Name: a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro
Uploaded: 2017-05-17T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

这项工作得到以下赠款的支持:中国自然科学基金（No. 81572198; 81260161; No. 81000460）;中国广东省自然科学基金（编号:2015A030313772）;中国博士后科学基金资助项目（No. 2013M530385）;中国广东省医学研究基金（No. A2016314）;深圳市科技项目（No.JCYJ20160301111338144; No.JSGG20151030140325149; No.JSGG20140519105550503; No.GJHZ20130412153906739; No.JCYJ20140414170821160; No.JCYJ20140414170821200）。

本研究由深圳市第二人民医院人类伦理委员会批准。 人关节软骨组织收集和软骨细胞培养<ol><li> 材料的制备 <ol><li>制备补充有10％胎牛血清和1％青霉素 - 链霉素的Dulbecco改良的Eagle培养基（DMEM）培养基。制备1mg / mL胶原酶II，0.25％胰蛋白酶-EDTA，磷酸盐缓冲盐水（PBS）和细胞过滤器（40μm尼龙）。 </li></ol></li><li> 人关节软骨组织收集 <ol><li>创伤后供体患者膝关节分离关节软骨。获得所有参与者的知情同意。 </li><li>使用无菌手术刀将软骨切成1-2毫米3片，并在含有1毫克/毫升胶原酶II的软骨细胞中，在DMEM中于37℃消化软骨细胞12-16小时。 </li><li>过滤所得的细胞l悬浮液通过细胞过滤器（40μm），并用PBS洗涤两次。 </li><li>用补充有10％胎牛血清（FBS）和1％青霉素 - 链霉素的DMEM中，用血细胞计数器和密度为20,000-30,000个细胞/ cm 2的细胞计数细胞。 </li><li>培养在37℃的培养箱中。 </li></ol></li><li> 单层软骨细胞扩张 <ol><li>使用0.25％胰蛋白酶-EDTA收获亚汇合细胞，并以6,600个细胞/ cm 2的密度重新平板。每周更换培养基两次。 </li><li>在单层中培养软骨细胞多达6代，并在第1,2,3,4和5代进行评估。 </li></ol></li></ol> 2.基因组DNA（gDNA）提取<ol><li>收集细胞，每1000万个细胞重悬于500μL裂解缓冲液（15mM Tris pH 8.0,10mM EDTA pH 8.0,0.5％SDS，200μg/ mL RNA酶A）中。通过移液和快速反转重悬细胞，然后在37℃下孵育1小时。 </li><li>一个dd蛋白酶K浓度为160μg/ mL的细胞裂解物，并剧烈反转混合物。在55℃下孵育6小时。 </li><li>向样品中加入一倍体积的Tris pH 7.9饱和酚:氯仿:异戊醇（25:24:1）。旋转或用手彻底摇动样品约20秒。 </li><li>将样品在室温下离心5分钟，时间为13,000×g。去除上层水相并将层转移到新管中。 </li><li>用等体积的氯仿萃取以除去苯酚并将顶部水相转移到新鲜管中。 </li><li>通过加入0.1体积的3M乙酸钠pH 8.0和2体积的100％乙醇沉淀DNA。 </li><li>将试管置于-20℃过夜，沉淀出gDNA。 </li><li>在4℃下将样品以16,000xg离心10分钟以沉淀gDNA。 </li><li>用70％乙醇洗涤样品三次，并以13,000 x g在4℃离心2分钟。 </li><li>取出上清液小心然后风干。将DNA重悬于10mM Tris pH 8.0,0.1mM EDTA中。 </li></ol> DNA甲基化分析<ol><li>根据制造商的方案，使用DNA甲基化试剂盒使用总共500ng的基因组DNA进行亚硫酸氢盐转化反应。在10μL洗脱缓冲液（50 ng /μL）洗脱。 </li><li>根据制造商的方案使用商业试剂盒进行DNA甲基化分析。 </li></ol>点斑分析<ol><li>将分离的DNA（每个样品1mg）在95℃下在0.1M NaOH中变性10分钟。在冰上用1M NH 4 OAc中和DNA，然后稀释两倍。在N +膜上点2μL连续稀释的基因组DNA。 </li><li>将膜在80℃下打印30分钟。 </li><li>通过将N +膜在TBS-T中的5％BSA中浸泡1小时来阻断非特异性抗体结合位点。使用10厘米培养皿作为反应离子室。 </li><li>在TBST中洗5分钟后，用TBS-T中的小鼠抗5-甲基胞嘧啶（5-mC）单克隆抗体（1:1,000）在4℃温育膜过夜。 </li><li>在TBS-T中洗涤膜5分钟3次，然后在室温下与TBS-T中的第二抗体HRP-缀合的绵羊抗小鼠免疫球蛋白-G（IgG）（1:5,000）孵育1小时。 </li><li>在TBS-T中将膜洗5分钟3次。 </li><li>将酶底物加入到膜中并孵育5-10分钟。使用化学发光试剂盒根据制造商的说明书显示二抗信号。 </li></ol>

