Name: validation of a mouse model to disrupt linc complexes in a cell-specific manner
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作者感谢分子遗传学核心和小鼠遗传学核心的华盛顿大学医学院圣路易斯学院（系眼科及视觉科学学院）的形态和成像的核心工作人员。作者是由麦克唐纳中心细胞与分子神经生物学，希望中心神经疾病的小额赠款计划的支持下，国家眼科研究所（＃R01EY022632卫生署），国家眼科研究所中心核心格兰特（＃P30EY002687）和不受限制的赠款防盲研究眼科及视觉科学系。

伦理声明:涉及动物个体程序批准的机构动物护理和使用委员会（IACUC）在圣路易斯华盛顿大学。 1.鼠标育种和基因分型<ol><li>养殖的Tg（CAG-的LacZ / EGFP-KASH2）小鼠的Tg（PCP2-CRE）小鼠产生的Tg（PCP2Cre CAG-EGFP / KASH2）19,20。注:虽然该协议的剩余部分的重点是使用的Tg（PCP2-CRE）鼠标线的小脑内限制EGFP-KASH2表达浦肯野细胞，类似的程序可以遵循与任何其他的Tg（CRE）鼠标（ 图2A）。 </li><li>进行基因分型，以确定幼仔窝藏两个绿色荧光蛋白和酶Cre转基因如前所述19。 </li></ol> 2.准备材料，进行解剖和组织收集<ol><li>制备100ml的PBS 30％蔗糖（30克蔗糖/ 100 ml的1×PBS中），并存储在-20℃。组织小时一天arvesting，解冻和负载的蔗糖溶液〜30〜35毫升到35毫升的注射器穿心灌注。 </li><li>准备40个ml的4％PFA的PBS中（10 ml的16％多聚甲醛和30ml PBS）中。负载毫升4％〜30-35的PFA入35毫升注射器穿心灌注。 </li><li>用10ml的PBS用于组织解剖填有10厘米组织培养板。 </li><li>准备一个氯胺酮/甲苯​​噻嗪鸡尾酒组成的16毫克/毫升氯胺酮和3毫克/毫升甲苯噻嗪。 </li><li>制备70％的乙醇对解剖的溶液。 </li></ol> 3.解剖和组织收集<ol><li>使用1ml注射器，注射500微升每25克体重的氯胺酮/甲苯​​噻嗪鸡尾酒和等待〜15分钟。或者，使用另一种机构上核准镇静方法适合于感兴趣的生物系统。 </li><li>镇静后，轻轻捏后爪以确认没有疼痛反应，PIN所有爪子上的泡沫塑料托盘的背侧鼠标朝下。 </li><li>喷用70％乙醇的腹部和做一个切口从下腹部到肋笼的顶部，以获得对心脏。 </li><li>做一个小切口心脏的右心房，插入包含30％的蔗糖溶液进入心脏的左心室注射器。 </li><li>灌注溶液在〜2-5毫升每分钟的速率。当空，取下注射器重复使用4％PFA解决方案。 </li><li>从泡沫塑料托盘取消固定鼠标和斩首访问大脑。 </li><li>除去使用镊子和剪刀围绕动物的头在皮肤上。戳一个洞的头骨用针后部并小心地用剪刀和镊子解剖取出头骨。 </li><li>一旦脑显然访问，使用镊子除去小脑，并将其放置于PBS中。平分在矢状平面小脑用手术刀和转移到30％蔗糖溶液，在4℃下过夜。注意:要预习是石蜡切片，在4％PFA固定过夜小脑在4℃下之前组织包埋和切片。 </li></ol> 4，组织处理和剖分<ol><li>制备干冰和2-甲基丁烷的浆料。允许浴的温度下平衡5分钟。 </li><li>填充OCT化合物中一个cryomold并将一半两段式小脑到模具中。 </li><li>使用解剖显微镜和一对钳子的位置，使得对分面（靠近小脑的中线）朝上小脑。 </li><li>所述cryomold转移到干冰浆液5分钟直至在OCT化合物已形成周围组织标本的固体白色基质。注意:将冷冻的样品现在可以储存在-20℃下，如果需要的。 </li><li>标签预先处理的聚赖氨酸的幻灯片，收集组织切片，并准备低温恒温器根据制造商的说明，以收集所述蜡膜的15微米的部分战争权。收集的部分中，小心地接触与滑动的多聚赖氨酸涂覆侧在OCT部分。为了保持部的方向，不旋转在该步骤中的幻灯片。 </li><li>储存组织切片在-20°C，直到继续进行染色和成像。 </li></ol> 5.染色和小脑部分的成像<ol><li>准备前的组织染色以下解决方案: 约50毫升的PBS 〜5-10毫升4％PFA的PBS 〜〜20毫升的0.5％的Triton X-100，10％驴血清的PBS </li><li>解冻小脑部分。重要的是，最大限度地减少他们接触到的连续和强烈的光线任何源头上防止EGFP的衰落。 </li><li>使用疏水屏障用笔，画周围的组织切片的矩形。 </li><li>后固定，用4％PFA的章节5分钟。使用足够的液体以覆盖由疏水性屏障（通常〜500微升）划定的整个区域。使用吸尘器或吸管，再移动固定剂是特别小心，不要打扰组织切片。 </li><li>用移液管，轻轻地在幻灯片上应用的PBS，而不会干扰组织切片。 5分钟后，使用真空或吸液管小心地取出液体。重复PBS中总共三次冲洗。 </li><li>使用移液管小心应用适当稀释的初级抗体（3微升钙结合蛋白和/或抗体Nesprin2每900微升溶液）在0.5％的Triton X-100，10％驴在PBS血清。孵育一小时，在室温下在黑暗中。 注意:EGFP-KASH2将通过对冷冻切片直接荧光显微镜可见的，这是没有必要利用抗EGFP抗体。然而，如果石蜡切片已经制备，EGFP-KASH2的成像需要免疫检测用抗EGFP抗体。 </li><li>除去抗体溶液和冲洗用PBS三次。 </li><li>制备1:1000稀释的Alexa-氟二次抗体在0.5％的Triton X-100，在PBS 10％驴血清并储存在黑暗中。 </li><li>轻轻地取出最终的PBS漂洗，并应用二抗稀释于暗处一小时。 </li><li>拆下二次抗体溶液，冲洗，洗节三次。如果需要的话，应用核染色剂，如DAPI（〜300nm的），在第一PBS涂布期间。 </li><li>从周围组织和地方〜5-7微升安装媒体上的组织切片完全除去PBS溶液。放置一个玻璃盖玻片上的组织切片的顶部并预留在室温下在黑暗中〜15-30分钟。 </li><li>使用配备有适当的过滤器在荧光显微镜可视化EGFP和适合于在步骤5.8 / 5.9中使用的第二抗体的任何其它波长。在小脑切片，用20倍的目标，查看EGFP-KASH2核边缘。 </li></ol>

