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.

全ての動物は、国立衛生研究所のガイドラインに従って処理されました。アラバマ大学バーミンガム校で動物実験委員会は、動物の使用を承認しました。 1.全脳組織からのアストロサイトの選別（FACS）、蛍光活性化細胞を使用してエンリッチドアストロサイトの集団からRNAとDNAの取得<ol><li>落ち着いた1分間のCO 2で、動物、その後急速に首を切ります。 アルバカーキらに記載されているように、皮質を解剖26。それは、髄膜を除去する必要はありません。 注意：S100β（アストロサイトマーカー）プロモーター下のeGFPを発現するトランスジェニックラットは27板倉らによって生成され、アストロサイトのFACSソーティングのために利用しました。 </li><li>製造業者のプロトコルに従って、非無菌条件下でパパイン解離キットを用いて全脳ホモジネートを準備します。 10分間、水浴中で37℃でパパインを熱活性化します。注意：パパイン溶液は、製造業者によって提供され、L-システインおよびEDTA（材料の表を参照）が含まれています。 <ol><li>閉じたコニカルチューブ（ 図1A）内に供給される5％のCO 2：95％のO 2を運ぶチューブを可能にするために50 mlのコニカルチューブの頂部に穴を開け、またはドレメル。解離培地を含む10ミリメートル培養皿に解剖皮質を置き（MEMは、20mMのグルコースおよびペニシリン/ストレプトマイシン（500 U / ml）を補充し、95％O 2で平衡化：5％のCO 2）で1に組織をミンチする清潔なカミソリの刃を使用しX 1ミリメートル2枚。 </li></ol></li><li>パパイン溶液を含有する50 mlのコニカルチューブに10 mlの手動ピペットマンを用いて転送組織。組織が引き継が解離媒体の量を最小限にするために排出する前に、転送ピペットの底に沈殿することを許可します。インキュベーションの間、表面ガス交換を経て、5％のCO 2：95％O 2に平衡化パパインソリューションをしてください。バブルパパインsolutiにしないでください上。 37℃の水浴中で20分間組織をインキュベートします。 </li><li>パパイン溶液中でのインキュベーション後、磨砕組織低速で10ミリリットルの転送ピペットで10回。 RTで5分間1,000×gで遠心濁った細胞懸濁液。 <ol><li>表面ガス交換を介して、（製造者によって提供される）、DNアーゼ/​​アルブミン阻害剤溶液を平衡化し、DNアーゼ/​​アルブミン阻害剤溶液3mlに細胞ペレットを再懸濁します。メーカーの指示に従って、商業不連続密度勾配を準備します。 </li></ol></li><li> 6分、1000×gで不連続密度勾配をスピン。ピペットを用いたボトムペレットを吸引してチューブの底から解離した細胞を分離します。 </li><li> 0.02％のウシ血清アルブミンおよび1mg / mlのDNアーゼまたは培地HEPES緩衝好適にDPBSの2-3 ml中に解離した細胞を再懸濁します。 FACS（ 図2A）の前に40μmのフィルターを通過。ソートするまで氷上で細胞を保管してください。 <ol><li> FACS 28を実行します 。&lt;/ LI&gt; <li> 4℃で5分間、2,000×gで1.5 mlの遠心管中で細胞をペレット化。 注：ペレット化した星状細胞は、直ちに利用またはDNA抽出まで-80℃で維持することができます。 </li></ol></li><li>好適な分離法29 30を使用して、RNAおよびDNAを抽出します。分光光度計とバイオアナライザ31を介して RNAとDNAの濃度と品質を評価します。 注意：以降の唯一の高品質のRNAとDNAを利用し、それぞれ280分の260 = 2.0〜2.2と1.8から1.9に、。バイオアナライザー分析は、RNAの分解またはDNA断片化のために評価することが不可欠です。 RNAまたはDNAは、それぞれ、その後の研究のため、すぐに利用または-80℃で保存するか、または-20℃できます。 </li></ol> 2.メチル化感受性高分解能融解分析（MS-HRMA）を用いた遺伝子のDNAメチル化状態を評価します<ol><li>遺伝子の任意のCpGアイランドを同定するために好適なオンラインメチルマッピングソフトウェアへの目的の遺伝子配列を入力します興味32の。 <ol><li>亜硫酸水素塩に対する設計プライマーは、DNA配列33に変換し、製造業者のプロトコルに従って、好ましいDNAポリメラーゼを用いて増幅します。 45分間、100Vで1％アガロースDNAゲルを実行することにより、増幅産物のサイズを確認します。 -20℃で20μMのストック濃度で保存するプライマー。 </li></ol></li><li>亜硫酸水素塩は、同じ動物種34から0〜100％の範囲で、各サンプルおよびメチル化DNA標準のDNAの500〜1,000 ngのを変換します。溶出サンプルは、20 ngの/μlの濃度を提供します。分光光度計31を介して重亜硫酸変換したDNAの濃度を確認してください。 </li><li> 表1および2に記載の 5μMの濃度で好ましいDNAポリメラーゼおよびプライマーを使用して、MS-HRM増幅のためのセットアップ20μlの反応は。