我们感谢祖卫国教授和加藤教授为试剂和小鼠以及Zu实验室的成员进行了有益的讨论。我们还感谢上海第九人民医院颅面异常数字化口腔学研究中心的帮助。这项工作部分得到了中国国家自然科学基金委员会（NSFC）的资助 [81570950，81870740，81800949]， 上海峰会与高原学科，上海精密医学研究所、上海市第九人民医院、上海交通大学医学院（JC201809）的SHIPM-mu基金，上海交通大学医学院高层次创新团队激励项目，上海第九人民医院跨学科研究基金，上海交通大学医学院[JYJC201902]。L.J.是上海"医学人才之星"青年发展计划和上海交通大学"陈星"项目的学者。

与动物有关的所有方法均得到上海交通大学医学院动物保护与使用委员会（IACUC）的批准。
1. 孕育骨质疏松特 异性Stat3 删除小鼠
注： 统计3fl/fl 小鼠是商业获得的。 克茨克-克里 小鼠由加藤（东京大学，东京，日本12）提供。这些小鼠是在标准化条件下在机构动物设施中特定的无病原体条件下繁殖和饲养的。
<ol><li>将性成熟的雄性小鼠与两只同龄雌性小鼠配对。18天后，检查新生儿的每日检查。分离怀孕的雌性小鼠，并在需要时单独保存。如果雌性小鼠在配对后1个月内未怀孕，则在不同的育种笼中更换雄性小鼠。</li>
	<li>将统计 3fl/fl鼠标交叉到克茨克-克里小鼠 （F0）。剪辑的尾巴为基因，并保持男性Stat3fl/+：Ctsk-Cre小鼠，直到他们性成熟，这是大约6周的年龄（F1）。使用以下引物：统计3 F-TT加特格特克特克塔卡：统计3 R-塔加塔格卡卡卡：克茨克 - 克雷 F - 加克特加特加卡;克茨克 - 克雷 R - 克塔卡克格特克特克特克特克。</li>
	<li>交叉 6 周大的男性统计 3fl/+：克茨克克里小鼠与雌性统计3fl/fl小鼠。夹住基因化的尾巴，并保持男性Stat3 fl/fl：Ctsk-Cre小鼠，直到他们性成熟，这是大约6周的年龄（F2）。</li>
	<li>交叉 6 周大的男性统计 3 fl/fl：克茨克克里小鼠与雌性Stat3fl/fl小鼠（F3）。剪断尾部进行基因化处理，并及时用幼鼠（F3+N）替换老繁殖小鼠。</li>
</ol>2. 标本收集
<ol><li>用二氧化碳窒息分别对六对8周大的雄性 Stat3fl/fl 和 Stat3Ctsk 垃圾小鼠进行安乐死。 注：CO2 流速取代了每分钟笼体积的 30%（例如，对于 45 厘米 x 30 厘米 x 30 厘米的笼子，CO2 流速为 40 L/min）。</li>
	<li>将鼠标置于超平位置。用手轻轻拆解双边臀部关节。使用眼科剪刀垂直切断皮肤从解剖头骨，然后解剖整个皮肤从后肢。</li>
	<li>用剪刀切断右臀部关节和膝盖关节的关节韧带，以分离后肢。切断木屑和纤维的结，然后将后肢浸入4%的副甲醛中。将右后肢保留为第 3 步。适当切开两端的骨骼，使骨髓完全浸入并修复4%的副甲醛。</li>
	<li>用剪刀切开左臀部关节和膝关节的关节韧带，轻轻取出软组织，小心地分离头骨和股骨。将头骨和股骨分别浸入75%乙醇中。将股骨保留为第 4 步，将蒂比留到第 6 步。确保保持木马完好无损。</li>
</ol>3. 石蜡部分准备
<ol><li>修复右后肢在4%副甲醛在4°C为48小时。</li>
	<li>贴花：用1倍PBS轻轻清洗标本10分钟3次。用超声波贴花器将标本在 15% EDTA（800 毫升的 ddH2O 中的 150 g EDTA 和 100 mL 的 10 倍 PBS 中除名 3 到 4 周，直到骨骼弯曲。每隔一天更换一次新鲜的贴花液。</li>
	<li>用 1 倍 PBS 轻轻清洗标本 3 倍，然后在 4 °C 过夜时将其浸入 75% 乙醇中。</li>
	<li>脱水：第二天，连续浸入95%乙醇、100%乙醇和二甲苯的标本中，每份浸入1小时两次。</li>
	<li>将标本浸入 1/2 xylene 1/2 石蜡中 30 分钟。将标本浸入石蜡中，在 65 °C 过夜。</li>
	<li>嵌入：选择合适的嵌入罐进行嵌入。将头骨均匀地放在下面。将股骨和头骨置于 90° 角。石蜡完全冷却和凝固后，将其从嵌入罐中取出。对标本进行编号，并在 -20 °C 过夜时将其存储。</li>