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作者什么都没有透露。

最关键的步骤，以成功地研究LINC配合物在体内使用的Tg（CAG-LacZ的/ EGFP-KASH2）模型是确定一个合适的Cre小鼠路线的作用。实际上，如果是的Cre活性在其他细胞类型参与类似的途径，它可以复杂化结果的解释。因此，为了检查附近小区是重要的，如在小脑（图2B）的分子和颗粒细胞层中所示此处。同样，为了最大限度地提高任何生理相关表型它以识别的Cre应变横跨感兴趣的细胞类型的广泛的Cre表达是重要的。同样重要的是关注系统的开发过程中进行了深入的刻画LINC成分复杂的表达模式。的确，Tg的（CAG-LacZ的/ EGFP-KASH2）小鼠模型中的一个缺点是，它不提供任何信息，其中太阳蛋白质或Nesprins变体都参与了生物系统到u考虑升气管。但是，这个问题可以通过采取严格的表征在转录和蛋白水平来克服。事实上，这样的研究已经进行了多次导致有关具体LINC复杂组件的作用的重要发现中枢神经系统发育期间和动态平衡21,22。 最后，虽然这种协议被集中在使用固定的中枢神经系统组织中，Tg的（CAG-LacZ的/ EGFP-KASH2）小鼠可用于分离和培养的原代细胞来研究扰乱LINC络合物在体外的作用。使用这种方案，可以分离和纯化EGFP-KASH2阳性细胞的原代培养物的应用，如时间推移的视频显微镜。这就免去了低转染效率或所需的原代培养体系的慢病毒生产，如神经元。 总之，的Tg（CAG-的LacZ / EGFP-KASH2）小鼠开辟新的和令人兴奋的可能性，研究的乐趣期间在多种组织和细胞类型的胚胎和出生后的哺乳动物发育过程LINC络合物在体内的ction以解决基本细胞生物学的问题。

