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，因为骨组织可以被分割。专注于股骨和头骨的研究人员应该将股骨和头骨置于嵌入步骤的 90° 角度。为了进行可靠和高质量的组学分析，我们选择了软骨层对称的下垂石蜡部分，并在H&E染色中显示了清晰的M形线，代表了这些连续部分的适当角度和深度。另一方面，如果您需要观察膝盖关节和相邻的关节韧带，股骨和头骨应放置在120°角。
数十年来，TRAP一直作为骨质疏松功能14的生化标志物，因为它主要表现在骨质疏松15、16。两个基板通常用于评估磷酸活性。纳普特霍尔-阿斯比磷酸盐（N-ASBI-P）是骨质极特异性TRAP等形5b的优良基板。然而，亚硝基磷酸盐（pNPP）也可以由非类型5 TRAP17水解。因此，用于TRAP组织化学演示的石脑油-ASBI磷酸-半生化方法在组织部分17、18、19中具有很强的特异性。巨噬细胞磷酸盐（酸磷酸盐）的pH值最佳值为5.0-6.0，其中大多数是耐酸酸盐20。因此，我们建议，在TRAP染色检测协议中，用血氧林重新染色的组织部分不应用盐酸去色。重要的是，虽然靠近骨骼改造区域的骨细胞和骨细胞也表达TRAP21，但只有具有三个以上核的巨大TRAP阳性细胞被认为是骨细胞。在这项研究中，Stat3Ctsk小鼠表现出抑制的骨质疏松活动（图4）。
钙素是一种荧光（绿色荧光），广泛用于指示钙化过程中的骨骼生长。它可以结合钙，并纳入新形成的碳酸钙晶体22。同样，钙素，阿利扎林红（红荧光）和四环素（黄色荧光）被内置到骨骼中，以估计从暴露时23，24的生长。过去的许多研究都使用单荧光素（通常是钙素）来标记骨骼，但观察者很容易混淆新形成的骨骼和旧骨骼，尤其是在不规则的血管骨中。因此，我们建议研究人员在小鼠体内注射两种不同类型的荧光素，以区分较新的骨骼和较旧的骨骼。根据我们的测试，钙素与阿利扎林可能达到最充分和持久的对比。在这项研究中，钙化素-阿利扎林红色标签用于检测骨组织的骨质成活率，结果发现，在Stat3Ctsk小鼠（图5）中，骨细胞活动没有受到影响。
简言之，为了了解控制骨质疏松活动的关键基因，本协议描述了生成转基因小鼠模型的规范方法。此协议还描述了一些分析骨骼表型的典型技术。在这项研究中，删除STAT3受损的骨质形成和增加的骨量。对于那些对骨骼组织研究较不感兴趣的人来说，这些技术可能很有趣。然而，骨架系统的更多特征有需要进一步研究，如机械特性25。我们总是需要关注新技术的发展。

