この作品は、APM [076566/Z/05/Z]、JCK [074318]と人類遺伝学[075491/Z/04]のためのウェルカムトラストセンターへのコアの賞への補助金を通じてウェルカムトラストによって資金を供給された。

1）細胞培養<ol><li>目的の遺伝子を発現する細胞の種類を選択してください。 </li><li>両方のDNAのリスクの亜種と目的の遺伝子（図1）内で転写されたコーディングの多型のヘテロ接合体細胞株を選択してください。 </li><li>文化サプライヤーの仕様に、それぞれDNA及びRNA抽出のために2 × 10cmペトリ皿を準備応じて選択された細胞株。 </li><li>細胞が80％コンフルエントに下記の手順に従ってまたは市販のキットを使ってどちらかの収穫量の細胞への手続きを開始し、DNAとRNAを単離に到達すると。 </li></ol> 2）DNAの抽出<ol><li>料理からメディアを削除する、PBSで洗浄し、溶解液（0.6％SDS、100mMのNaCl、50mMトリス、20mMのEDTA、50μg/ mLのRNase Aを）の料理500μLに追加します。 </li><li>室温で20分間穏やかにプレートを揺らし。 </li><li> 37℃で一晩マイクロ遠心チューブやインキュベートに細胞ライセートをこすり落とす。 </li><li> 100μg/ mlの終濃度にプロテイナーゼKを追加し、37℃で一晩インキュベートする。 </li><li>フェノール - クロロホルムpHを8、繰り返し、同量のDNAを抽出し、クロロホルム/イソアミルアルコールの等量を抽出する。 </li><li> 1 / 10量の3 M NaClおよび100％エタノールを2.5倍量でDNAを沈殿させる。 </li><li> 4℃で30分間、13,000 rpmで回転して氷上で5分間のままにして、 </li><li> 70％エタノールでDNAを洗浄、空気乾燥し、TE200μlに再懸濁させることができます。 </li><li>室温で2日間に向けて出発して65℃でインキュベートする1​​0分間と。 </li><li> 4℃-20℃または長期℃に</li></ol> 3）RNA抽出<ol><li> PBSで洗い、皿からメディアを削除し、室温で10分間TRIZOLとインキュベートの1 mLを加える。 </li><li>スクレイプの細胞は、マイクロ遠心チューブに数回、転送をピペットで5分間インキュベートする。 </li><li> 4℃で10,000 rpmで10分間スピン℃に</li><li>新しいチューブに上清を移し、クロロホルム200μLを加える。 </li><li>振ると4℃10,000 rpmで10分間スピン℃に</li><li>新しいチューブに水相を移し、70％エタノールの等量を追加します。 </li><li> 1または2（ボリュームに応じて）最高速度で30秒間RNeasyカラムとスピンのソリューションを読み込みます。 </li><li>ここからRNeasyキット（キアゲン）の指示に従ってください。 </li></ol> 4）核酸試料の調製<ol><li>光度計分光光度計（Thermo Scientific社）を用いてDNAとRNAの濃度を定量化する。 </li><li>製造元の指示に従ってランダムdecamersとのSuperScript III逆転写酵素（Invitrogen）を用いてRNAの500から1000 ngからcDNAを準備します。 </li></ol> 5）PCRとプライマー伸長アッセイ<ol><li>各DNAマーカーのためのPCRプライマーの設計つのペアを、ゲノムDNAの増幅とcDNAの増幅（ 図2）のための他のペアに対して1つのペア。前方に配置し、ゲノムの混入を避けるために、異なるエクソンにcDNAの増幅用のプライマーを逆に。 100から500 bpの長さの範囲でPCR産物を得るための設計プライマー。 </li><li> 0.5 U Immolase Taqポリメラーゼ（Bioline）0.8 mMのdNTPsを、2mMのMgCl 2、0.2Mの各プライマーとcDNAまたはDNAのいずれかの20ngのを含む10μLのPCR反応を準備する。つの独立した反応で各サンプルを増幅する。 </li><li>増幅の線形位相でサイクル数を保ち、各プライマーのペア用に最適化された条件下でのPCR反応を実行します。 </li><li>エキソヌクレアーゼでPCR産物を培養することにより過剰なPCRプライマーおよびdNTPを除去する私、37でエビアルカリホスファターゼ（SAP）℃で20分間。 </li><li> 8測定は各サンプルに対して使用可能になるように、384ウェルプレートに二回、各PCR産物を見つける。 </li><li>同じプライマーがゲノムおよびcDNAの両方のPCR産物のために使用できるように、アッセイデザイン（シーケノム）ソフトウェアと拡張子のプライマーを設計します。 3ddNTPsの組み合わせ（図2）を含めて14から18塩基対と拡張ミックスの範囲内にプライマーの長さを指定します。 </li><li>メーカーの指示に従ってMassARRAYシステム（シーケノム）を使用して、プライマー伸長反応とMALDI-TOF/MS分析を行う。 </li><li>典型的なプライマー伸長反応は94℃で開始するステップ℃で2分間、5秒、52℃で5秒と72℃、94℃の40サイクルが続く5秒のために含まれています</li><li>プライマー伸長産物はSpectroCLEAN樹脂を用いて洗浄し、SpectroPOINTナノリットルディスペンサーによるマイクロアレイ（チップ）上に転写されています。 </li><li>拡張されたオリゴヌクレオチドはMALDI - TOFはSpectroREADER質量分析計を用いて定量化されています。 </li></ol> 6）統計解析<ol><li>実験の一般的な品質のためのMassARRAYのタイパーソフトウェアとのMALDI-TOF/MSの結果を確認してください。 </li><li>ピーク面積のデータを含むアレロタイプ化のレポートを抽出します。 </li><li>個々のスペクトルで計算アレル1およびアレル2のピーク面積（ピーク面積比）の比率。 </li><li>各cDNAサンプルは8測定を越え、Excelで対応する関数を使用して、ピーク面積比の平均値と標準偏差を導出する。 </li><li>ゲノムDNAのための8 measurmentsで同じ平均ピーク面積比を求めよ。 </li><li>予想される1:1の比率に偏差を評価するためにゲノムの平均の比によりcDNAの平均比率を割ります。 </li></ol> 7）実験のコントロール<ol><li>実験的なアーティファクトを除外するために、少なくとも3回の実験を繰り返す。 </li><li>追加のDNAマーカーの可能な限り、設計のアッセイ。 </li><li>順方向および逆方向の両方の鎖にアニーリング複数のPCRのプライマーペアと拡張子のプライマーを設計します。 </li><li>可能な限りのDNAマーカーのヘテロ接合体であるが表現型に関連付けられているDNAのリスクの亜種を運ぶしないネガティブコントロールの細胞株などのテスト（図1及び図3） </li></ol> 8）代表の結果アレル特異的発現解析の例は、質量分析のトレースの例を図3に示します。図は、4つの異なるテンプレートで実施4対立遺伝子特異的プライマー伸長産物の分析結果を示しています。上部のグラフは、転写されたコーディングの多型についてヘテロ両方とも二つの異なる細胞株のゲノムDNAの解析から得られた結果を表します。しかし、唯一の細胞株は、リスクのDNAの変異体（図1）のヘテロ接合体である。下のグラフは、同じ細胞株から生成されたcDNAの解析から得られた結果を表します。実験的なアーティファクトの結果として、最初のピークは常にさえゲノム分析の第2ピークよりも高いです。そのため、ゲノム解析の結果を、cDNAのデータを正規化するために使用されます。正規化した後、アレル比は細胞株で1から有​​意に異なっている（第二のピークは他のスペクトルに比べて低く表示される場所）、データはそのセルの行を示唆B.それは、細胞株では1に非常に近いしている間には大電流が流れる解析で遺伝子の発現を減少させる番目のピークで測定された対立遺伝子との相におけるDNAの変異、。セルラインBは、非常に便利な負の制御を提供します。 <img src="/files/ftp_upload/2279/2279fig1.jpg" alt="図1" /> 図1。細胞株の選択の戦略。細胞株とBのリスクのDNAバリアント（丸）のヘテロ接合体である転写コーディングマーカー（三角形）だけ細胞株に対してヘテロ接合されています。リスクバリアントの青対立遺伝子は、転写レベルが低いと（実際の機能的変異体との密接な相関関係に直接的な影響を持つため、またはどちらか）られています。 <img src="/files/ftp_upload/2279/2279fig2.jpg" alt="図2" /> 図2。対立遺伝子特異的プライマー伸長アッセイ。この図は、cDNA（左）とゲノムDNA（右）テンプレートの両方を搭載し、実験手順の作業の流れを示す。 PCRプライマーは（灰色の長方形）ヘテロ接合体をコードする多型（三角形）を増幅するように設計されています。ゲノムコンタミネーションPCRプライマーは、増幅はcDNAを鋳型に行われている異なるエクソン（水色のバー）に配置されたシーケンスをアニール避けるために。 PCR産物は、多型の隣に一つの基地をアニーリング、エクステンションのプライマーを用いて行ったプライマー伸長反応の鋳型として使用され、3つのジデオキシヌクレオチドの適切な組合せ（ddNTPsは）ターミネーター（四角）一デオキシヌクレオチド（円が）延長にそれぞれ1つまたは2つの基礎の入門書。結果として得られるプライマー伸長産物はMALDI - TOF質量分析法による分離と定量化を可能にするさまざまな質量を持っている。 DNAマーカーの青対立遺伝子に対応する製品の量は、赤対立遺伝子の相対的に低くなります。 <img src="/files/ftp_upload/2279/2279fig3.jpg" alt="図3" /> 図3。対立遺伝子特異的発現アッセイの結果。図は、分析から質量分析の結果は、ゲノムDNAと細胞株のcDNAおよび細胞株のB（図1）で実施を示す。

