我々はpTSO2を構築するための純新王に感謝：：GUSと図1の写真、デトレフワイゲル有益なコメントをGUS -フールプルーフプロトコル、Paja Sijacic、とコートニーホランダーを共有するための。 Z. Lの研究室の研究は米国国立科学財団（IOB0616096とMCB0744752）によってサポートされています。 ZLは部分的にメリーランド州農業試験場の大学によってサポートされています。

I.フローラルディップシロイヌナズナの形質転換<ol><li>変換の前に約1ヶ月、ポット当たり約10〜15工場で4〜8ポット（5インチ平方のポット）にシロイヌナズナの種子をまく。正常な受精能を持つ植物のために、プラスミド構築あたりの植物の4鉢で十分です。一低受精率と変異体シロイヌナズナを変換する必要がある場合、それに応じてポットの数を増やす。 </li><li>植物は標準条件（16時間明、22℃で暗所8時間）で栽培されています。植物が短い日またはより低い温度下で栽培されている場合、それらは変換の準備をして花と長い時間がかかるようになります。良い植物の世話は、成功した変換のために不可欠な健全な植物を、保証されます。 </li><li>植物は、単にボルトで固定し、花に始めているときに。彼らは今、変革のための準備が整いました。形質転換効率、変換前の3-4日を増やすために、可及的速やかに多くの二次シュート形成を促進するためにボルトで固定しているとして、主花序の枝を切り落とす。植物形質転換の前日に水。これは土が花、浸漬の間にあまり浸透メディアを吸収しないことが保証されます。 </li><li> アグロバクテリウムツメファシエンス菌株GV3101はpTSO2を運んで：（GUS、50μg/ mlのカナマイシン：pTSO2の場合）：2日前の変換に、適切な抗生物質を含む40ミリリットルLBに接種する：GUSコンストラクト 。 pTSO2：：GUSはカナマイシン3に抵抗性を付与するNPTII遺伝子を有するpBIN20ベクターにクローン化されています。 28〜30℃の振盪インキュベーターで一晩成長</li><li>翌日、50μg/ mlのカナマイシンを含むLBの400ミリリットルに一晩培養の転送8ミリリットル。 28〜30℃の振盪インキュベーターで一晩成長</li><li>変革の日に、 アグロバクテリウム培養のOD 600が約0.8（0.5 TO1の間にOD 600が許容される）に達すると、5000rpmで8分間でアグロバクテリウム一晩培養をスピンダウン。 </li><li>浸透培地1 L（リットル）（10mMのMgCl 2を 、5％スクロース、0.44mM 6 -ベンジルアデニン（BA）、0.3％シルウェットL - 77、1X Gamborgのビタミン溶液とオートクレーブ水）にアグロバクテリウムペレットを再懸濁する。培地はオートクレーブする必要がありますが、新鮮させる必要がありますされません。料理（8など&quot;X15&quot;X2&quot;パイレックスガラス皿）に1 L アグロバクテリウムを含有する浸透の培地を注ぐ。 </li><li>ポットを反転させる（植物が落ちることはありません）と浸透溶液中に植物のすべての地上部を浸し、一度に5分ワンポットのために保持する。植物のすべての地上部が浸透媒体に浸漬されていますが、土壌は土壌に真菌の増殖の可能性を減らすために浸透のメディアのいずれかを吸収させることは避けてください。 </li><li>浸漬した後、トレイにペーパータオルのいくつかの層に彼らの側にあるすべての浸漬ポットを置きます。ペーパータオルは、浸潤のメディアのアクセスの量を浴びる。高湿度を確保するためにプラスチック製のラップとトレイをカバーしています。植物の成長室にトレイを置きます。 </li><li>次の日に、直立ラップと紙タオル、と場所の鉢を削除します。四から五日以上の水、これらの植物をしないでください。その後、彼らは種を設定するまで、通常の植物を維持し、大量の種子を収集する。 </li><li> 4グラムのクールな寒天、オートクレーブ、およびを追加、1M NaOHで5.8に水500ml、pH値で溶解、MS培地は、50μg/ mlのカナマイシン（重量2.2グラムムラシゲを含有し、ビタミンなしで基礎塩をスクーグとプレート（150x15mmペトリ皿を）注ぐ50μg/ mlの最終濃度）にカナマイシンを加える。必要とされるまでプレートを4℃に保管されています。 </li><li>抗生物質耐性トランスジェニック植物のために選択するためには、最小で20,000種をスクリーニングする必要があります。 