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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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。?...

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

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

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

科研

生物学

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

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 ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

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 ...

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 ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

腰椎不稳定的鼠标模型

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

原代培养中分化的小鼠脂肪细胞：胰岛素抵抗模型

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...