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作者宣称他们没有竞争的经济利益。

软骨细胞去分化在体外严重损害ACI治疗软骨缺损修复的结果11,12 。为了优化ACI结果，避免使用去分化软骨细胞13至关重要。研究表明，一般的DNA甲基化水平与软骨细胞去分化的程度有关。因此，在临床应用前，建立可靠，快速的方法来检测软骨细胞的一般DNA甲基化状态。 为了检测去分化的人软骨细胞中的一般DNA甲基化水平，我...

自体软骨细胞植入（ACI）是一种相对较新的，最先进的手术来治疗关节软骨缺损1,2 。 ACI的关键步骤之一是通过单层培养体外扩增软骨细胞 。在扩增期间，透明软骨细胞容易失去其表型并变得去分化，这对于ACI治疗3,4是不合需要的。为了优化ACI治疗的结果，软骨细胞去分化的程度应在再植之前确定。建立经济，快速的方法来确定软骨细胞的状态是至关重要的。最近，DNA甲基化与软骨细胞去分化之间的关联已经引起了很多关注4，5，6 。 DNA甲基化是一个过程将甲基添加到DNA中，导致胞嘧啶残基转化为5-甲基胞嘧啶（5-mC）。 为了阐明软骨细胞去分化中DNA甲基化的生物学，第一步是评估软骨细胞的DNA甲基化水平，迄今证明是有挑战性的。亚硫酸氢盐基因组测序是分析DNA甲基化最广泛使用的技术7,8 。在该测定中，亚硫酸氢盐转化引起DNA降解，因此必须提供大量的样品用于测定。此外，高效液相色谱（HPLC）已被用于量化全球DNA甲基化水平9,10 。然而，HPLC分析需要基因组DNA消化。此外，需要先进和昂贵的实验仪器。因此，除了高成本之外，这些实验程序是时间短的uming。抗-5-mC抗体现在已经成为可商购的，这已经产生了来自复杂基因组的含有5-mC的基因组DNA的免疫印迹的可能性。 在本报告中，我们从一系列单层培养物中生长的软骨细胞中提取基因组DNA。我们使用斑点印迹测定来评估具有不同数量通道的人软骨细胞中的5-mC含量。我们发现，与低级去分化的软骨细胞相比，高度去分化的软骨细胞中5-mC含量增加。此外，我们确定了去分化状态和5-mC水平之间的关系。最后，我们报道了5-mC含量的变化与软骨细胞表型有关。因此，5-mC斑点印迹测定是检测软骨细胞中DNA甲基化水平的可靠，简单和快速的方法。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco Inc.</td><td>11965&amp;ndash;092</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>HyClone Inc.</td><td>SH30256.01B</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>FBS</td><td>Gibco Inc.</td><td>10099-141</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco Inc.</td><td>25200-056</td><td></td></tr><tr><td>1% Penicillin-Streptomycin</td><td>Gibco Inc.</td><td>15140-122</td><td></td></tr><tr><td>Chloroform</td><td>Mallinckrodt</td><td>4440</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Sigma</td><td>I-3643</td><td></td></tr><tr><td>Phenol</td><td>Gibco BRL</td><td>15513-039</td><td></td></tr><tr><td>Proteinase K</td><td>Gibco BRL</td><td>24568-2</td><td></td></tr><tr><td>TAE buffer&amp;nbsp;</td><td>Bio Whittaker</td><td>16-011V</td><td></td></tr><tr><td>Distilled Water</td><td>Gibco BRL</td><td>15230-170</td><td></td></tr><tr><td>1 M Tris-HCl</td><td>Biosharp Inc.</td><td>BL514A</td><td></td></tr><tr><td>Tween20</td><td>Biotopped Inc.</td><td>C58H114O26</td><td></td></tr><tr><td>BSA</td><td>Proliant Inc.</td><td>68700</td><td></td></tr><tr><td>Collagenase, Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>ISOLAB Inc.</td><td>075.03.001</td><td></td></tr><tr><td>Falcon 100 mm dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>TPP AG</td><td>91050</td><td>Gamma-sterilized</td></tr><tr><td>High-speed centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>ThermoMixer</td><td>MIULAB</td><td>MTH-100</td><td></td></tr><tr><td>Carbon dioxide cell incubator</td><td>Thermo scientific</td><td>3111</td><td></td></tr><tr><td>Chemi-imaging Analyse System</td><td>UVITEC Cambridge</td><td>ALLIANCE</td><td></td></tr></tbody></table>

