Name: correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)
Uploaded: 2015-09-26T15:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

所有动物均按照健康准则全国学院处理。动物护理和使用委员会在阿拉巴马大学伯明翰分校批准的动物使用。 1.使用荧光激活细胞从全脑组织排序星形胶质细胞（FACS）从富集的星形胶质细胞人口获得的RNA和DNA <ol><li>稳重动物用CO 2 1分钟，然后迅速斩首。在阿尔布开克等人解剖皮层，26。这是没有必要去除脑膜。 注:表示在S100β绿色荧光蛋白（星形胶质细胞标记物）子转基因大鼠由板仓等 27产生和利用的星形胶质细胞的FACS分选。 </li><li>使用根据制造商的协议在非无菌条件下用木瓜蛋白酶的解离试剂盒制备全脑匀浆。在水浴热激活的木瓜蛋白酶在37℃下进行10分钟。注意:木瓜蛋白酶溶液通过制造商提供，并包含L-半胱氨酸和EDTA（见表材料）。 <ol><li>切割或DREMEL空穴到50毫升锥形管的顶部，以允许管道携带95％O 2:5％的CO 2被送入一个封闭的圆锥管（图1A）。放置在10毫米培养皿含有解离媒体解剖皮层（MEM补充有20mM的葡萄糖和青霉素/链霉素（500U / ml）和平衡至95％O 2，5％CO 2），并使用干净的刀片剁碎组织成1- ×1毫米2件 。 </li></ol></li><li>用10ml手册的Pipetman至50ml锥形管含有木瓜蛋白酶溶液转移组织。让组织来履行，以尽量减少分离介质的结转额前下沉到移液管的底部。保持平衡至95％ 的 O 2的木瓜蛋白酶溶液:5％的CO 2经由该孵育期间表面气体交换。不要泡木瓜蛋白酶soluti上。孵育组织为在37℃水浴20分钟。 </li><li>孵育后在木瓜蛋白酶溶液，磨碎的组织的10倍以低速10ml的移液管。离心浑浊细胞悬浮液在1000×g离心5分钟，室温。 <ol><li>平衡经由表面气体交换的DNase /白蛋白抑制剂溶液（由制造商提供的），并在3ml的DNA酶/清蛋白抑制剂溶液重新悬浮沉淀的细胞。制备商业不连续密度梯度依照制造商的指令。 </li></ol></li><li>自旋不连续密度梯度，在1000×g离心6分钟。通过抽吸底部沉淀用吸管隔离管的底部离解的细胞。 </li><li>重悬离解的细胞在2-3毫升的DPBS用0.02％牛血清白蛋白和1毫克/毫升DNA酶或缓冲培养基优选HEPES。流式细胞仪（ 图2A），然后通过40微米的过滤器传递。保持细胞在冰上，直到排序。 <ol><li>执行流式细胞仪28。&lt;/ LI&gt; <li>沉淀细胞在1.5ml的离心管中，在2000×g离心5分钟，在4℃。 注意:丸粒化星形胶质细胞可以立即使用或保存在-80℃直至DNA提取。 </li></ol></li><li> RNA和DNA使用优选的分离方法29 30提取。通过分光光度计和生物分析仪31评估RNA和DNA的浓度和质量。 注意:利用仅高质量RNA和DNA在随后的步骤中，260个/ 280个= 2.0-2.2和1.8-1.9，分别。生物分析仪分析是必不可少的，以评估RNA降解或DNA片段。 RNA或DNA可以立即使用或储存于-80℃或-20℃，分别用于随后的研究。 </li></ol> 2.评估使用甲基化敏感高分辨率熔解分析（MS-HRMA）的基因的DNA甲基化状态<ol><li>进入首选网上甲基化地图软件感兴趣的基因序列，以识别任何CpG岛的基因关注32。 <ol><li>亚硫酸氢盐对设计的引物转化DNA序列33和扩增按照制造商的方案使用优选的DNA聚合酶。通过运行在100伏的1％琼脂糖凝胶的DNA 45分钟确认扩增产物的大小。商店引物储备浓度20μM的在-20℃。 </li></ol></li><li>亚硫酸氢盐转化500-1000毫微克的每个样品和甲基化DNA的标准范围从0-100％来自同一动物种类34的DNA。洗脱样本以提供20毫微克/微升的浓度。通过分光光度计31验证的亚硫酸氢盐转化的DNA浓度。 </li><li>设置20微升反应为根据表1和2使用优选的DNA聚合酶和引物在5微米浓度的MS-HRM扩增。运行所有的样品，包括流式细胞仪DNA和甲基化的标准，以一式三份。 </li><li>根据分析软件，设置前置和后置启动和停止参数周围的熔解曲线的过渡。组预先熔融开始和预熔融停止参数，使两者之间的差为0.2 - 0.5℃。