	<li>使用微原子连续切割 5 μm 厚的部分。切 20–40 节。将部分铺在 37 °C 的水面上，粘附在显微镜滑梯上，并在 42 °C 下烘烤过夜。</li>
</ol>4. 微CT扫描和分析
<ol><li>使用微型 CT 扫描仪扫描左股骨。分辨率： 10μm;电压：70千伏：当前： 114 μA;飞： 0.5 毫米阿尔：旋转步骤：0.5°。</li>
	<li>按照制造商的指示，使用扫描仪的辅助软件重建皮质骨骼和血管骨的 3D 图像。投资回报率在靠近腹腔生长板的血管骨中，在股骨中间总共有1毫米宽的皮质骨。</li>
	<li>计算定量微结构参数：骨矿物质密度（BMD）、骨体积分数（BV/TV）、血管厚度（Tb.Th）、血管数（Tb.N.）、血管分离（Tb.Sp.）和皮质骨厚度（Ct.Th）。</li>
</ol>5. 陷阱染色
<ol><li>在 65 °C 下烘烤石蜡部分 30 分钟。</li>
	<li>德瓦克斯：将部分浸入二甲苯中10分钟。用新鲜的二甲苯执行此步骤 3 倍。</li>
	<li>补充水分：将各部分按顺序浸入100%乙醇、95%乙醇、70%乙醇和ddH2O中，每段浸入5分钟两次。</li>
	<li>使用 TRAP 染色套件按照制造商的说明准备染色解决方案，并加热至 37 °C。 注意：TRAP 染色解决方案应在每次检测前立即准备。</li>
	<li>在每个样品中加入 50+100 μL 染色溶液，在 37 °C 潮湿的室内孵育 20–30 分钟。每5分钟在光显微镜下检查骨质疏松器的染色状态，直到可以看到红色多核骨质疏松剂。用ddH2O结束反应。</li>
	<li>30年代血氧林溶液中的防污剂。通过浸入 1% 氨溶液中 1 分钟创建稳定的蓝色。在缓慢运行的自来水中冲洗。</li>
	<li>使用中性香膏安装带盖片的部分，并在一夜之间干燥。</li>
	<li>通过显微镜捕捉 3–5 个感兴趣的领域。通过图像 J 分析 trabee 周长：使用"直线"工具测量比例杆 （Ls） 的长度为 L1，然后使用"分段线"工具测量轨道周长为 L2，物理长度 （Lp）= Ls*L2 /L1。计算具有三个以上核的 TRAP 阳性细胞的数量。</li>
</ol>6. 卡尔辛和阿利扎林红色双标签
<ol><li>标本制备：第0天内注射20毫克/千克钙素（2%NaHCO3 溶液中的1毫克/毫升），第4天注射25毫克/千克阿利扎林红S（AL，H2O中的2毫克/毫升）。牺牲老鼠在第7天。小心地分离头骨，在4°C的48小时固定在4%的副甲醛中。</li>
	<li>脱水：固定后，用1倍PBS轻轻清洗头骨3倍。连续将标本分别浸入 95% 乙醇、100% 乙醇和二甲苯中 5 分钟两次。</li>
	<li>将标本浸入 Aceton 12 h 中，在 1/2 Atone 1/2 树脂中浸入 2 小时，并在干燥烤箱中浸入纯树脂中过夜。 注：树脂由市售树脂（如嵌入812树脂）根据制造商的说明制备。</li>
	<li>嵌入：将纯树脂加入合适的硅胶嵌入罐中，轻轻放置标本以避免气泡。在 60 °C 的干燥烤箱中将树脂聚合 48 小时。</li>
	<li>用旋转微原子连续将标本切成 5 μm 厚的部分。在室温下用干燥剂储存其余样品。</li>
	<li>在酒精下降 75% 时，用钳子粘附部分。使用中性香膏安装带盖片的部分。用荧光显微镜捕捉红色和绿色荧光标签。</li>
	<li>测量两条标记线（Ir.L.Wi）、单标记圆周（sL.Pm）、双标记圆周（dL.Pm）和总轨道周长（Tb.Pm）之间的宽度。计算矿物应用率 （MAR） 和骨形成率 （BFR/BS）。MAR = Ir.L.Wi/间隔天数。BFR/BS= （dL.Pm±下午2点）*MAR/Tb.pm*100%。</li>
</ol>

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作者没有什么可透露的。

基因工程小鼠模型常用于研究人类疾病的机理和药物治疗。Ctsk-Cre小鼠已广泛应用于骨质疏松6的功能研究。本研究描述了分析骨骼表型和研究控制体内骨质活性的关键基因的方法的协议。
神学分析是检测骨代谢的最佳直观方法。石蜡部分的质量是组织学分析的基础。足够的时间对骨组织进行完全固定和贴花是非常必要的，因为骨组?...