核信封（NE）分隔细胞质核质。它是由内和外核膜（INM和ONM，分别）连接在核孔。由两种膜划定的管腔被称为核周腔（PNS）。所述ONM是粗面内质网（ER）的延伸，并且INM粘附到核层，核型-V的中间体长丝的小梁由A-和B-型表示核纤层蛋白1,2-。的核骨架，骨架（LINC）配合的接头是跨越整个核膜物理细胞核的内部连接到细胞骨架细丝和分子马达（图1A）的大分子装配体。它们由进化上高度保守性表征的网元积分跨膜蛋白的两个家庭之间的相互作用的:太阳（Sad1在每个/ Unc84）的蛋白质和Nesprins（核膜SPectRINS）。在哺乳动物中，SUN1和SUN2芳在INM，其N端核质区域直接与A-和B型的核纤层蛋白3-5交互电子跨膜蛋白。在INM的另一边，PNS内，孙海港蛋白质进化保守舒展〜150℃末端氨基酸称为孙域。孙域直接交互与进化保守KASH（Klarsicht / ANC-1，地久天长同源）域，Nesprins的分子信号。 KASH域由一个伸展的30〜C末端氨基酸突入PNS后跟一个跨膜结构域6的。至少有四个不同的Nesprin基因（Nesprin1-4）编码KASH-包含定位于东北7蛋白质。 Nesprins，其大小各不相同的50kDa〜（Nesprin4）的一个惊人1000 kDa的（Nesprin1巨型）胞质区，包含多个血影重复以及特定的图案使它们的相互作用与细胞骨架组分如肌动蛋白，网蛋白和分子马达8- 13。 &lt;P类="j​​ove_content"&gt;研究脊椎动物和无脊椎动物表明，核纤层蛋白/阳光/ Nesprin /分子马达构成一个进化上保守的"轴"控制核迁移和锚地。几个敲除（KO）小鼠的LINC复杂的组件的模型已经描述并均有助于提供一个框架哺乳动物发育9,14,15期间理解Sun和Nesprin蛋白质在网元的角色。然而，这些模型本几个显著缺陷，最明显的是:1）难以在解释由于细胞中区分的表型贡献非自主的效果，2）表型困难KASH含与KASH少Nesprin异构体16，3）的Sun和Nesprin蛋白在多种细胞类型的网元功能冗余需要复杂的育种方案来灭活所有SUN-KASH相互作用小鼠17只小鼠中4）围产期杀伤力不足两个的KASH结构域Nesprins1和2排除成人的分析表型18。 这个协议描述了一种新的小鼠模型旨在破坏体内所有SUN-KASH相互作用，在一个细胞中自主和发育调节的方式，从而绕过许多上面列出的缺点。此酶Cre /液氧系小鼠模型依赖于两个重要的概念:1）任何已知Nesprin蛋白的KASH域足以靶向EGFP到在细胞培养系统中的网元，2），SUN域相互作用混乱与KASH域，从而过度任何KASH结构域将饱和所有的内源性SUN结构域和灭活LINC配合的显性负方式17（ 图1B）。本协议描述的组织采伐和用于确认在小脑浦肯野细胞都SUN-KASH相互作用的中断处理步骤。

<table><tbody><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S0389</td><td></td></tr><tr><td>10x PBS</td><td>Gibco</td><td>14200-075</td><td></td></tr><tr><td>16% Paraformaldehyde Solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>OCT Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Adhesion Slides</td><td>StatLab</td><td>M1000W</td><td></td></tr><tr><td>Donkey Serum</td><td>Sigma Aldrich</td><td>D9663</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T9284</td><td></td></tr><tr><td>ImmuEdge Pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Anti-Calbindin Antibody</td><td>Sigma Aldrich</td><td>C9848</td><td></td></tr><tr><td>Anti-EGFP Antibody</td><td>Abcam</td><td>ab13970</td><td></td></tr><tr><td>Anti-Nesprin2 Antibody</td><td></td><td></td><td>Previously described in Ref. 21</td></tr><tr><td>Fluorescent Mounting Media</td><td>Dako</td><td>S3023</td><td></td></tr><tr><td>2-methyl butane</td><td>Sigma Aldrich</td><td>O3551</td><td></td></tr></tbody></table>

validation of a mouse model to disrupt linc complexes in a cell-specific manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

