この作品は、ヒューマン·フロンティア·サイエンス·プログラム助成RGP0024によって資金を供給された。

1。 DNAテンプレートの調製とin vitro転写 <ol><li>興味のある遺伝子はT7および/または例えばSP6転写プロモーターの下に複製されます。 </li><li> in vitro転写用テンプレートDNAは、ORFの終止コドンおよび/ ​​またはmRNAの3 &#39;末端の下流を切断し、適切な制限酵素で直線化されています。一つは、アガロースゲル電気泳動で制限酵素消化製品を実行することにより、プラスミドDNAの完全な線形化を確認する必要があります。 </li><li>線状プラスミドは 、in vitro 転写反応に使用されます。テンプレートDNAの異なる濃度は 、in vitro転写のために必要な最適なDNA濃度を識別するためにテストすることができます。一般的には、AmbionのMEGAscript高利回り転写キット（Ambion社/ライフテクノロジーズ、ニューヨーク州グランドアイランド）で、1μgの直鎖状DNA、mRNAの40から60μgを得られます。in vitro転写メーカーの指示（Ambion社/ライフテクノロジーズ、グランドIslan後に行われますD、NY）。 </li><li> in vitro転写で以下の mRNAは、製造元の指示（AmbionのMEGAscript高利回り転写キット）によると塩化リチウム沈殿により精製されています。 </li><li> mRNAの整合性は、さらに、アクリルアミドまたはアガロースゲル上で電気泳動によって確認されています。 </li></ol> （2）in vitroのセルフリー翻訳以下の手順に従ってくださいR abbit R eticulocyte溶解、RRL（プロメガ、マディソン、WI）を用いたin vitro翻訳のために： <ol><li>メーカーの指示に続くin vitro翻訳反応で 100μlを準備します。簡単に言えば、ヌクレアーゼフリーの水に2μlのアミノ酸混合物を引いたメチオニン（1mM）を、RNase阻害剤（Invitrogen社/ライフテクノロジーズ、グランドアイランド、NY）、放射性の20μCiの[35 S] -メチオニン（MPのバイオメディカルの10単位を追加ソロン、オハイオ州）、ウサギ網状赤血球ライセート（プロメガ社、マジソン、WI）の70μlの。 </li><li>目をインキュベート暖かい翻訳反応を事前に5分間30℃eの反応。 5分間30℃での翻訳反応とインキュベートするmRNAを2μgのを追加します。 </li><li>氷の上で翻訳反応を置くことによって反応を停止します。 </li></ol> 3。 in vitro翻訳反応でから新生ポリペプチドの分離<ol><li>リボソーム（ポリソーム）にバインドされた新生ポリペプチドを単離するためには、翻訳反応は、100mM KCl、10mMのMgCI 2を含む10 mM Tris-HCl緩衝液のpH 7.6の30％グリセロール4.5 mlの上に重ね、100,000×gで遠心分離し4℃で1時間TLA-110ローター（ベックマン·コールター社、ブレア、カリフォルニア州）で</li><li>上清をさらに慎重に削除されます。 </li><li>新生ポリペプチドを含有するポリソームペレットを0.5 mg / mlのリボヌクレアーゼ（Invitrogen社/ライフテクノロジーズ、ニューヨーク州グランドアイランド）とインキュベートを含む、1 mMのTris-HCl緩衝液pH 7.6の少量（10-15μL）に再懸濁している37℃で30分間で順番にペプチジル-tRNAをエステル結合の加水分解を高めるために、水酸化ナトリウムを10mMの最終濃度まで添加し、インキュベーションをさらに30分間継続されます。 </li></ol> in vitroで翻訳反応を発現することなく組み立てられたと同じ条件（記載されたように新生ペプチドの単離に続いて）の下で行われ、主要なコントロールとして機能するでしょう。密度勾配遠心分離抽出を施す前にピューロマイシンによる翻訳反応（s）の治療は、追加のコントロールとして機能することができる。 4。トリス - トリシンSDS PAGE上での新生ポリペプチドを解決する<ol><li>孤立した新生ポリペプチドは、トリス - トリシンSDS-PAGEで分離し、分析されます。ゲルにロードされた材料の量は、タンパク質の標識、タンパク質翻訳の効率や他の多くのパラメータの効率に依存しますのでご注意ください。ロードされた材料の量は、BESを可能にするために経験的に調整する必要がありますトンの可視化と個々の新生ポリペプチド鎖の分離。 </li><li>電気泳動ゲルは固定された後、真空ゲル染色を使用して、オートラジオグラフィーにかけ乾燥させた。新生ポリペプチドの分布はphosphoimagingを用いて観察しています。 </li></ol> 5。代表的な結果ウシγ-Bクリスタリンは、同様にE.のように、ウサギ網状赤血球ライセートで翻訳された大腸菌 S30抽出システム（プロメガ社、マジソン、WI）新生ポリペプチド鎖の分離に続いて、 図1は、リボソーム結合した新生鎖の分離に必要な手順を示しています。本研究での目的は、tRNAの翻訳すなわちレパートリーの環境を（（ 大腸菌 ）生物間のコドンバイアスの違いに起因するシステムを実質的哺乳類で異なる（RRL）と原核生物であることが知られて）変更すると、可能であれば、テストすることでしたmRNAに沿ってリボソームの動きを変更して、翻訳の一時停止部位の分布に影響を与える。