设置后开始融化，后熔停止类似。提取峰值温度的差数据的每个样本。 </li><li>使用百分之甲基标准（y值）和它们的相应的平均峰温度差（x值）生成的线性回归方程（图3A-B，表3-4）。使用该线性回归方程来估算未知样品35的甲基化状态。 </li></ol> 3.通过使用荧光素酶测定的评估超甲基化的启动子活性<ol><li>确定目标基因（步骤2.1）的CpG岛。 PCR扩增感兴趣36和克隆区域luc2萤火虫萤光素酶报道基因的上游，以产生的CpG岛luc2质粒37。 </li><li>线性化30微克的通过限制的CpG岛luc2质粒酶消化38。验证网站的限制消化，避免使用首选刀具软件38双削减。热灭活根据制造商的协议如下消化在适当的温度和持续时间的酶。在线性化步骤是DNA的损失降到最低。 </li><li>使用的CpG甲基化酶（M.Sssl）O / N在30°C或将未经处理的，按照制造商的协议，除了以下调整甲醇线性质粒。 </li><li>执行50微升反应。 </li><li>使用5个单位的CpG甲基对甲基化700纳克线性化的质粒。 </li><li>运行反应O / N为13-19小时。 </li><li>以下的CpG甲基化酶反应，使用标准的，根据制造商的协议市售硅胶膜上清理试剂盒进行DNA的清理。 DNA的显著损耗发生以下的CpG甲基化酶反应，30-60％的损失。 </li><li>通过中Hpa II酶切验证质粒甲基化。 </l我&gt; <li>取1微克的甲基化或非甲基化DNA和限制在37℃下消化用的Hpa II 1小时。使用5-10个单位的Hpa II为每微克DNA。继中Hpa II消化，使用的是首选，市售的硅胶膜清洁套装进行DNA清理。运行在100V在TAE或TBE缓冲液的1％琼脂糖凝胶的DNA在两个CpG的甲基化和非甲基化的质粒45分钟进行可视化（图4B）。 </li><li>受试者都甲基化和非甲基化的质粒双重消化用合适的限制性酶释放的全长的luc 2（矢量）和CpG岛（插入） 片 38。以下，根据制造商的协议消化进行双酶切O / N在适当的温度和热灭活酶。在双酶切出现DNA浓度的损失降到最低。 </li><li>运行在1％琼脂糖凝胶双酶切质粒在100V 1小时，以允许分离ö˚F载体和插入（图4C）。注意事项。戴防护紫外线面罩，放置在桌面黑光DNA凝胶可视化的乐队。基于大小，消费甲基化和非甲基化的插入和非甲基化的载体用干净的手术刀。 </li><li>凝胶提取DNA用饱和酚，pH值6.6。简单地说，衡量含载体或插入DNA凝胶。用100微升苯酚每0.1克DNA凝胶。用玻璃Dounce匀浆均质DNA凝胶苯酚。 </li><li>加氯仿（以苯酚量的1/5），并摇动样品20秒。孵育的样品在RT 2-3分钟。离心15分钟以最大速度（16.1×1,000 XG）在4℃。 </li><li>除去水溶液，并添加3M乙酸钠和乙醇2.5X体积0.1X体积。孵育样品1小时，在-80℃下。孵育后，除去乙醇并于30μl优选缓冲液重悬的DNA。 DNA的显著损失发生的插入和载体以下隔离，30-50％LOSS。 </li><li>再结扎甲基化和非甲基化的插入物，使用T4 DNA连接酶38未甲基矢量。使用1:4的比例矢量的插入用于连接反应。根据制造商的说明书设置反应。使用50微升的总体积的反应和孵化O / N在-20°C或在冰上用盖在冰桶。 </li><li>连接后，使用的是首选，市售的硅胶膜清洁套装进行DNA清理。的DNA显著损失发生以下的连接反应，DNA的近似30-50％的损失。验证重新结扎通过在1％琼脂糖凝胶的DNA 45分钟运行，在100 V和评估的重新连接质粒（图4D）的浓度。 </li><li>染甲基化或非甲基化的质粒导入使用根据制造商的协议的市售的转染试剂D54细胞。种子D54细胞到12孔板，在一个0.14×10 6个细胞/孔。 </li><li>继24小时，转染细胞无线个相等的浓度，无论（1）非methyated CpG的luc2质粒+海肾或其他控制荧光素酶载体或（2）甲基化的CpG-luc2质粒+海肾或其他控制荧光素酶载体。 </li><li>让细胞进行双荧光素酶检测前转24小时。根据制造商的说明进行检测。执行用光度计读数。一式三份阅读每口井。 </li><li>计算萤火虫荧光素酶活性的比为海肾或其他控制的荧光素酶的活性。通过将甲基化荧光素酶活性非甲基化的荧光素酶控制的荧光素酶:规范化甲基化萤火虫:控制荧光素酶的活性，以非甲基化的萤火虫 </li></ol>

