每天延长。 注意:记得在适应期间奖励动物。例如，在本研究中，猪获得了无盐爆米花的奖励。</li>
</ol>3.运动试验
注意:猪应在运动测试前至少 2 小时禁食或在跑步前只喂一小部分食物。
<ol><li>打开跑步机并将坡度设置为5%-10%。</li>
	<li>一旦动物在跑步机上，以 2 公里/小时的启动速度启动跑步机。</li>
	<li>每 60 秒将速度提高 0.5 公里/小时，直到达到 5 公里/小时。总运行时间为15分钟。</li>
	<li>如果动物无法以最大速度全程奔跑，请执行以下步骤。<ol><li>如果猪没有跑到选定的速度，从后面轻轻地推它，因为这可能会给动物一种感觉，它需要跑得更快而不会减速。</li>
		<li>尝试轻轻推动动物最多三次;之后，一次将速度减慢 0.5 公里/小时，直到猪能够处理速度。不要减速到2公里/小时以下。</li>
		<li>如果动物即使以低速也拒绝跑步，请关闭跑步机并停止测试。</li>
	</ol></li>
</ol>4. 运动测试期间的心电图监测
<ol><li>将心电图电极（见 材料表）放置在跑步过程中运动最小的解剖位置，例如肩胛骨或胸部。 注意:使用专为运动测试设计的心电图电极，以获得更好的皮肤粘附。请记住从放置ECG电极的区域剃掉头发。</li>
	<li>记录跑步过程中的心率变化。 注意:我们的经验表明，ST段分析通常由于移动和其他伪影而变得复杂。节律监测也可以使用植入式循环记录器或起搏器来完成。</li>
</ol>5. 数据收集
<ol><li>记录每次更改速度时的运行距离、总时间和速度。 注意:现代跑步机可能会收集许多其他数据，因此必须熟悉跑步机手册以充分发挥设备的潜力。</li>
	<li>注意动物行为的可能变化，例如跛行。 注意:如果需要，请联系兽医并确保动物接受必要的镇痛。将动物从未来的锻炼中移开，直到它完全恢复。</li>
</ol>6. 术后护理
<ol><li>确保动物可以进入供水点。</li>
	<li>例如，用零食或玩具奖励动物。</li>
	<li>跑步后监测动物30分钟是否有可能的不良反应。</li>
</ol>

猪运动耐力评价

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+anatomic+and+physiologic+overview+of+the+porcine+heart.">A comparative anatomic and physiologic overview of the porcine heart.</a> Journal of the American Association for Laboratory Animal Science. 53 (5), 432-438 (2014).</li><li>Korpela, 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=AAV2-VEGF-B+gene+therapy+failed+to+induce+angiogenesis+in+ischemic+porcine+myocardium+due+to+inflammatory+responses.">AAV2-VEGF-B gene therapy failed to induce angiogenesis in ischemic porcine myocardium due to inflammatory responses.</a> Gene Therapy. 29 (10-11), 643-652 (2022).</li><li>Swindle, M. 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作者声明不存在利益冲突。

这种大型动物运动试验模仿了临床中使用的试验，缩小了临床前研究和临床试验之间终点的差距。它可用于评估严重心血管疾病的新疗法的疗效，例如闭塞性动脉硬化，心力衰竭和缺血性心脏病。本协议中应用的时间点可能因测试的治疗而异。该方案已基于与大型动物合作的长期经验进行标准化，可用于评估心血管基因治疗和其他新型治疗方法的安全性和有效性。
猪的心脏?...