変更リボソームの動きは翻訳中に蓄積新生ポリペプチド、並びにそれらの相対強度の大きさの分布の変化につながるはずだ。バンドの相対強度は一時停止の期間を反映しています。新生ポリペプチドは単離され、16.5％T、6％のTris-CトリシンSDS PAGE（図2）で解決されました。 γ-Bクリスタリンは20 kDaの蛋白質である。 RRLとE.非同一の長さと強度の新生ポリペプチドの観察大腸菌のシステムは、これらの2つのシステムのmRNAに沿ってリボソームの動きが異なる翻訳速度を、以下のことを示しています。たとえば、我々はE. 17.7 kDaの顕著なバンドが観察さRRLシステムの大腸菌ライセートとしない、それはE.に翻訳されるmRNAの3&#39;-末端領域（ガンマBクリスタリンのエンコーディングのC末端領域）に追加の翻訳停止部位が存在することを示唆している大腸菌システム。我々は注意して、γ-Bクリスタリン港タンデムのArgコドン（AGG 1哺乳類のシステムで頻繁にありますが、非常にまれであることが知られてゆっくりとEに変換されます53、AGA 154） 大腸菌、E.に蓄積小説新生ペプチド大腸菌のシステム（〜17.7 kDaの図2.2）は、おそらくこれらのタンデム稀なコドンで停止リボソームによって引き起こされます。全くバンドがmRNAの不在（ 図2.3）で観察されず、そのピューロマイシン治療がポリソーム（ 図2.3）からの新生鎖の大部分を削除することができることに注意してください。ピューロマイシン（&gt; 15分）による長期治療は、すべての新生鎖（データは示さず）を削除します。これは観測されたポリペプチド産物ではなくポリソームでcosedimenting mRNPsよりリボソーム関連した新生鎖であることを確認します。 上記のように、コドンバイアスは、生物だけでなく、組織固有のものです。代表的な例は、ここで説明する、明らかにするtRNA /コドンの翻訳すなわちレパートリーの環境を変更することを示します。バイアスは、mRNAに沿ってリボソームの変化した動きにつながる可能性があります。明らかに、この単純なテクニックだけでmRNAに沿って並進一時停止部位の同定を可能にするだけでなく、異なるシステム間での翻訳停止部位の迅速な比較を可能にすることはできません。 <img alt="図1" src="/files/ftp_upload/4026/4026fig1.jpg" /> 図1。リボソーム結合した新生ポリペプチドを単離に必要な手順を説明する回路図は、ウシ γ-BクリスタリンのORF配列（528塩基対）がpBSKSで下流にT7プロモーターのクローニングした+。 in vitro転写用テンプレートDNAをEcoRIで処理することにより線形化されました。線形化されたテンプレートは、in vitro転写のために使用されていました。 DNAテンプレートのT7プロモーター下流に、γ-Bクリスタリン遺伝子を転写するT7 RNAポリメラーゼによって認識されている。 mRNAは、塩化リチウム沈澱法を用いて精製されています。精製したmRNAは、in vitro翻訳のために使用されます。以下インキュベーション、翻訳反応は、30％グリセロール溶液の上に重層し、4℃で1時間遠心分離されるリボソーム結合した新生ポリペプチドを単離するための100,000 x gで°C。 <img alt="図2" src="/files/ftp_upload/4026/4026fig2.jpg" /> 図2。ウサギ網状赤血球ライセート（RRL）とE.に翻訳ウシγ-Bクリスタリンの新生ポリペプチドの分離大腸菌ライセートを 2μgのmRNAは、RRL または E を含有する100μlの反応混合した5分20μCiの[35 S] -メチオニン、30℃でインキュベートしたメーカーの指示に従って大腸菌 S30抽出システム（Promega、Madison、WI）を。以下のインキュベーションは、翻訳反応は、100mM KCl、10mMのMgCI 2を含む10 mM Tris-HCl緩衝液のpH 7.6で30％グリセロール4.5 mlの上に階層化し、で4℃、100,000×gで1時間遠心分離したTLA-110ローター（ベックマン·コールター社、ブレア、カリフォルニア州）。隔離されたポリリボソームペレットresuでした（Invitrogen社/ライフテクノロジーズ、ニューヨーク州グランドアイランド）のNaOH（などのプロトコルのセクションで説明）で処理、続いては0.5 mg / mlのリボヌクレアーゼを含む、1 mMのトリス-HCl pH 7.6の20μlにspended。新生ポリペプチドは、16.5パーセントT、6％のTris-CトリシンSDS PAGEゲル上で解決されました。ゲルを乾燥し、オートラジオグラフィー用に公開されました。暴露後のゲルは、GEヘルスケアの台風イメージングスキャナを使用してスキャンしました。 図2.1は、翻訳反応は、2つの異なるシステム（RRLと大腸菌 ）で行われた後、分離し、解決する新生ポリペプチドゲルを示しています。 図2.2は、ゲルがイメージクワントTLにより解析した、v2005ソフトウェア、2つのシステムに蓄積新生ポリペプチドの分子量と強度を簡単に分析し、比較することができることを示すための例としてここで使用する図2.3は、コントロールの2つのセットを示しています。翻訳後、SDS PAGEで分離し、解決する新生ポリペプチドを反応が組み立てられた反応は、遠心分離に供した前に、mRNAおよび/または後にすることなく翻訳反応は、ピューロマイシン（5分、最終濃度1 mM）で処理した。