此协议说明的Tg（CAG-的LacZ / EGFP-KASH2）小鼠模型的有效性，以限制EGFP-KASH2表达使用的Tg（PCP2-CRE）小鼠小脑浦肯野细胞。在的Tg（PCP2Cre CAG-EGFP / KASH2）的后代，所述的LacZ / V5开放读框是通过Cre重组酶从而导致EGFP-KASH2的专门针对浦肯野细胞（图2A）的表达切下的P6。正如所料，EGFP-KASH2靶向核膜通过围绕原子核（图2B）观察到的绿色荧光蛋白阳性的缘状图案所指示的。为了最大限度地提高任何生理学表型，以确保通过计算EGFP-KASH2阳性细胞被共染色的细胞特异性抗体标记物的百分比EGFP-KASH2表达于大多数靶细胞是重要的。在这个例子中，EGFP-KASH2表达钙结合蛋白阳性浦肯野细胞（图2B）的70％以上进行检测。在这些康迪系统蒸发散，我们没有观察到10月龄的Tg（PCP2Cre CAG-EGFP / KASH2）小鼠20任显著组织学或行为表型。确保任何形态或行为表型不是由于EGFP-KASH2的在非预期的细胞类型/组织异位表达是重要的。到这方面，EGFP-KASH2未在颗粒细胞层或Tg的（PCP2Cre CAG-EGFP-KASH2）小脑中（图2B）的分子层观察。 最后，这是至关重要的，以确认LINC复杂破坏的细胞中表达EGFP-KASH2的程度。这可以通过使用高质量的防Nesprin抗体强调从细胞中表达EGFP-KASH2的NE的位移来实现。 如图3，内源性Nesprin2从浦肯野细胞的核膜表达EGFP-KASH2（图3顶板，黄色箭头）移动，而浦肯野细胞不表达EGFP-KASH2 RETA内源性Nesprin2在核膜（图3顶板，白色箭头）。正如所料，对照同窝，不表达Cre重组酶，显示Nesprin2在整个浦肯野细胞群体（ 图3底白色箭头）的核膜。现在几个报告已经证实EGFP-KASH2的表达，伴随着内源性Nesprins，在附加的组织和细胞类型的位移如骨骼肌纤维和锥光感受器19,21。 <img alt="图1" src="/files/ftp_upload/53318/53318fig1.jpg" /> 图1. LINC络合物核连接到细胞骨架和由EGFP-KASH2的过表达被打乱。LINC络合物核的内部连接到胞质细胞骨架的（A）的描写。需要注意的是SUN结构域相互作用混乱与KASH结构域所有Nesprins（Nesprins 1-4）。 EGFP-KASH2（B）的过表达内源置换Nesprins从核膜到急诊室，以显性负时尚，从而断开从周围胞质细胞骨架的核。 <img alt="图2" src="/files/ftp_upload/53318/53318fig2.jpg" /> 的Tg（CAG-的LacZ / EGFP-KASH2） 图 2. 表达EGFP-KASH2的专门针对小脑浦肯野细胞。（A）选育的Tg（PCP2-CRE）的小鼠产生的Tg（PCP2Cre CAG-EGFP-KASH2）的后代其中Cre重组酶在小脑蒲肯野细胞中特异性表达。当表达式（P6），Cre重组酶转位进入它切除了LacZ的ORF并诱导EGFP-KASH2表达的核心。 （B）的Tg（PCP2Cre CAG-EGFP-KASH2）小鼠特异性表达EGFP-KASH2小脑浦肯野细胞IDENTIF灭蝇灯与钙结合蛋白（红色彩色）。注意，分子层的两个小脑颗粒神经元和中间是阴性的EGFP-KASH2表达。简称:MOL:分子层，PCL:浦肯野细胞层和保利协鑫:颗粒细胞层。 <img alt="图3" src="/files/ftp_upload/53318/53318fig3.jpg" /> 图3.破坏的小脑浦肯野细胞内源性的LINC络合物。EGFP-KASH2单共聚焦平面表示表达式置换内源Nesprin2（着色为红色）从浦肯野细胞的核膜（黄色箭头顶部）。需要注意的是浦肯野细胞缺乏EGFP-KASH2表达维持内源性Nesprin2在他们的核膜（白色箭头顶部）。在的Tg（CAG-的LacZ / EGFP-KASH2）对照同窝，没有EGFP的信号观察和Nesprin2是在所有Purki的核膜检测这个协议说明的有用的T g（CAG-的LacZ / EGFP-KASH2）小鼠模型来限制EGFP-KASH2表达使用的Tg（PCP2-CRE）小鼠小脑浦肯野细胞。在的Tg（PCP2Cre CAG-EGFP / KASH2）的后代，所述的LacZ / V5开放读框是通过Cre重组酶从而导致EGFP-KASH2的专门针对浦肯野细胞（图2A）的表达切下的P6。正如所料，EGFP-KASH2靶向核膜通过围绕原子核（图2B）观察到的绿色荧光蛋白阳性的缘状图案所指示的。为了最大限度地提高任何生理学表型，以确保通过计算EGFP-KASH2阳性细胞被共染色的细胞特异性抗体标记物的百分比EGFP-KASH2表达于大多数靶细胞是重要的。在这个例子中，EGFP-KASH2表达钙结合蛋白阳性浦肯野细胞（图2B）的70％以上进行检测。在这种情况下，我们没有观察到任何显著组织或behaviora升表型在10个月龄的Tg（PCP2Cre CAG-EGFP / KASH2）小鼠20。确保任何形态或行为表型不是由于EGFP-KASH2的在非预期的细胞类型/组织异位表达是重要的。到这方面，EGFP-KASH2未在颗粒细胞层或Tg的（PCP2Cre CAG-EGFP-KASH2）小脑中（图2B）的分子层观察。 最后，这是至关重要的，以确认LINC复杂破坏的细胞中表达EGFP-KASH2的程度。这可以通过使用高质量的防Nesprin抗体强调从细胞中表达EGFP-KASH2的NE的位移来实现。 如图3，内源性Nesprin2从浦肯野细胞的核膜表达EGFP-KASH2（图3顶板，黄色箭头）移动，而浦肯野细胞不表达EGFP-KASH2保留内源Nesprin2在核膜（图3 </s仲&gt;顶部面板，白色箭头）。正如所料，对照同窝，不表达Cre重组酶，显示Nesprin2在整个浦肯野细胞群体（ 图3底白色箭头）的核膜。