私たちは、透過型電子顕微鏡で助けステファンウィルキンスに感謝。この作品は、健康グラントR01AG023731の国立研究所によってサポートされていました。

これまでの研究では、Mgm101の最初のアミノ末端22残基がミトコンドリア19へのインポート後に切断されることが示されている。 大腸菌における発現のために、最初の22コドンを欠くMGM101オープンリーディングフレームをPCRで増幅された発現ベクター物pMAL-C2Eの修正版でマルトース結合タンパク質（MBP）をコードする雄配列の下流に配置された。これは、プレシジョンプロテアーゼ（ 図3）のための切断部位を含むリンカーとMBP-Mgm101融合を生成します。プラスミドは、第E.を選択することにより構成されているIPTGおよびXgal白/青選択せずに形質転換体。得られたプラスミド物pMAL-C2E-MGM101その後、E.に導入され、アンピシリンとクロラムフェニコール耐性コロニーを選択することにより、 大腸菌株BL21-CodonPlus（DE3）-RIL。 1。表現、誘導、細胞溶解とのDNase I処理<ol><li> INOCグルコース（0.2％）、アンピシリン（100μg/mlの/ ml）およびクロラムフェニコール（50μgの/ ml）を添加したLB培地10ml（1％バクトトリプトン、0.5％酵母エキス、1％NaCl）中に新鮮な形質転換体をulate。 200 rpmで振とうしながら37℃CO / Nでインキュベートする。 </li><li>上記のように添加したLB培地を2リットルに10ミリリットルの前培養に接種。 OD 600が 0.5に達するまで振とうしながら37℃で細胞を成長させる。 </li><li> 0.3mMの最終濃度イソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を添加することによりMBP-Mgm101融合タンパク質の発現を誘導する。 5時間30℃で振とうしながら細胞を育てる。 </li><li>ベックマンJA-10ローター（5,500×gで、4℃、10分）を使用して、遠心分離により細胞を回収。上清を廃棄した後に、プロテアーゼ阻害剤を含む溶解緩衝液（20mMのトリス-HCl、pHが7.4および1mM EDTA、pH7.4）中（25μMロイペプチン、1μMのペプスタチン及び1mM PMSF）30ml中に細胞ペレットを再懸濁。 </li><li> 20氷上で細胞懸濁液を超音波処理秒超音波プロセッサ（熱機関;モデルW-385）を用いて50％のデューティサイクルで、30秒間、氷上で冷却し、そして2×繰り返すことができる。 </li><li> 2 mg / mlのDNアーゼで1株を1 mlを加える。 2時間4℃で細胞溶解物を揺する。 </li><li> 500mMの最終濃度のNaClを調整します。 </li><li> 30分間、4℃で10,000×gで遠心分離することにより細胞の破片を取り除きます。 </li></ol> 2。アミロースアフ​​ィニティークロマトグラフィーによる精製<ol><li>溶解緩衝液中のアミロース樹​​脂を1.5ml（50％スラリー）を平衡化する。細胞ライセートに平衡化アミロース樹​​脂を追加します。 4℃のCO / Nで優しく揺らし</li><li>寒い部屋に設置エコノカラムクロマトグラフィーカラムにライセート樹脂ミックスをロードします。非結合タンパク質は重力によって列を渡すことができます。 </li><li>洗浄バッファーI（; 400のNaCl、1mMのEDTA、pHが7.4、および0.2 mMのPMSF、20mMのトリス-HCl、pH7.4）で300mlでアミロース樹​​脂を洗ってください。 </li><li>洗浄バッファーII（20mMのT 150mlで洗浄を繰り返し立上り - 塩酸、pHが7.4、NaClが200 mM、1 mMのEDTA、pHは7.4であり、0.2 mMのPMSF）。 </li><li>カラムに、4℃で10分間インキュベートする、収集溶出緩衝液5mlの（; 200mMのNaClを、図1 mMのEDTA、pHが7.4および0.2 mMのPMSF、10mMのマルトース20mMのトリス-HCl、pHが7.4）を追加もっと回溶出を繰り返し。 </li><li>溶出液を組み合わせ、クマシー染色（ 図4）に続いて、SDS-PAGEゲル上で溶出液のアリコートをロードすることにより、MBP-Mgm101の収率および純度を決定します。 </li></ol> 3。プレシジョンプロテアーゼ切断及び陽イオン交換クロマトグラフィー<ol><li> MBP-Mgm101溶出液にプレシジョンプロテアーゼの50単位を追加します。 4℃での切断を行うCO / Nし、透析緩衝液に対して透析中継続することができ（20mMのトリス-HCl、pH7.4の、100mMのNaCl、2mMのDTT、1mMのEDTA、pHは7.4であり、0.2 mMのPMSF） </li><li> SDS-PAGEゲル（ 図5A）の切断の効率を確認してください。 </li><li>複数の注入によって切断MBP-Mgm101を読み込むバイオスケールミニマクロプレップハイSカートリッジのイオンは、Bio-Rad社の生物学的DuoFlow FPLCシステムに接続。 </li><li> 300mMの、5mMの工程の塩勾配を適用することによって、陽イオン交換クロマトグラフィーを開始し、500mMの、混合液（5 mMの塩化ナトリウムによって作成され750mMのNaClを、1,000 mMの、10mMのリン酸ナトリウム、pH7.2の図1 mMのPMSF）とバッファーB（1MのNaCl、10mMのリン酸ナトリウム、pHは7.2、1mMのPMSF）。 0.5ml /分で、流量を設定してください。 Mgm101画分を回収し、SDS-PAGEゲル（ 図5B）上のタンパク質の純度を確認してください。 </li></ol> 4。サイズ排除クロマトグラフィー<ol><li>陽イオン交換クロマトグラフィーからのタンパク質画分を兼ね備えています。 GF平衡緩衝液（; 150mMのNaCl、1mMのEDTA、pH7.4の、5mM 2 - メルカプトエタノール、0.2mMのPMSFを20mM MOPS、pH7.0）中でのタンパク質の透析。 </li><li> 4℃で3,000 xgで回転させて〜1ミリリットルの容積を減らすためにビバスピン15Rの限外ろ過スピンカラムにタンパク質を集中</li><li> Mgm101 OのロードNAはクロマトグラフィバッファー（; 150mMのNaCl、5mMのβ-メルカプトエタノール、1mMのEDTA、pH7.4の、0.2 mMのPMSFを20mM MOPS、pH7.0）で平衡化したスーパーロース6プレップグレードカラムをキャリブレーション。 </li><li> 0.5ml /分の流速でクロマトグラフィーを実行する緩衝液を用いてサイズ排除を開始する。 </li><li>精製Mgm101ピークの画分を集め。タンパク質の最終品質をチェックするための12％SDS-PAGE上の画分のアリコートをロードします。 </li><li>ビバスピン6タンパク質を集中し、その後速やかに-80℃で液体窒素とストアでサンプルを凍結℃に</li><li>一本鎖DNA結合アッセイ（ 図8）および透過型電子顕微鏡（ 図9）による構造可視化のための6-12カ月以内にMgm101サンプルを使用。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