三重で、FACSのDNAメチル化規格を含むすべてのサンプルを実行します。 </li><li>解析ソフトウェアによっては、事前設定したパラメータを開始および停止後融解曲線の遷移の周り。 0.5℃ - 両者の差が0.2であるので、事前に溶融開始とプリメルト停止パラメータを設定します。ポスト溶融開始を設定し、同様に停止後の溶融。各サンプルのピーク温度差データを抽出します。 </li><li> ％のメチル化標準（y値）とそれに対応する平均ピーク温度差（x値）を使用すると、線形回帰方程式（ 図3A-B、表3-4）を生成します 。未知試料35のメチル化状態を推定するために、この線形回帰式を使用してください。 </li></ol> 3.ルシフェラーゼアッセイの使用を介してハイパーメチル化プロモーター活性を評価します<ol><li>標的遺伝子（ステップ2.1）のCpGアイランドを識別します。 PCRは、CpGアイランド-luc2プラスミド37を生成するために luc2ホタルルシフェラーゼレポーター遺伝子の上流に興味36クローンの領域を増幅します。 </li><li>制限経由のCpGアイランドluc2プラスミド30μgのを線形酵素消化38。制限消化のための部位を確認し、好ましいカッターソフトウェア 38 を使用して二重のカットを避けます。製造業者のプロトコルに従って消化後に、適切な温度および時間で加熱不活性化する酵素。 DNAの最小限の損失が線形化工程の間に発生します。 </li><li> 30℃でのCpGメチラーゼ（M.Sssl）O / Nを使用するか、次の調整を除き、未処理の次のメーカーのプロトコルを残すメチル化線​​状化したプラスミド。 </li><li> 50μlの反応を行います。 </li><li>線状プラスミドの700 ngのをメチル化するのCpGメチラーゼの5単位を使用してください。 </li><li> 13-19時間の反応O / Nを実行します。 </li><li> CpGメチラーゼ反応に続いて、標準を用いてDNAのクリーンアップを実行し、市販のシリカゲル膜製造業者のプロトコルに従ってキットをクリーンアップします。 DNAの大幅な損失が30〜60％の損失を以下のCpGメチラーゼ反応を生じます。 </li><li> Hpa II制限消化を介しプラスミドのメチル化を確認してください。 </lI&gt; <li>メチル化または非メチル化DNAの1μgのを取り、制限が37℃で1時間のHpa IIで消化。各μgのDNAのためのHpa IIの5〜10単位を使用してください。 Hpa II消化後、好適な、市販のシリカゲル膜クリーンアップキットを用いてDNAのクリーンアップを実行します。可視化のために45分（ 図4B）を100 VでTAEまたはTBE緩衝液中、1％アガロースゲル上でのDNAのCpGメチル化および非メチル化プラスミドの両方を実行します。 </li><li>適切な制限酵素で二重消化の対象と両方メチル化および非メチル化プラスミドは、全長LUC 2（ベクトル）とCpGアイランド（挿入）38を断片化を解除します。製造業者のプロトコルに従って消化後に、適切な温度と熱不活性化する酵素で二重消化O / Nを実行します。 DNA濃度の最小の損失は、二重制限消化中に発生します。 </li><li> O分離を可能にするために1時間、100Vで1％のDNAのアガロースゲル上の二重消化したプラスミドを実行します。Fベクターと挿入した （図4C）。注意。保護のUVフェイスシールドを着用、バンドを可視化するために卓上ブラックライトにDNAゲルを配置します。きれいな外科用メスを用いたサイズに基づいて、消費税メチル化および非メチル化インサートと非メチル化ベクトル。 </li><li>飽和フェノール、pHが6.6を使用してゲル抽出DNA。簡単に述べると、ベクターまたはインサートを含有するDNAゲルの重量を量ります。 DNAゲルの0.1グラムあたりフェノール100μlのを使用してください。ガラスダウンスホモジナイザーを用いてフェノールにDNAゲルをホモジナイズします。 </li><li> （フェノール量の1/5を使用して）、クロロホルムを加え、20秒間のサンプルを横に振ります。 2-3分間、室温でサンプルをインキュベートします。 4℃での最大速度（16.1 X千XG）で15分間遠心。 </li><li>水溶液を削除し、3 M酢酸ナトリウムとエタノールの2.5倍の量の0.1Xボリュームを追加。 -80℃で1時間サンプルをインキュベートします。インキュベーション後、エタノールを除去し、好適な緩衝液30μlにDNAを再懸濁。 DNAの大幅な損失を挿入し、ベクトルの次の分離を発生し、30〜50％のLOssです。 </li><li> T4 DNAリガーゼ38を用いて、非メチル化ベクターを再ライゲーションメチル化及び非メチル化インサート。ライゲーション反応に挿入するためのベクターの4：1の比を使用してください。メーカーの指示に従ってセットアップ反応。反応のための50μlの総容量を使用してC°またはアイスバケットの上に蓋を氷上で-20℃でO / Nインキュベートします。 </li><li>連結後、好適な、市販のシリカゲル膜クリーンアップキットを用いてDNAのクリーンアップを実行します。 DNAの有意な損失は、次のライゲーション反応、DNAのおよそ30-50％の損失を生じます。 