Name: skeletal phenotype analysis of a conditional stat3 deletion mouse model
Uploaded: 2020-07-03T14:30:00
Description: Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

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?...

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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。

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Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

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从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

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 ...

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

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

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

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

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

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 ...

A Mouse Model of Intestinal Partial Obstruction

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

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