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	<li>Cookson, W., Liang, L., Abecasis, G., Moffatt, M., Lathrop, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+complex+disease+traits+with+global+gene+expression.">Mapping complex disease traits with global gene expression.</a> Nat Rev Genet. 10, 184-194 (2009).</li><li>Singer-Sam, J., LeBon, J. M., Dai, A., Riggs, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+quantitative+assay+for+measurement+of+allele-specific+transcripts+differing+by+a+single+nucleotide.">A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.</a> PCR Methods Appl. 1, 160-163 (1992).</li><li>Yan, H., Yuan, W., Velculescu, V. E., Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+human+gene+expression.">Allelic variation in human gene expression.</a> Science. 297, 1143-11 (2002).</li><li>Ding, C., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+expression+analysis+technique+using+competitive+PCR+and+matrix-assisted+laser+desorption+ionization+time-of-flight+MS.">A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.</a> Proc Natl Acad Sci U S A. 100, 3059-3064 (2003).</li><li>Gabriel, S., Ziaugra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+using+Sequenom+MassARRAY+7K+platform.">SNP genotyping using Sequenom MassARRAY 7K platform.</a> Curr Protoc Hum Genet Chapter. Chapter 2 (Unit 2), 12-12 (2004).</li><li>Lo, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+gene+expression+is+common+in+the+human+genome.">Allelic variation in gene expression is common in the human genome.</a> Genome Res. 13 (8), 1855-1862 (2003).</li><li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-based+discovery+of+variation+in+the+human+glutathione+S-transferase+M3+promoter+and+functional+analysis+in+a+glioma+cell+line+using+allele-specific+chromatin+immunoprecipitation.">Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.</a> Cancer Res. 65, 99-104 (2005).</li><li>Knight, J. C., Keating, B. J., Rockett, K. A., Kwiatkowski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+regulatory+polymorphisms+by+allele-specific+quantification+of+RNA+polymerase+loading.">In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.</a> Nat Genet. 33 (4), 469-475 (2003).</li><li>Pastinen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+genetic+and+epigenetic+variation+affecting+human+gene+expression.">A survey of genetic and epigenetic variation affecting human gene expression.</a> Physiol Genomics. 16 (2), 184-193 (2004).</li><li>Francks, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+77-kilobase+region+of+chromosome+6p22.2+is+associated+with+dyslexia+in+families+from+the+United+Kingdom+and+from+the+United+States.">A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States.</a> Am J Hum Genet. 75 (6), 1046-1058 (2004).</li><li>Paracchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromosome+6p22+haplotype+associated+with+dyslexia+reduces+the+expression+of+KIAA0319,+a+novel+gene+involved+in+neuronal+migration.">The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration.</a> Hum Mol Genet. 15 (10), 1659-1666 (2006).</li></ol>