45分間、100Vで1％アガロースゲル上でのDNA実行することにより、再連結を確認し、再連結したプラスミド（ 図4D）の濃度を評価します。 </li><li>製造業者のプロトコルに従って商業トランスフェクション試薬を用いて、D54細胞にメチル化または非メチル化プラスミドをトランスフェクトします。よく0.14×10 6細胞 /で12ウェルプレート上にシードD54細胞。 </li><li> 24時間後、トランスフェクト細胞のwi（1）非methyatedのCpG-luc2プラスミド+ウミシイタケまたは他の制御ルシフェラーゼベクターまたは（2）メチル化CpG-luc2プラスミド+ウミシイタケまたは他の制御ルシフェラーゼベクターのいずれかの目等しい濃度。 </li><li>細胞は、デュアルルシフェラーゼアッセイを行う前に24時間トランスフェクトすることを許可します。製造者の指示に従ってアッセイを行います。ルミノメーターを用いて測定値を実行します。三連で各ウェルをお読みください。 </li><li> ウミシイタケまたは他の制御ルシフェラーゼ活性に対してホタルルシフェラーゼ活性の比率を計算します。非メチルホタルに制御ルシフェラーゼ活性：メチル化されたホタルを正規化対照ルシフェラーゼ活性を、非メチル化ルシフェラーゼ活性によってメチル化ルシフェラーゼ活性を分割して</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.

アストロサイトの濃縮集団は、eGFPの-S100βトランスジェニック動物27のFACSソーティングを介して取得しました。生後50日目（P50）より古い動物から単離された細胞や分子の分子の品質を低下させるには、P0〜P40老化した動物は、そのような実験のために最適です。皮質組織は、これらの実験に使用しました。 2〜6匹の動物からの皮質を一緒にプールしました。 FACSは、UAB包括的フローサ...

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.

アストロサイトの発現および転写活性を有する遺伝子特異的DNAメチル化の変化の相関<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

Research

JoVE Journal

Biology

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

7.9K ビュー.  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. 

ビデオ: 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.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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

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.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

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.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

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.

のインプリント遺伝子のDNAメチル化の定量シロイヌナズナ胚乳

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

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メチル化：重亜硫酸修飾と分析

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

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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.

遺伝子発現のDNA相互作用、ディスカバー転写レギュレータ、および新規抗腫瘍剤を同定：タンパク質を研究するために、定量的アッセイ

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.

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

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.

塩基対解像度でDNAメチル化の評価のための強化されたの減少表現重亜硫酸塩シークエンシング

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.

次世代シーケンシングによりDNAメチル化解析をターゲット

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 ...