0.1グラムは約5000種です。重さと種子の量を見積もる。 </li><li> 30秒間70％エタノールで15 mlのFalconチューブ中でそれらを洗うことで種子を滅菌する。一度滅菌水で種を洗ってください。市販の5％の漂白剤（クロロックス）の1:10希釈液を含む溶液中で30秒間の種子を浸す。滅菌水で3〜6倍と種子をすすぎます。無菌フードでは、各MS（カナマイシン）プレート上にピペット約5000種が、汚染を避けるために細孔3Mテープでプレートをシールし、プレート上に均一に種を広げる。 </li><li> 4でプレートをインキュベート° Cで3〜4日間暗所で、その後、チャンバー（同じ植物の成長チャンバーかもしれない）にプレートを転送する。約14日後、トランスジェニック実生は緑色のままですが、非トランスジェニックものは薄いと死んで回っている。減速は、培地から根を引き抜いて、土壌にそれらを配置することにより土壌へのトランスジェニック苗を移す。 </li></ol> II。調べるpTSO2：：GUSの発現パターン<ol><li>収穫pTSO2：：GUSトランスジェニック苗やガラスシンチレーションバイアルやマイクロチューブに冷90％アセトン中に置いてくださいそのは、氷上で保存されています。いずれかの大量のサンプルを分析する場合ポリスチレンマイクロタイタープレート（ではないポリプロピレン）を使用することもできます。 </li><li>すべてのサンプルが採取された後、20分間室温でバイアルを置きます。この時間の間にX - Gluc（0.2％トリトンX - 100、50mmのNaHPO 4緩衝液pH7.2、2mMのフェロシアン化カリウム、2mmのフェリシアン化カリウム）と氷上に置きますなく新鮮な染色バッファーを構成しています。 </li><li>試料からアセトンを除去し、染色バッファーを追加します。 </li><li> X - Gluc染色液（0.2％トリトンX - 100、50mmのNaHPO 4緩衝液pH7.2、2mmのフェロシアン化カリウム、2mmのフェリシアン化カリウムの2mm x - gluc）を構成します。サンプルから染色バッファーを取り除き、試料にX - Glucを含む染色バッファーを追加します。 </li><li> 15〜20分間真空下で試料を浸潤する。徐々に真空を解放し、すべてのサンプルは、染色液の表面の下に沈むことを確認してください。必要に応じて真空が解放された後、すべてのサンプルのシンクまで浸潤を繰り返します。 </li><li> 37インキュベートサンプル℃でX - Glucと染色バッファーで一晩。 </li><li>インキュベーターからサンプルを取り出して、染色バッファーを除去する。各変更室温で30分間連続するエタノールシリーズ（20％、35％、50％エタノール）でサンプルを洗浄する。 </li><li>室温で30分間FAA固定液（50％エタノール、5％のホルムアルデヒド、10％酢酸、残りの水）で組織を修正。 FAAを削除し、70％エタノールを加える。この時点で組織を可視化し、デジタルカメラを搭載した解剖顕微鏡下で撮影することができます。 TSO2が （図1）に発現されている場所濃い青色を示します。また、組織を° C後で表示するために4℃で保存することができます。 <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="図1" /> 図1 pTSO2：。：トランスジェニック実生におけるGUS発現（）、側根（B）、および根端（C）。 TSO2プロモーターが若いと割って組織中の高活性であることに注意してください。 （C）に示すように散発的な発現パターンは、特定の細胞周期の段階で発現する遺伝子の典型です。 </li></ol>代表的な結果正しく行われると、形質転換効率は約0.1から0.2パーセントにする必要があります。別の言葉で、人はすべての5000種をスクリーニングすることにより、50〜10トランスジェニック実生を得る必要があります。平均で、約100トランスジェニック系統は、野生型植物の4鉢を変換することによって得ることができます。 ：pTSO2を含有するトランスジェニック実生の場合：GUSコンストラクト3、GUS活性を反映して濃い青の色は若い葉、茎頂、根端、及び側根の原基（図1）を含めて積極的に分裂細胞に含まれています。不均一な散発的なパターンは、細胞周期段階特異的発現の特徴です。