利害の衝突は宣言されません。

このメソッドは、in vivoでの対立遺伝子特異的転写レベルの違いで評価することによって、遺伝子発現に疾患関連のDNA変異体の効果の評価が可能になります。このプロトコルでは相対的な転写産物量はMALDI - TOFが、そのようなのTaqMan 6,7などのアレル特異的定量を、許可する他の技術によって測定され、使用することができます。このアプローチの主要な制限は、転写されたコーディングマーカーの可用性です。原理に基づく方法はここで説明するが、多型の異なるクラスを利用して、記載されている。 haploChipアッセイ対策の対立遺伝子特異的ポリメラーゼII 8を RNAに特異的な抗体で分離したクロマチン免疫沈降された物質中に存在するであろう遺伝子の転写開始点または終了点のサイトの1 kb以内に位置するマーカーを用いて表現。鋳型がRNA 9異種の場合に別の方法として、イントロンのSNPは、使用することができます。 特定のプロトコルは、haploChipアッセイと組み合わせて、ここで説明した失読症（または障害を読み込む）するための候補遺伝子を同定するために成功裏に使用されています。最初に遺伝的関連の研究では、失読症とつの遺伝子10をスパニングされたに関連付けられているDNA配列を同定した。対立遺伝子特異的遺伝子発現アッセイは、失読症関連DNA変異の発現変動の存在下で3つの遺伝子の一方のみが観察ではなく、他の二つの11歳のことを示した。

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>PBS</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>P-4417-100TAB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>&nbsp;</td>
			<td>Biorad</td>
			<td>161-0416</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>VWR</td>
			<td>27788.366</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>E5134-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>&nbsp;</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>P2308-500MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenol: chloroform</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>77617</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>&nbsp;</td>
			<td>VWR</td>
			<td>100777C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>32221-2.5L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>&nbsp;</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy kit</td>
			<td>&nbsp;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>&nbsp;</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Immolase Taq</td>
			<td>&nbsp;</td>
			<td>Bioline</td>
			<td>BIO-21048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>DNTP100A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Exonuclease 1</td>
			<td>&nbsp;</td>
			<td>NEB</td>
			<td>M0293S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shrimp Alkaline Phosphatase</td>
			<td>&nbsp;</td>
			<td>GE Healthcare</td>
			<td>E70092Z</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroCLEAN resin</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>10053</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MassEXTEND ddNTP/dNTP mix </td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>Varies according to assay design</td>
		</tr>
		<tr>
			<td>MassEXTEND thermosequenase </td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>10052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chilled microcentrifuge</td>
			<td>&nbsp;</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>&nbsp;</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroPOINT nanolitre dispenser</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroREADER mass spectrometer</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an allele-specific gene expression assay to test the functional basis of genetic associations