これは、Eから安定した、ネイティブ換えMgm101タンパク質を産生するために課題となっている細菌細胞におけるその不溶性が原因の可能性大腸菌 。本稿では、MBP融合戦略はタンパク質が適度に高いレベルで発現されることができることを示している。ネガティブ染色透過型電子顕微鏡およびサイズ排除クロマトグラフィーを用いて、我々は以前にMBP-融合タンパク質は、インビ...

相同組換えは、二本鎖切断（DSBの）と鎖間架橋の修復のための、および縮小複製フォーク1からDNA複製の再開始のために重要である。従来の相同組換えでは、中央の反応は、原核生物においてRecAタンパク質、及びRad51の1-3および真核生物におけるDMC1含むATP依存性リコンビナーゼにより触媒される。これらのリコンビナーゼは、二本鎖DNAテンプレート（ 図1、左パネル）4-7内の相同性検索と鎖侵入を開始するために不可欠である一本鎖DNA上で核タンパク質フィラメントを形成。従来方式に加えて、相同組換えはまたRecA/Rad51-independent方法（ 図1、右パネル）で行うことができる。例えば、酵母RAD52とRad59タンパク質は直接二本鎖DNA切断の切除によって公開され、相補鎖DNA鎖のアニーリングを触媒することができます。歌うと呼ばれるこの再結合過程、ル鎖がアニーリング、一般的に相同な二本鎖DNAテンプレートとのペアリングは含まれません。アニール後、異種尾はエキソヌクレアーゼとニックゲノム継続8-10を復元するために連結されることにより除去される。一本鎖アニーリング機構により修復は、しばしば直接繰り返さ地域間のゲノム配列の欠失を伴っている。 RAD52はバクテリオ11の間で普及している組換えタンパク質の多様なグループに属しています。これらのタンパク質はまた、相同な一本鎖DNA分子のアニーリングを促進するそれらの活性に基づいて、一本鎖アニーリングタンパク質（SSAPs）として知られている。最高の特徴バクテリオファージSSAPsはRedβとラクトコッカスファージul36からプロファージRACおよびサックタンパク質からバクテリオファージλとP22、RecTをからErfにある。類似性が事実上undetectですがSSAPsは、構造的、典型的なβ-β-β-α倍によって特徴付けられる彼らの主要なシーケンスでできる。 10彼らは、すべてのフォーム大ホモオリゴマーリング- 体外 12-14 で 14回対称。この特徴的な高次構造、組織の機能的な意味はよく理解されていない。 私たちは、ミトコンドリアゲノムに相同組換えのメカニズムを理解することに興味を持っています。我々は以前に、サッカロミセス·セレビシエ 15内のmtDNAの維持に必須であるMGM101遺伝子を同定した。MGM101は 、その後、ミトコンドリア核様体と関連することが見出され、DNA損傷剤に対するミトコンドリアの許容16に必要とされる。しかし、Mgm101の研究は、組換えMgm101を生産する難しさによって10年で戻って開催されています。我々は最近、E.から大量に可溶Mgm101を生産することに成功しましたMBP融合戦略を使用して大腸菌 。これは、Mgm101株式を発揮することができましたタンパク質17,18のRAD52-家族と生化学的および構造的類似性。本報告では、三段階精製法は均質Mgm101for生化学的および構造解析します（ 図2）を生成し、これに記載されています。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Expression vector pMAL-c2E</td>
			<td>New England Biolabs</td>
			<td>#N8066S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PreScission Protease</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#27-0843-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus(DE3)-RIL cells</td>
			<td>Strategene</td>
			<td>#230245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche Applied Science</td>
			<td>#11034626001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Roche Applied Science</td>
			<td>#11359053001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche Applied Science</td>
			<td>#10837091001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>#DN25-1G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roche Applied Science</td>
			<td>#11411446001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>#E8021L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Econo-Column chromatography column</td>
			<td>BIO-RAD</td>
			<td>#7372512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td>
			<td>BIO-RAD</td>
			<td>#732-4130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS15RH02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 prep grade column</td>
			<td>Amersham Bioscirnces</td>
			<td>#17-0489-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS0611</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