The authors have no disclosures.

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge in studying the epigenome of the CNS is the diversity of cell populations. As genes of interest may be expressed to varying degrees with both temporally and spatially unique patterns in the CNS, the use of whole tissue often confounds data from both transcription and DNA methylation studies. 
Kir4.1, the protein of interest, is spatially and temporally regulated in astrocytes, oligodendrocytes, and ependymal cells 13,14,40-47. Thus, the use of eGFP-S100&#946; transgenic animals 27 for FACS provided an enriched population of astrocytes 15, and therefore a more accurate assessment of the level of gene transcription and DNA methylation specific to this cell population. Despite this advantage, the use of FACS sorted astrocytes presents several challenges. Preparation of a cell suspension for FACS requires significant disruption of whole tissue and several changes in the temperature and external media environment. While pH and temperature are maintained to promote cell viability and stabilize cellular machinery, it is reasonable to assume that some changes in DNA methylation occur subsequent to the mechanical and enzymatic disruption of cells. Additionally, in comparison to the use of whole tissue, FACS sorting of astrocytes yields significantly lower amounts of RNA and DNA for downstream molecular studies. This low ratio of yield to labor/reagent may limit the feasibility of some studies depending on the number and complexity of subsequent molecular applications as well as brain regions under study. Taking time to maintain proper pH and temperature as well as working efficiently to reduce total protocol time will aid in isolating healthy cells and thus high quality DNA/RNA. 
In regards to methylation studies, a variety of applications are currently available for detection of DNA methylation 48,49. Methylation sensitive high resolution melt analysis (MS-HRMA) was utilized to study KCNJ10, the gene of interest. MS-HRMA is useful in screening changes in DNA methylation of any given region of a gene of interest. This technique incorporates many of the same basic concepts and procedures as standard PCR amplification 50. Thus, reactions can be setup using a number of thermal cyclers and data analysis performed in most laboratories familiar with standard molecular techniques. One caveat to MS-HRMA is that the method provides estimates of the methylation status of a given region of DNA. This is in contrast to pyrosequencing which provides exact percentages of methylation of specific CpG sites. Additionally, depending on the number of CpG sites assessed within one region, using a linear regression may skew results for those regions with limited numbers of CpG sites. 
In studying the ability of DNA methylation to regulate KCNJ10 gene expression, we supplemented correlative studies with a dual luciferase assay to directly assess changes in the transcriptional activity of the KCNJ10 gene following hypermethylation. Use of the luciferase assay allows for alteration of the methylation status of a specific region of the gene and subsequent assessment of transcriptional activity directly related to these changes. Two major challenges of the luciferase assay are the laborious nature of the protocol and the loss of DNA input through each sequential step. Validation of the quantity and quality of DNA following each reaction as well as starting with a sufficient amount of DNA is essential to acquiring adequate amounts of DNA for transfection. Overall, the use of these techniques can be performed using tools found in most molecular laboratories and be can performed without the use of a large genomic sequencing facility, making these techniques widely accessible to investigators interested in DNA methylation.