慢性心血管疾病是全世界死亡和发病的重要原因1,2。尽管目前的治疗方法对大多数患者有效，但由于弥漫性慢性疾病或合并症等原因，许多人仍然无法从目前的治疗中受益。此外，在一些患者中，可用的治疗并不能缓解心脏症状，尽管进行了最佳药物治疗，他们的心血管疾病仍会进展3。因此，显然需要为严重的心血管疾病开发新的治疗方案。
在过去的几年中，已经发现了新的分子途径和操纵这些靶点的方法，使基因疗法，细胞疗法和其他新疗法成为治疗严重心血管疾病的现实选择4。然而，在有希望的临床前结果之后，许多心血管应用未能满足临床试验的期望。尽管临床试验疗效不佳，但一些试验已经建立了新疗法的良好安全性5,6,7,8,9。因此，为患者带来新的心血管疗法将需要改进的方法和更好的临床前模型、研究环境和可以预测临床疗效的临床前研究的终点。
在动物模型中，重点通常放在易于量化的终点上，例如从组织学切片计算的毛细血管的数量和面积，或从静息和药理应激下的左心室成像参数。在临床试验中，许多终点更加主观，例如运动耐量或症状缓解4。因此，临床前研究和临床试验中的终点很可能测量应用疗法的不同方面。例如，血管数量的增加并不总是与更好的灌注、心脏功能或运动耐量相关。然而，开发成功的治疗方法需要两种类型的终点10。尽管如此，主要目标始终是缓解症状并改善患者的预后和生活质量。为了实现这一目标，终点测量必须在临床前和临床研究之间更好地匹配4。
心肺健康反映了循环和呼吸系统在持续体力活动期间提供氧气的能力，因此它量化了个体的功能能力。功能能力是关键的预后标志，因为它是心血管和全因死亡风险的强有力的独立预测指标11。心肺健康的改善与死亡风险降低有关12。运动测试适用于评估心血管疾病的有氧表现和治疗反应。根据可用性，在自行车测力计或跑步机上进行测试。通常使用每分钟工作量的逐渐增加，避免突然增加;这导致线性生理反应。运动测试中最重要的变量包括总运动时间、达到的代谢当量 （MET）、心率以及 QRS 波群（Q、R 和 S 波）和 T 波（ST 段）之间心电图 （ECG） 线的变化。临床压力测试成本低，易于获得13.由于这些原因，压力测试，如6分钟步行测试，已被广泛用于临床，也应该用于新疗法的临床前评估。
据我们所知，没有描述良好的大型动物模型来评估基因疗法或其他新疗法的功能疗效。因此，临床相关的运动测试为评估这些新疗法在临床前环境中的效率提供了一个极好的视角。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Defibrillator</td>
			<td>Zoll M series</td>
			<td>TO9K116790</td>
			<td>All portable defribrillators will work</td>
		</tr>
		<tr>
			<td>Defibrillator pads</td>
			<td>Philips</td>
			<td>M3713A</td>
			<td>All pads work, as long as the pads are compatible with the defibrillator</td>
		</tr>
		<tr>
			<td>ECG electrodes</td>
			<td>Several providers</td>
			<td></td>
			<td>Prefer ECG electrodes designed for exercise tests</td>
		</tr>
		<tr>
			<td>Loop recorder</td>
			<td>Abbott Oy</td>
			<td>DM3500</td>
			<td>Optional for rhythm monitoring</td>
		</tr>
		<tr>
			<td>Patient monitor</td>
			<td>Schiller Argus LCM Plus</td>
			<td>7,80,05,935</td>
			<td>All portable ecg monitors will work</td>
		</tr>
		<tr>
			<td>Pigs</td>
			<td>Emolandia Oy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>NordicTrack</td>
			<td></td>
			<td>All treadmills with adjustable incline and speed are suitable for the exercise test.&#160; The treadmill should be as long and wide as possible.</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips EPIQ 7 ultrasound</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Various building materials</td>
			<td>Several providers</td>
			<td></td>
			<td>For building fences, ramps and gates according to the Figure 1 and Figure 2</td>
		</tr>
		<tr>
			<td>Various treats for the animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

exercise test for evaluation of the functional efficacy of the pig cardiovascular system