MGM101遺伝子はミトコンドリアDNAの維持におけるその役割のために20年前に同定された。いくつかのグループからの研究がMgm101タンパク質は、ミトコンドリアDNAの組換え修復に関与することが示唆されている。最近の研究は、Mgm101がRAD52型組換えタンパク質ファミリーに関連することが示されている。これらのタンパク質は、大きなオリゴマーの環を形成し、相同な一本鎖DNA分子のアニーリングを促進する。しかし、Mgm101の特徴は、組換えタンパク質を産生することが困難によって妨げられてきた。ここでは、組換えMgm101の調製のための信頼性のある手順を説明する。マルトース結合タンパク質（MBP）タグ付きMgm101は、第大腸菌で発現される。融合タンパク質は、最初アミロースアフ​​ィニティークロマトグラフィーにより精製する。タンパク質分解切断によって放出された後、Mgm101は、陽イオン交換クロマトグラフィーによってMBPから分離される。単分散Mgm101その後、得られたサイズ排除クロマトグラフィーによる。細菌培養のリットルあたりMgm101の〜0.87ミリグラムの収量は日常的に得ることができる。換えMgm101は、DNAの最小の汚染されています。調製したサンプルが正常にMgm101の、生化学的構造及び単粒子像分析のために使用される。このプロトコルは、ミスフォールドおよび細菌細胞に対して毒性することができる他の大きなオリゴマーDNA結合タンパク質の調製のために使用することができる。

Mgm101はミトコンドリアにおけるRAD52関連の組換えタンパク質である。 RAD52が広くミトコンドリアDNA組換え（ 図1）におけるその役割のために研究されてきた。組換えMgm101は、3段階の手順（ 図2）により調製することができる。これは、Mgm101が可溶型で発現することができ、その後、タンパク質分解的切断（ 図3）により、タグから放出MBP-タグ付け戦略の?...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

酵母ミトコンドリア核様体タンパク質、Mgm101は、大きなオリゴマーの環を形成するRAD52型組換えタンパク質である。プロトコルは、陽イオン交換およびサイズ​​排除クロマトグラフィーと連結マルトース結合タンパク質（MBP）タギング方式を使用して可溶性組換えMgm101を調製するために記載されている。

MBPベースタギング戦略によってMgm101組換えタンパク質の調製

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

preparation-mgm101-recombination-protein-mbp-based-tagging

Research

JoVE Journal

Biology

12.0K ビュー.  State University of New York Upstate Medical University. 酵母ミトコンドリア核様体タンパク質、Mgm101は、大きなオリゴマーの環を形成するRAD52型組換えタンパク質である。プロトコルは、陽イオン交換およびサイズ​​排除クロマトグラフィーと連結マルトース結合タンパク質（MBP）タギング方式を使用して可溶性組換えMgm101を調製するために記載されている。

ビデオ: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

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 GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

唾液腺器官培養の遺伝子改変と再結合

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

における相同組換えコンストラクトを遺伝子改変<em&gt;ショウジョウバエ</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

ランダムMicroseedマトリックススクリーニングによってタンパク質結晶化の成功率を向上させる

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4&#039;,6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

緑色蛍光タンパク質タグと 4', 6-Diamidino-2-phenylindole線虫の染色によるタンパク質細胞内局在を検出

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

フローサイトメトリーによる生体内におけるタンパク質自己集合体の検出と特性特性

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...