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table><tbody><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Methyl Primer</td><td>Applied Biosystems</td><td>online</td><td>software to localize CpG Islands</td></tr><tr><td>EZ DNA methylation Kit</td><td>Zymo Research</td><td>D5001</td><td></td></tr><tr><td>Rat Premixed Calibration Standard</td><td>EpiGenDx</td><td>80-8060R-Premix</td><td></td></tr><tr><td>CpG Methylase (M.Sssl)</td><td>Zymo Research</td><td>E2010</td><td></td></tr><tr><td>QIAquick Gel Extraction&amp;nbsp;</td><td>QIagen</td><td>28704</td><td>Used for gel extraction and DNA cleanup</td></tr><tr><td>Restriction enzymes</td><td>New England BioLabs</td><td></td><td></td></tr><tr><td>NEB cutter</td><td>New England BioLabs</td><td>online</td><td>verify restriction digest sites</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Luc2 vector, pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>renilla vector, pGL4.74</td><td>Promega</td><td>E2241</td><td></td></tr><tr><td>TD-20/20 Luminometer</td><td>Turner Designs</td><td></td><td></td></tr><tr><td>Lipofectamine LTX and Plus Reagent</td><td>Life Technologies</td><td>A12621</td><td></td></tr><tr><td>Phenol, saturated pH 6.6/6.9</td><td>Fisher Scientific</td><td>BP 17501-100</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophtometer</td><td>ThermoScientific</td><td></td><td></td></tr><tr><td>MeltDoctor Master Mix</td><td>Life Technologies</td><td>4415440</td><td></td></tr><tr><td>High Resolution Melt (HRM) Software v2.0</td><td>Life Technologies</td><td>4397808</td><td></td></tr><tr><td>AB SDS software v2.3</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB High Resolution Melting Getting Started Guide&amp;nbsp;</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB 7900HT Fast Real-Time System</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