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利害の衝突が宣言されません。

再現可能な結果を得るためには、in vitro転写·翻訳反応に使用されるコンポーネントの品質と濃度が非常に重要です。現在の研究では、慎重に処理した場合、再現性の高いデータを提供する市販のキット及び抽出物を使用しています。ただし、必要であれば、翻訳、有能な抽出物は、自分の選択した細胞から調製することができる。 mRNAの品質は翻訳に影響を与えることがないの?...

<table border="1"><tbody><tr><td> 試薬/キットの名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> MEGAscript T7高収率転写キット</td><td> Ambionの</td><td> AM1333 </td></tr><tr><td>リボヌクレアーゼ阻害剤</td><td>インビトロジェン</td><td> 15518012 </td></tr><tr><td>トランス[35 S] -ラベル</td><td> MPのバイオメディカル</td><td> 0151006 </td></tr><tr><td>リボヌクレアーゼ</td><td>インビトロジェン</td><td> 12091 </td></tr><tr><td>ウサギ網状赤血球ライセートシステム、ヌクレアーゼは、治療</td><td>プロメガ</td><td> L4960 </td></tr><tr><td>リニアテンプレートの大腸菌 S30抽出システム</td><td>プロメガ</td><td> L1030 </td></tr><tr><td>遠心分離</td><td>ベックマン·コールター</td><td>オプティマTLX超遠心</td></tr><tr><td>貯蔵燐光体オートラジオグラフY </td><td> GEヘルスケア</td><td>台風9410可変モードイメージャ</td></tr><tr><td>新生ポリペプチド分析用ソフトウェア</td><td> GEヘルスケア</td><td>画像クワントTL、v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