尽管在治疗方面取得了进展，但心血管疾病仍然是全世界死亡和发病的最大原因之一。基于基因治疗的治疗性血管生成是一种有前途的方法，用于治疗有明显症状的患者，尽管有最佳的药物治疗和侵入性操作。然而，许多有前途的心血管基因治疗技术未能在临床试验中达到预期。一种解释是用于衡量疗效的临床前终点和临床终点不匹配。在动物模型中，重点通常放在易于量化的终点上，例如从组织学切片计算的毛细血管的数量和面积。除了死亡率和发病率外，临床试验的终点是主观的，例如运动耐量和生活质量。然而，临床前和临床终点可能测量应用疗法的不同方面。然而，开发成功的治疗方法需要这两种类型的终点。在诊所，主要目标始终是缓解患者的症状并改善他们的预后和生活质量。为了从临床前研究中获得更好的预测数据，终点测量必须与临床研究中的测量更好地匹配。在这里，我们介绍了临床相关的猪跑步机运动测试方案。本研究旨在:（1）提供可靠的猪运动测试，可用于评估基因疗法和其他新疗法的安全性和功能有效性，以及（2）更好地匹配临床前和临床研究之间的终点。

必须具有与大型动物合作的经验才能成功使用此协议。研究人员需要能够评估动物是否由于疲劳或缺乏动力而停止奔跑。记录速度和距离可能有助于评估这一点，像往常一样，缺乏动力的动物完全停止奔跑，而疲劳的动物在放慢速度后继续奔跑（图3）。如有必要，如果结果似乎不可靠，可以在第二天重复该方案。
腺相关病毒（AAV）处理动物的代表性时?...

Watch this Scientific Journal Video about Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System at JoVE.com

作者聚焦:用于评估猪心血管系统功能功效的运动试验  

本协议描述了一种大型动物运动试验模型，用于评估心血管系统在临床前环境中评估新疗法效率的功能能力。它与临床运动测试相当。

猪心血管系统功能功效评价的运动试验

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based ...

该研究的目的是为猪提供可靠的运动测试，可用于评估基因疗法和其他新疗法的安全性和功能有效性。通常，心血管动物研究强调易于量化的终点，就像从组织学切片计算毛细血管面积一样。相比之下，临床研究使用更主观的终点，如过度耐受。该方案更好地匹配了临床前和临床研究之间的终点。先前发表的具有较长时间跑步会话的方法旨在评估慢性运动适应，而所提出的方法更适合评估心肺健康。

exercise-test-for-evaluation-functional-efficacy-pig-cardiovascular

Research

JoVE Journal

Genetics

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

1.2K 次观看.  University of Eastern Finland. 该研究的目的是为猪提供可靠的运动测试，可用于评估基因疗法和其他新疗法的安全性和功能有效性。通常，心血管动物研究强调易于量化的终点，就像从组织学切片计算毛细血管面积一样。相比之下，临床研究使用更主观的终点，如过度耐受。该方案更好地匹配了临床前和临床研究之间的终点。先前发表的具有较长时间跑步会话的方法旨在评估慢性运动适应，而所提?...

视频: 作者聚焦:用于评估猪心血管系统功能功效的运动试验  

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

科研

遗传学

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

使用旋转运动量化系统测量果蝇中的运动水平 (满足要求)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

TreadWheel:果蝇轻度诱导运动的间歇训练协议

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model organism. However, little is known about the sex-determination mechanisms found in other non-model hymenopteran taxa, such as ants. Because of the complex nature of the life cycles that have evolved in hymenopteran species, it is difficult to maintain and conduct experimental crosses between these organisms in the laboratory. Here, we describe the methods for conducting inbreeding crosses and for evaluating the success of those crosses in ant Vollenhovia emeryi. Inducing inbreeding in the laboratory using V. emeryi, is relatively simple because of the unique biology of the species. Specifically, this species produces androgenetic males, and female reproductives exhibit wing polymorphism, which simplifies identification of the phenotypes in genetic crosses. In addition, evaluating the success of inbreeding is straightforward as males can be produced continuously by inbreeding crosses, while normal males only appear during a well-defined reproductive season in the field. Our protocol allow for using V. emeryi as a model to investigate the genetic and molecular basis of the sex determination system in ant species.

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

In this protocol, methods for conducting inbreeding crosses, and for evaluating the success of those crosses, are described for the ant Vollenhovia emeryi. These protocols are important for experiments aimed at understanding the genetic basis of sex determination systems in Hymenoptera.

利用蚂蚁Vollenhovia Emeryi对近亲繁殖杂交的诱导和评价

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...