经由的eGFP-S100β转基因动物27的 FACS分选被收购的星形胶质细胞的富集群。由于降低年龄P0-P40细胞和年龄超过出生后第50天（P50）动物中分离分子的分子，动物的质量是最适合这样的实验。皮质组织被用于这些实验。从两个到六个动物皮质合并在一起。流式细胞仪在UAB综合流式细胞仪核心设施进行。在Becton Dickinson公司FacsAria II进行排序。采用488 nm激光获得绿色荧光蛋白的激励;没有补偿是必要的。基于前向和侧向散射（ 图1B）门控人口目标。阿活细胞群体，用溴化乙锭死细胞指示器确定和门（图1C）。基于所述的eGFP轮廓（ 图1D），观察两个不同的种群。通过经验性测试，细胞群更高的eGFP信息被确定为的eGFP阳性细胞群体（图1F-F&#39;&#39;）。第二群体与低级eGFP的轮廓的视觉分析显示表达eGFP的细胞碎片，但不含细胞核，可能代表来自星形细胞过程（ 图1G-G&#39;&#39;）碎片。解离的eGFP阳性星形细胞的以下的解离的代表性图像，但之前的分选（ 图2A）和分选（ 图2B）示出后。孤立的eGFP阳性细胞群表现出40倍的增加的mRNA ALDH1L1，一个星形细胞特异性标记物39（图2C）。此外，尽管S100β的NG2 + OPCs的星形胶质细胞和39共享表达，我们观察到4倍的降低NG2 mRNA的，标记为少突胶质细胞前体细胞39，说明富集星形细胞的细胞群体（图2C-D）的隔离。 RNA和DNA从分离的FACS分类的星形胶质细胞的足够质量和数量，以用于后续甲基化研究。总RNA和DNA从不同年龄和动物的数目分离被列为分子分子以下的FACS（表5）预测合格率的基准。应当指出的是，产率也将变化的基础上的动物使用的，潜伏期，和研究者的经验。 表6表明根据动物使用和潜伏期的一些事件的变异性获得的。 对于甲基化研究，开始以高品质和足够数量的DNA是成功的下游应用是必不可少的。以确保组织充分裂解为RNA或DNA提取，组织或细胞的所有均质化使用23G针7倍，同时注意避免裂解过程中起泡，随后通过研磨。一旦高品质的DNA提取和亚硫酸氢转换版，感兴趣的区域可以有针对性的MS-HRMA。需要注意的是评估和质量的亚硫酸氢盐转化的DNA的浓度时，分光光度计应设置为读取在33的OD因子如亚硫酸氢盐转化的DNA是单链的。 260/280值亚硫酸氢盐转化的DNA介于2.20-2.70。 之前开始甲基化研究，关键是要适当地选择和校准的MS-HRMA研究的热循环仪。此外，必须确定如何导出和使用从MS-HRMA生成的原始数据。 美国应用生物系统高分辨率熔解入门指南被用于帮助校准7900HT热循环仪。最后，数据提取并导入到HRM分析软件进行随后的数据分析。详见协议，前后的启动和停止参数设定接近熔解曲线的过渡。峰温度的差异被用来推断未知Sa的甲基化状态mples。从MS-HRMA获得估计甲基化数据密切对应于从焦磷酸测序产生的甲基化数据（焦磷酸测序数据在内部使用靶向相同的区域的MS-HRM引物的引物产生）（图3C）15。 双萤光素酶测定法用于评估感兴趣（图4A）的基因的超甲基化区域的转录活性。每一个的CpG岛luc2质粒线性化，然后甲基化。但是，由于这两个插入物（CpG岛）和载体（luc2）在反应过程中被甲基化，就必须切除甲基化插入件和重新结扎到非甲基化载体。由于大量操纵质粒，出现显著损失，必须首先足够的原料。的HpaⅡ消化用来验证每个质粒的甲基化状态为HpaII的消化仅非甲基化DNA（图4B）。应当指出的是如果O / N甲基化不足，SAM的和1x的CpG甲基化酶的另外1个可加入以下1小时的温育在30℃。继续O / N培养下加入补充SAM和CpG甲基化酶。甲基化和非甲基化的CpG岛-luc2质粒代允许DNA甲基化和转录活性之间的变化通过萤光素酶活性的测量直接评估。 <img alt="图1" src="/files/ftp_upload/52406/52406fig1.jpg" /> 图1. FACS分选分离绿色荧光蛋白+细胞popoulation。（A）使用50毫升的锥形管，孔切顶部允许木瓜蛋白酶消化过程中表面气体交换。 （B）的分选的细胞是基于前向和侧向散点图门控。 （C）一种溴化乙锭基于死细胞指示剂用于栅极的活细胞群（红色）。 （四）现场CEL升人口表明两个种群 - 一个具有高的eGFP轮廓（绿色）和低的eGFP轮廓（紫色）。对于所有的图表，y轴表示侧向散射面积（SSC-A）; x轴表示前侧散射区域（FSC-A）中，绿色荧光蛋白区域（GFP-A）中，或溴化乙锭（EtBr的）。 （EG）解离细胞孵育用Hoechst和20μl解离的细胞置于盖玻片和成像。 （EE&#39;&#39;），绿色荧光蛋白+星形胶质细胞，具有广泛的进程排序（NS），然后进行观察。 （FF&#39;）经过分拣，高绿色荧光蛋白+人口（POS）展示绿色荧光蛋白+细胞胞体其共定位与Hoechst染色。 （GG&#39;&#39;）低绿色荧光蛋白人口展示了绿色荧光蛋白+染色而没有共定位与Hoechst染色，可能较星形细胞碎片（所有图像，规模= 20微米）。 <a href="https://www.jove.com/files/ftp_upload/52406/52406fig1large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52406/52406fig2.jpg" /> 图2. FACS的eGFP-S100β转基因动物的排序提供星形细胞的富集群。（A）的20微升解离的细胞的FACS（NS）之前被示出，过程保持不变（标度= 50微米）。 （B）的代表性的图像后的FACS（FACS）细胞被示出;星形胶质细胞的过程被删除，胞体遗迹（规模= ​​20微米）。 （C）的qPCR数据表明相比没有排序人口（NS）的40倍增加ALDH1L1 mRNA的FACS分选的细胞（FACS）。 （四）NG2表达减少了4倍FACS分选的细胞相比，没有排序人口（NS）。 <a href="https://www.jove.com/files/ftp_upload/52406/52406fig2large.jpg" target="_blank">请点击此处</a><html>查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52406/52406fig3.jpg" /> 图3.