现在几个报告已经证实EGFP-KASH2的表达，伴随着内源性Nesprins，在附加的组织和细胞类型的位移如骨骼肌纤维和锥光感受器19,21。 <img alt="图1" src="/files/ftp_upload/53318/53318fig1.jpg" /> 图1. LINC络合物核连接到细胞骨架和由EGFP-KASH2的过表达被打乱。LINC络合物核的内部连接到胞质细胞骨架的（A）的描写。需要注意的是SUN结构域相互作用混乱与所有Nesprins（Nesprins 1-4）KASH结构域。 （二）过表达Ø˚FEGFP-KASH2取代内生Nesprins从核膜到急诊室，在显性负时尚，从而切断从周围的细胞质细胞骨架的核心。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53318/53318fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53318/53318fig2.jpg" /> 的Tg（CAG-的LacZ / EGFP-KASH2） 图 2. 表达EGFP-KASH2的专门针对小脑浦肯野细胞。（A）选育的Tg（PCP2-CRE）的小鼠产生的Tg（PCP2Cre CAG-EGFP-KASH2）的后代其中Cre重组酶在小脑蒲肯野细胞中特异性表达。当表达式（P6），Cre重组酶转位进入它切除了LacZ的ORF并诱导EGFP-KASH2表达的核心。 （B）的Tg（PCP2Cre CAG-EGFP-KASH2）小鼠特异性表达EGFP-KASH2在确定与钙结合蛋白小脑浦肯野细胞（红色彩色）。注意，分子层的两个小脑颗粒神经元和中间是阴性的EGFP-KASH2表达。简称:MOL:分子层，PCL:浦肯野细胞层和保利协鑫:颗粒细胞层<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53318/53318fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53318/53318fig3.jpg" /> 图3.破坏的小脑浦肯野细胞内源性的LINC络合物。EGFP-KASH2单共聚焦平面表示表达式置换内源Nesprin2（着色为红色）从浦肯野细胞的核膜（黄色箭头顶部）。需要注意的是浦肯野细胞缺乏EGFP-KASH2表达维持内源性Nesprin2在他们的核膜（白衣e箭头顶部）。在的Tg（CAG-的LacZ / EGFP-KASH2）对照同窝，没有EGFP的信号观察和Nesprin2是在所有浦肯野细胞（白色箭头底部）的核膜检测。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53318/53318fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

validation-mouse-model-to-disrupt-linc-complexes-cell-specific

Nanyang Technological University

Research

JoVE Journal

Biology

7.3K 次观看.  Washington University in St. Louis School of Medicine. Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

视频: Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

诱导蛋白缺失的通过<em&gt;在子宫内</em&gt;电穿孔来定义赤字的神经细胞迁移的转基因模型

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

科研

发育生物学

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

行为学

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

遗传学

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

免疫与感染

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

神经科学

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

基因工程小鼠模型中的前列腺癌建模:小鼠前前列腺叶细胞中的 CRISPR/Cas9 介导的局部基因编辑技术

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

JoVE Encyclopedia of Experiments

癌症研究

Prostate Cancer

Animal model studies

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Source: Pang, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64059">A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice.</a> J. Vis. Exp. (2022)
This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. ...

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

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