一般的な複雑な形質で有意な遺伝的関連の数は絶えず増加しています。しかし、これらの団体のほとんどは、分子レベルで理解されていない。表現型上のDNA変異の効果を仲介するメカニズムの一つは、複雑な形質1の特に重要であることが示されている遺伝子発現、です。 このメソッドは、細胞のコンテキスト遺伝子発現上の特定のDNA配列の効果のテスト。原則は、疾患（リスクの亜種）2,3に関連付けられているDNA配列のコピーを運ぶ細胞を解析、遺伝子の二つの対立遺伝子に起因する転写物の相対量を測定することです。したがって、この方法で使用される細胞は二つの基本的な遺伝子型の要件を満たす必要があります：彼らは、DNAのリスクの亜種のためにとその染色体の起源（図1）に基づいて転写産物を区別できるDNAマーカー、一般的にコーディングする遺伝子多型、両方のヘテロ接合体でなければならない。 DNAのリスクの亜種とDNAマーカーは、同じ対立遺伝子頻度を持つ必要はありませんが、遺伝子マーカーの相（haplotypic）の関係を理解し​​ておく必要があります。目的の遺伝子を発現する細胞の種類を選択することも重要です。このプロトコルは、線維芽細胞から核酸を抽出するために採用された手順のことを指しますが、この方法では、主要な細胞を含む他の細胞タイプにも等しく適用可能である。 DNAとRNAを選択した細胞株から抽出され、cDNAが生成されます。 DNAとcDNAをコードするDNAマーカー4を対象とするように設計されたプライマー伸長アッセイで分析されています。プライマー伸長アッセイは、製造業者の仕様に従ってMassARRAY（シーケノム）5プラットフォームを用いて行われる。プライマー伸長産物を質量分析（MALDI-TOF/MS） - 飛行のマトリックス支援レーザー脱離/イオン化時間で分析されています。選択したマーカーがヘテロ接合体であるので、彼らはMSのプロファイル上の2つのピークが生成されます。各ピークの面積は、転写産物量に比例し、アレル比（アレル1：対立遺伝子2）生成するMassARRAYのタイパーソフトウェアの機能を測定することができる計算に。 cDNAに得られる対立遺伝子比率は対立遺伝子比が1:1の技術的なアーチファクトを補正すると予想されるゲノムDNAから測定されたものを使用して正規化されます。 1〜大幅に異なる正規化された対立遺伝子の比を有するマーカーは、表現型に関連付けられているDNA変異が遺伝子発現に影響を​​与えることを示唆し、同じセルに2つの染色体から生成された転写物の量が異なることを示している。実験的なコントロールは、結果を確認するために使用する必要があります。

Watch this Scientific Journal Video about An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations at JoVE.com

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

遺伝的関連は、多くの場合、機能レベルで解明されないままである。このメソッドは、転写されたSNPについてヘテロ接合体の細胞を分析することによって遺伝子発現の表現型に関連する遺伝子マーカーの効果を評価することを目的とする。技術は、アレル特異的なプライマー伸長産物を定量化するためにMALDI - TOF質量分析法による正確な測定が可能になります。

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

an-allele-specific-gene-expression-assay-to-test-functional-basis

Research

JoVE Journal

Biology

22.7K ビュー.  University of Oxford. 遺伝的関連は、多くの場合、機能レベルで解明されないままである。このメソッドは、転写されたSNPについてヘテロ接合体の細胞を分析することによって遺伝子発現の表現型に関連する遺伝子マーカーの効果を評価することを目的とする。技術は、アレル特異的なプライマー伸長産物を定量化するためにMALDI - TOF質量分析法による正確な測定が可能になり...

ビデオ: An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

マウス用ダーク/ライト遷移テスト

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

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

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髄電気生理学

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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

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 ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

胚形成の間に機能的な冗長性をテストするための逆遺伝学的アプローチ

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

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

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

プラナリアの再生を操作するために薬理学的および機能的遺伝学的アッセイ<emジャポニカ> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

遺伝子発現用機械の機能的アッセイのための小規模の核抽出物

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Methods Article

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