数据和分析为MS-HRM研究，从甲基化的标准产生的温差地块​​（A）为例。相应百分比的甲基化标记为从大鼠甲基化的标准生成线性回归方程的每个曲线（B）的实施例。从焦磷酸测序（pyroseq，绿柱线）和MS-HRMA（灰色条）所获得的百分比甲基化（C）的比较表明甲基化水平相似的不同地区KCNJ10的。使用靶向相同的区域MS-HRM引物内部产生的焦磷酸测序数据分析， <a href="https://www.jove.com/files/ftp_upload/52406/52406fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 帐篷"FO:保持together.within页="总是"&gt; <img alt="图4" src="/files/ftp_upload/52406/52406fig4.jpg" /> 图4.双荧光素酶测定允许差异甲基化基因区域的转录活性的评估。对荧光素酶转录活性测定（A）图说明协议的工作流程。在甲基化与未甲基化的质粒以下的HpaⅡ消化（B）的 DNA凝胶显示差异。非甲基化的DNA可容易地消化以下的HpaⅡ消化相比甲基化的DNA，证实DNA质粒的成功甲基​​化。 （ 三）DNA凝胶演示从插入下面的双酶切载体的分离。下列DNA分离，载体和插入的凝胶中提取。 （ 四）DNA凝胶展示了成功的重新连接载体和插入的。重新连接质粒具有类似分子量为未处理质粒（UnTx上）。 NM，非会见hylated;男，甲基化; RL，再连接; UnTx上，未经处理的。 <a href="https://www.jove.com/files/ftp_upload/52406/52406fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 。 </td><td>卷（微升） </td><td> ×3 + 10％过量</td><td> x＃样品</td></tr><tr><td> MeltDoc MM </td><td> 10微升</td><td> 33微升</td><td></td></tr><tr><td>底漆1 </td><td> 1.2微升</td><td> 3.96微升</td><td></td></tr><tr><td>底漆2 </td><td> 1.2微升</td><td> 3.96微升</td><td></td></tr><tr><td>基因组DNA </td><td> 1微升</td><td> --- </td><td></td></tr><tr><td> 卫生署2 0 </td><td> 6.6微升</td><td> 21.78微升</td><td></td></tr></tbody></table> 表1.样本计算MeltDoctor反应体系的。每个运行MS-HRM REAction一式三份，用10％过量，以弥补移液错误。最后一列表示需要多个号码样品的计算。总体积为每个反应是20微升。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>周期</td><td>温度（℃） </td><td>时间（秒） </td><td>斜坡率（％） </td></tr><tr><td></td><td> 95℃ </td><td> :15 </td><td> 100 </td></tr><tr><td> 40X </td><td> 95℃ </td><td> :15 </td><td> 100 </td></tr><tr><td></td><td> 60℃ </td><td> :60 </td><td> 100 </td></tr><tr><td>熔参数</td><td> 95℃ </td><td> :10 </td><td> 100 </td></tr><tr><td></td><td> 60℃ </td><td> :60 </td><td> 100 </td></tr><tr><td></td><td> 95℃ </td><td> :15 </td><td> 1 </td></tr><tr><td></td><td> 60℃ </td><td> :15 </td><td> 100 </td></tr><tr><td>抱</td><td> 4<html> 76的温度</td><td></td><td></td></tr></tbody></table> 表2:用于扩增样品协议变性样品在95℃下进行10分钟，随后扩增熔解曲线的产生的40个循环。熔化参数列表内。注意改变升温速率，允许收购高分辨率熔解温度。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>百分比甲基化（鼠标） </td><td>峰值温度差</td></tr><tr><td> ÿ </td><td> x </td></tr><tr><td> 0 </td><td> 5.670116 </td></tr><tr><td>五</td><td> 10.70448 </td></tr><tr><td> 10 </td><td> 15.5044 </td></tr><tr><td> 25 </td><td> 25.52295 </td></tr><tr><td> 50 </td><td> 34.43047 </td></tr><tr><td> 75 </td><td> 43.80894 </td></tr><tr><td> 100 </td><td> 53.88717 </td></tr></tbody></table>核苷酸"&gt; 表3.实施例的峰值温度的差数据。百分比甲基标准标记为y坐标。峰值温度差数据被标记为x坐标。使用MS-HRM从一个基因区获得代表峰温度差数据。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>峰值温度差异</td><td>预计甲基化</td></tr><tr><td> X </td><td> ÿ </td></tr><tr><td>样品1（4例） </td><td></td></tr><tr><td> 47.450 </td><td> 81.115 </td></tr><tr><td> 46.