翻訳伸長率は、非均一である。二次構造、コドン使用頻度をmRNAと1を参照して 、関連するタンパク質はレビューのためにメッセージにリボソームの動きを変更することがmRNAを。しかし、現在広く同義コドンの使用率が不均一な翻訳伸長率が1の主な原因であることを受け入れている。同義コドンは、同一の周波数で使用されていません。バイアスは、他の2よりも頻繁に使用されるいくつかのコドンを同義コドンの使用に存在しています。コドンバイアスは、生物と同様に2,3 -特定の組織である。また、コドンの使用頻度は、同族のtRNA 4の濃度に直接比例します。したがって、頻繁に使用されるコドンは、さらに頻繁なコドンが速くまれなものよりも翻訳されることを意味し、対応するtRNAの高い多数を持っています。したがって、使用頻度の低いコドン（潜在的な休止サイト）に富んでmRNA上の領域は、原則としてミサにリボソームの動きが遅くなりますそれぞれのサイズ5-8の新生ペプチドのGEの原因と蓄積。これらのポーズのサイトはレビューのために機能的なタンパク質の発現に及ぼす影響は、mRNAの安定性とタンパク質の折りたたみを持っている9を参照することができます。確かに、そのようなポーズサイトの緩和は、mRNAにリボソームの動きを変更することができ、その後、共同翻訳（ インビボ ）タンパク質のフォールディング1,7,10,11の効率に影響を与える可能性があることが示された。最終的には翻訳伸長中のmRNAに沿ってリボソームの移動に関するコドン使用頻度/ tRNAのコンテンツの影響に包括的な洞察を得ることが不可欠であるタンパク質合成のプロセスに結合されている細胞で、 生体内でのタンパク質の折り畳み過程を理解するために、 。 ここでは、様々な無細胞系6-8に翻訳与えられたmRNAの主要な翻訳停止部位を見つけるために使用することができる簡単なテクニックについて説明します。この手順は、新生ポリペプチドaccumulatiの分離に基づいています標的mRNAのin vitro翻訳中のリボソーム上でNG。根拠は、低周波のコドンでは、対応するサイズの新生ペプチドの増加量のリボソーム結果の滞留時間の増加が。in vitroで転写されたmRNA には放射性標識アミノ酸の存在下でin vitroでの翻訳反応に使用されていることです。新生鎖の検出を可能にします。リボソーム結合した新生ポリペプチド複合体を単離するために翻訳反応は、遠心分離に続いて、30％グリセロール溶液の上に階層化されています。ポリソームペレットの新生ポリペプチドはさらにリボヌクレアーゼで処理し、SDS PAGEで解決されます。このテクニックは、潜在的に任意のタンパク質で使用され、リボソームのmRNAに沿って動き、主要な休止部位の検出の分析を可能にすることができます。さらに、このプロトコルは、リボソームの動きを変えることができるので、潜在的にも目を変えることができる要素や条件を研究するために適応させることができます電子機能/タンパク質のコンフォメーション。

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

mRNA上の翻訳停止部位を同定する技術が記載されている。この手順は、変性ゲル電気泳動を用いた初期のチェーンの大きさの分析に続いて標的mRNAのin vitro翻訳、イン時にリボソーム上で蓄積新生ポリペプチドの分離に基づいています。

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

isolation-ribosome-bound-nascent-polypeptides-vitro-to-identify

Research

JoVE Journal

Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K ビュー.  Cleveland State University. mRNA上の翻訳停止部位を同定する技術が記載されている。この手順は、変性ゲル電気泳動を用いた初期のチェーンの大きさの分析に続いて標的mRNAのin vitro翻訳、イン時にリボソーム上で蓄積新生ポリペプチドの分離に基づいています。

ビデオ: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR -クリップ - RNA結合タンパク質のトランスクリプトーム全体の結合部位を同定する方法

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated遺伝子の潜在的な規制モチーフを特定するためにスコープの使用

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

ヒト細胞におけるタンパク質発現の誘導のために修飾されたmRNAのインビトロ合成における

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

の酸化ストレス抵抗を測定<em&gt;線虫（Caenorhabditis elegans）</em&gt; 96ウェルのマイクロタイタープレート中

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

BrdUでのChIP-スロット西洋技術を用いて哺乳動物細胞中で新生DNAに結合したタンパク質の検討

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

遺伝子を使用するプロトコル設定トランスレーショナルリサーチの適切な動物モデルを識別するために濃縮分析

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

体外相撲 E3 リガーゼ活性を研究する sumo 化アッセイ

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...