骨骼骨是人体的主要承重器官，在行走和锻炼过程中受到来自内外环境的压力。在整个生命中，骨骼不断经历自我更新，由骨细胞和骨细胞平衡。骨细胞清除旧骨骼和形成新骨的骨骼的过程，保持了骨骼系统2的平衡和机械功能。平衡中的干扰可能会诱发骨代谢疾病，如骨质疏松症。骨质疏松症是由过度的骨质疏松症活动引起的，在全球普遍存在，给社会造成重大经济损失。根据可用于骨质疏松症治疗的药物数量有限及其不良反应的风险4，必须公布骨质疏松症形成和活动的细节。
从单核细胞/巨噬细胞血细胞系中提取的骨细胞具有多个核（可能有2至50个核），并且很大（通常直径大于100μm）2。虽然通过体外骨质疏松培养，对骨质疏松症药物的探索和药物筛选得到了广泛的改进，但复杂的有机反应使得体内证据成为靶向治疗不可或缺的。由于小鼠与人类的遗传和病理生理学相似性，基因工程小鼠模型常用于研究体内人类疾病的机理和药物治疗。Cre-loxP系统是一种广泛使用的小鼠基因编辑技术，使研究人员能够以组织/细胞特异性的方式研究基因功能。导热素K（CSTK）是一种由骨细胞体分泌的半胱氨酸蛋白酶，可以降解骨胶原蛋白8。众所周知，CTSK有选择地用成熟的骨细胞表达：因此，Ctsk-Cre小鼠被认为是骨细胞功能研究的有用工具，并已被使用6。
转录（STAT）家族的信号传感器和活化剂是经典的，在免疫力和癌症进展和发展7，8具有非常重要的意义。据报道，在7个STSTAT中，STAT3与骨平衡9、10最相关。一些体内研究报告说，在骨细胞中特定的STAT3灭活会减少骨骼形成9，10。然而，关于STAT3参与骨质形成和骨代谢在体内的可靠证据仍然有限。最近，我们提供了体内证据与骨质疏松特异性Stat3删除鼠标模型（统计3fl/fl：Ctsk-Cre，以下简称Stat3Ctsk），STAT3参与骨质疏松和骨代谢11。在本研究中，我们描述了我们用来分析Stat3Ctsk小鼠骨量、骨组织形态、骨代谢和催化变化的方法和协议，以研究骨质疏松特异性STAT3缺失对骨质疏松的影响。

<table>
	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

转基因小鼠模型对于了解控制骨质疏松症分化和活动的关键基因，以及研究骨质疏松症的机制和药物治疗具有强大的作用。导热素K（Ctsk）-Cre小鼠已广泛用于骨质疏松的功能研究。转录3（STAT3）的信号传感器和活化剂与骨平衡有关，但它在体内骨质疏松中的作用仍然定义不明确。为了提供STAT3参与骨质疏松和骨代谢的体内证据，我们生成了一个骨质疏松特异性Stat3删除小鼠模型（Stat3 fl/fl：克茨克-克雷）并分析了其骨骼表型。微CT扫描和3D重建意味着有条件的淘汰小鼠的骨量增加。进行了H&E染色、钙素和阿利扎林红色双染色，以及耐酸磷酸（TRAP）染色，以检测骨骼新陈代谢。简言之，本协议描述了一些规范的方法和技术来分析骨骼表型和研究控制体内骨质活性的关键基因。

使用本协议，生成了骨质疏松特异性Stat3删除小鼠，以研究STAT3删除对骨质疏离的影响。Stat3Ctsk小鼠及其野生类型 （WT） 垃圾在基因化后繁殖和保存。骨髓巨噬细胞被分离并培养成骨细胞，并演示了Stat3Ctsk小鼠的STAT3缺失（图1）。
与WT小鼠（图2）相比，Stat3Ctsk...

Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

此协议描述了一种规范的方法，以了解控制体内骨质疏松活动的关键基因。该方法采用转基因小鼠模型和一些规范技术来分析骨骼表型。

有条件 统计3 删除鼠标模型的骨骼表型分析

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Research

JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K 次观看.  National Clinical Research center of Stomatology. 此协议描述了一种规范的方法，以了解控制体内骨质疏松活动的关键基因。该方法采用转基因小鼠模型和一些规范技术来分析骨骼表型。

视频: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

有条件 统计3 删除鼠标模型的骨骼表型分析

有条件统计3 删除鼠标模型的骨骼表型分析