​​292 </td><td> 78.657 </td></tr><tr><td> 41.694 </td><td> 68.894 </td></tr><tr><td> 47.292 </td><td> 80.779 </td></tr><tr><td>样品2（N = 4） </td><td></td></tr><tr><td> 29.925 </td><td> 43.904 </td></tr><tr><td> 24.269 </td><td> 31.894 </td></tr><tr><td> 25.061 </td><td> 33.575 </td></tr><tr><td> 25.389 </td><td> 34.272</td></tr></tbody></table> 表计算估计％的甲基化4例。不明的甲基化状态样品的峰值温度差时采用线性回归方程来估算％的甲基化。从两个不同的条件下，样品1和样品2的代表性数据，示出; n = 4时为每个条件。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>龄</td><td>动物的数目</td><td>总RNA（NG） </td><td>总DNA（NG） </td></tr><tr><td> P4-P10 </td><td> 3-6 </td><td> 455-868 </td><td> 265-1523 </td></tr><tr><td> P19-P22 </td><td> 1-2 </td><td> 220-680 </td><td> 583-1483 </td></tr><tr><td> P40-P50 </td><td> 3-4 </td><td> 198-260 </td><td> 578-1700 </td></tr></tbody></table> 表5总RNA和DNA从FACS获得的分选的细胞。年龄和使用的FA的动物数CS实验上市。注意，无论是皮质用于每一奈瑟总RNA和DNA被列为预期收率的参考。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>龄</td><td>动物的数目</td><td>波什活动</td><td> NEG活​​动</td><td>孵育时间（37℃） </td></tr><tr><td> P26 </td><td> 1 </td><td> 2.18X10 ^ 6 </td><td> 1.15X10 ^ 6 </td><td> 20:00分</td></tr><tr><td> P26 </td><td> 2 </td><td> 4.4X10 ^ 6 </td><td> 2.78X10 ^ 6 </td><td> 20:00分</td></tr><tr><td> P26 </td><td> 2 </td><td> 9.2X10 ^ 6 </td><td> 3.3×10 ^ 6 </td><td> 30:00分</td></tr></tbody></table> 从流式细胞仪获得在26日龄时的正（+ eGFP的）事件和负面事件的p26 数量的事件表6数量取决于动物使用和潜伏期的数目。

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

correlating-gene-specific-dna-methylation-changes-with-expression

Nanyang Technological University

Research

JoVE Journal

Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

7.9K 次观看.  University of Alabama at Birmingham. DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. 

视频: Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

科研

神经科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

发育生物学

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

成像细胞内Ca<sup&gt; 2+</sup&gt;从成年小鼠使用基因编码的钙指标纹状体星形胶质细胞信号

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

生物化学

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

耦合星形胶质细胞网络的大小、形状和方向性分析

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性

摘要

探索更多视频

此视频中的章节

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

成像细胞内Ca

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

耦合星形胶质细胞网络的大小、形状和方向性分析

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性

摘要

探索更多视频

此视频中的章节

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

成像细胞内Ca

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

耦合星形胶质细胞网络的大小、形状和方向性分析

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

在多地址基因组集成颜色（MAGIC）标记的 Utero 电冲中，以个性化皮质小鼠星形细胞