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>&lt;em&gt;Ctsk-Cre&lt;/em&gt; 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>&amp;mu;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>&lt;em&gt;Stat3&lt;sup&gt;fl/fl&lt;/sup&gt;&lt;/em&gt; 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小鼠的骨质增加。
通过H&E染色（图3）检查了WT和Stat3Ctsk小鼠股骨的组织形态。
TRAP染色检测出小鼠的骨质原活性。骨质疏松是TRAP+（葡萄酒红色或紫色）细胞与多个核和巨大的大小（图4A）。与WT小鼠（图4B）相比，Stat3Ctsk小鼠的TRAP+骨细胞数量较低，这表明STAT3缺乏会损害骨质疏松形成。
小鼠的骨质形成以矿物吸收率（MAR）表示，由钙化物和阿利扎林红色双标（图5）测量。卡尔塞因和阿利扎林红被连续注射在腹中。因此，钙素（绿色）和阿利扎林红（红）荧光线之间的区域代表新形成的骨骼在四天内。如图5所示，骨细胞中删除的STAT3不影响骨骼代谢。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61390/61390fig01.jpg"> 图1:说明骨质疏松特异性Stat3删除小鼠模型生成和转基因小鼠。（A） 通过 Cre-loxP 系统在导管 K （Ctsk） 表达骨质体中删除Stat3的示意图图。（B） 骨质疏松特异性Stat3删除小鼠的繁殖进展。统计3fl/fl小鼠被交叉到克茨克-克里小鼠（F0）产生异质统计3fl/+:克茨克克里小鼠 （F1） 。男性统计3fl/+;Ctsk-Cre小鼠被保存并交叉到雌性Stat3 fl/fl小鼠，以产生同源统计3fl/fl:克茨克克里小鼠 （F2） 。男性统计3fl/fl:Ctsk-Cre小鼠被保存并交叉到雌性Stat3 fl/fl小鼠以生成统计3fl/fl:克茨克克里小鼠和统计3 fl/fl小鼠。（C） 小鼠各种基因型的基因型。统计3突变体:187 bp，Stat3野生类型:146 bp，Cre:200bp.（D）在骨细胞培养的STAT3的西斑，由Stat3fl/fl和Stat3Ctsk小鼠的骨髓巨噬细胞培养。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61390/61390fig01large.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/61390/61390fig02.jpg"> 图2:利用微CT扫描对小鼠股骨进行三维重建和定量微结构参数分析。（A） 从8周大的WT和 Stat3Ctsk 小鼠的股骨的三维重建微CT图像。感兴趣的区域 （ROI） 位于靠近解剖生长板的血管骨的共 1 mm 宽度中。（B） 3D重建了8周大的WT和 Stat3Ctsk 小鼠的股骨皮质骨骼的微CT图像。投资回报率是总共1毫米宽的部分皮质骨从股骨中间。（C–H）微CT的定量微结构参数:骨矿物质密度（BMD）、骨量分数（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）。错误栏表示平均± SD，n=4，*P<0.05。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61390/61390fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61390/61390fig03.jpg"> 图3:通过H&E染色展示的WT和 Stat3Ctsk 小鼠的股骨的组织形态。M 形虚线曲线表示软骨层。皮质骨的相对大功率场用虚线装箱，在下面展示。相对大功率的血管骨场用虚线圈起来，并在底部展示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61390/61390fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61390/61390fig04.jpg"> 图4:通过TRAP染色表示的小鼠股骨骨质疏松。（A） 从 8 周大的 Wt 和 Stat3Ctsk 小鼠身上染上股骨的陷阱污渍。TRAP+ 多核骨细胞由黑色三角形表示。下面展示了用虚线圈成的相对大功率骨质体场，展示了多个原子核和巨大的大小。（B） 计算TRAP和 多核骨细胞的数量。错误栏表示平均± SD，n=5，*P<0.05。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61390/61390fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61390/61390fig05.jpg"> 图5:通过钙素和阿利扎林红色双重标签表示的小鼠股骨骨质疏松。（A） 七周大的老鼠在第0天依次注射钙素，第4天注射阿利扎林红，然后在第7天牺牲。钙素（绿色）和阿利扎林红（红色）荧光线之间的区域代表新形成的皮质骨在四天内。（B） 以两条线的距离计算的矿物附着率 （MAR） 和骨形成率 （BFR/BS） 表示皮质骨的骨质活性。（C） 钙素（绿色）和阿利扎林红（红）荧光线之间的区域在四天内代表新形成的血管骨。（D） MAR 和 BFR/BS 代表血管骨的骨质活性。错误栏表示平均± SD，n=5，*P<0.05。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61390/61390fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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

Universidad San Sebastian

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

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

科研

发育生物学

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

行为学

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

医学

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

神经科学

分析牙槽骨重塑以探索骨骼力学生物学

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

作者聚焦：比较牙槽和长骨重塑以探索 OTM 模型的潜力 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

有条件统计3 删除鼠标模型的骨骼表型分析

摘要

探索更多视频

此视频中的章节

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

通过子代检测改变斑马鱼致死性骨骼突变显

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

一个简单的临界尺寸股骨缺损模型小鼠

一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

在体内病态的人类基因组建模使用斑马鱼

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

有条件 统计3 删除鼠标模型的骨骼表型分析

摘要

探索更多视频

此视频中的章节

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

通过子代检测改变斑马鱼致死性骨骼突变显

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

一个简单的临界尺寸股骨缺损模型小鼠

一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

在体内病态的人类基因组建模使用斑马鱼

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

有条件统计3 删除鼠标模型的骨骼表型分析