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 i...

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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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&#160;</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?...

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

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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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...