a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro

透明软骨细胞去分化成纤维细胞软骨细胞通常伴随体外软骨细胞的单层扩张。软骨细胞的全球DNA甲基化水平被认为是软骨细胞表型丧失的合适的生物标志物。然而，基于不同实验方法的结果可能不一致。因此，建立一种精确，简单，快速的方法来量化软骨细胞去分化中的全局DNA甲基化水平是非常重要的。 目前的全基因组甲基化分析技术主要依靠亚硫酸氢盐基因组测序。由于亚硫酸氢盐转化期间的DNA降解，这些方法通常需要较大的样品体积。用于定量全球DNA甲基化水平的其他方法包括高效液相色谱（HPLC）。然而，HPLC需要完全消化基因组DNA。另外，HPLC的成本高昂结构限制了HPLC的广泛应用。 在本研究中，从用不同数量传代培养的人软骨细胞中提取基因组DNA（gDNA）。使用甲基化特异性斑点印迹法检测gDNA甲基化水平。在该斑点印迹方法中，将含有待检测的甲基化DNA的gDNA混合物直接点在N +膜上，作为先前绘制的圆形模板图案内的点。与其他基于凝胶电泳的印迹方法和其他复杂印迹方法相比，斑点印迹法节省了大量时间。此外，斑点印迹可以使用市售的5-mC抗体检测总DNA甲基化水平。我们发现DNA甲基化水平在单层亚文化之间不同，因此可能在软骨细胞去分化中起关键作用。 5-mC斑点印迹是检测一般DNA甲基化水平进行评估的可靠，简单和快速的方法e软骨细胞表型。

软骨细胞在单层培养直至第6代（P6）。软骨细胞随着单层培养物的连续传代显示进行性表型变化。 P0软骨细胞形态是圆形的，而细胞变成高度夹持和平坦化，连续传代直到P6（ 图1 ）。这种形态变化是软骨细胞去分化过程的典型。同时，热图的结果表明CpG位点的一般甲基化水平随着传代培养的增加而增加。 5-mC斑点印迹分析进一步表明，较高的CpG甲基化水平伴?...

Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

我们提出了一种基于5-甲基胞嘧啶（5-mC）斑点印迹定量DNA甲基化的方法。我们在软骨细胞去分化中测定了5-mC的水平。这种简单的技术可用于快速测定ACI治疗中的软骨细胞表型。

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

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Universidad de Cartagena UDEC

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Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

16.8K 次观看.  Guangzhou Medical University. 我们提出了一种基于5-甲基胞嘧啶（5-mC）斑点印迹定量DNA甲基化的方法。我们在软骨细胞去分化中测定了5-mC的水平。这种简单的技术可用于快速测定ACI治疗中的软骨细胞表型。 

视频: A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

科研

遗传学

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

用酮抑制剂 PD0325901 和维生素 C 维持小鼠胚胎干细胞基因组低甲基化的一种替代培养方法

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

生物化学

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

发育生物学

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

化学

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 ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

神经科学

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.

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 ...

生物学

<meta charset="utf8"></head><body>从人关节软骨中分离软骨细胞

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

<meta charset="utf8"></head><body>使用纤连蛋白粘附和迁移测定分离软骨细胞和软骨生成器

This protocol details the isolation of chondrocytes, Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and Migratory Chondroprogenitors (MCPs) from human articular cartilage. It covers enzymatic digestion, fibronectin adhesion, and migration-based assays for isolating and characterizing these cells.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平

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