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

利害の衝突は宣言されません。

形質転換の効率は以下に説明されているさまざまな要素によって決定されます。 <ol><li>植物の健康と自分の年齢が最も重要です。多数の未熟な花芽を生産する約1ヵ月の古い植物は理想的です。変換する前に二次シュート形成3-4日奨励するために、主枝をオフにトリミング形質転換効率を向上します。古い植物は、形質転換することではなく、より低いレートで上昇を与える。 </li><li>植物?...

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		<th>Comment</th>
		</tr>
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<td>6-benzyladenine</td>
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<td>Gamborg's vitamin solution 1000X</td>
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<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
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floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

生物に外来遺伝子を導入する能力は、現代生物学とバイオテクノロジーの基盤となります。モデルの顕花植物の<em&gt;シロイヌナズナ</em&gt;、フローラルディップ変換法<sup&gt; 1月2日</sup&gt;は、その単純さ、効率、および低コストのすべての以前のメソッドを置き換えています。特に、若い開花の芽<em&gt;シロイヌナズナ</em&gt;植物はの溶液に浸漬されています<em&gt;アグロバクテリウムツメファシエンス</em&gt;特定のプラスミド構築物を運ぶ。浸漬した後、植物が正常な成長と収量の種子、外来遺伝子で形質転換され、抗生物質を含む培地上でのために選択できるのわずかな割合に戻されます。著しく促進このフローラルディップ法<em&gt;シロイヌナズナ</em&gt;研究と植物の遺伝子機能の理解に大きく貢献した。本研究では、我々は、レポーター遺伝子を変換するフローラルディップ法を使用してください<em&gt;β-グルクロニダーゼ（GUS）</em&gt;、の制御下に<em&gt; TSO2</em&gt;プロモーター。<em&gt; TSO2</em&gt;、リボヌクレオチド還元酵素（RNR）小サブユニットをコードする<sup&gt; 3</sup&gt;、細胞周期のS期にdNDP生合成に不可欠な細胞周期調節遺伝子である。の検討<em&gt; GUS</emトランスジェニックの&gt;表現<em&gt;シロイヌナズナ</em&gt;苗がいることを示しています<em&gt; TSO2</em&gt;活発に分裂組織で発現される。報告された実験方法と材料は容易に高校や大学レベルでの研究のためだけでなく、教育のためだけでなく、適合させることができます。

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

この記事はのフローラルディップ法を示しています。<em&gt;アグロバクテリウムツメファシエンス</em&gt;媒介形質転換の<em&gt;シロイヌナズナ</em&gt;。細胞周期調節プロモーター - レポーターを導入することにより、<em&gt; pTSO2：：β-グルクロニダーゼ（GUS）</em&gt;、へ<em&gt;シロイヌナズナ</em&gt;、我々は1つがトランスジェニック実生におけるGUSレポーターの発現を検出する方法を示しています。

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

Research

JoVE Journal

Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

36.1K ビュー.  University of Maryland College Park. この記事はのフローラルディップ法を示しています。アグロバクテリウムツメファシエンス媒介形質転換のシロイヌナズナ。細胞周期調節プロモーター - レポーターを導入することにより、 pTSO2：：β-グルクロニダーゼ（GUS）、へシロイヌナズナ、我々は1つがトランスジェニック実生におけるGUSレポーターの?...

ビデオ: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

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 ...

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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 ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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 ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

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でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ

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

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

から初期段階の胚の効率的かつ迅速な単離

の組織化学的染色

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析