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1。子のう殻を作り出す<ol><li> Fの肥沃なひずみで6.0 cmの直径のペトリ皿の中心接種ニンジン寒天10 graminearum種 （PH-1を推奨）。 </li><li>場所は、室温（18-24℃）で明るい蛍光灯の下で料理を接種し、菌糸体は、ペトリ皿（3-5日）の外側の端に達するまで成長することができます。市販の家庭用蛍光灯では、子実体の形成と放出を刺激するために正常に動作します。 </li><li>穏やかに滅菌爪楊枝で菌糸を削除します。 </li><li>滅菌ガラス又は滅菌hockeystickガラスロッドの丸みを帯びた端と表面に1.0ミリリットル2.5パーセントのTween 60水溶液を配布しています。 </li><li>光板を返します。プレートにパラフィルムを追加しないでください。 </li><li> 24時間後、プレートの表面は光沢のある外観を持つ必要があります。もし菌糸が再び表示され、スクレープ表面および再適用のTween-60ソリューションを提供します。 </li><li>来週上の子の​​う殻の発達を観察します。の中間段階被子器の開発が静かにRNA抽出（ 図1）凍結寒天とフラッシュの表面をこすることによって収穫することができます。 </li><li>成熟した胞子は7日からプレートの蓋に蓄積されます。当初はこれらの唯一の実体顕微鏡の下に表示されますが、9日目で、彼らは肉眼で見えるとなるように十分な密度に蓄積されているでしょう。 </li></ol> 2。子嚢胞子の放出アッセイ<ol><li>ツイーン溶液の塗布後6日目に、木のボーラーで寒天の1 cmの直径の円を切り取る。また、メスは、同様のサイズのセグメントを削除するために使用することができます。円を半分にスライスして各半分はガラス顕微鏡スライド上に配置することができます。 </li><li>オリエントは断片ので、子実体を含んでいる面がスライドの表面に垂直であり、胞子は、スライドの長さをダウンして起動されます。 </li><li>照明の下で一晩湿度チャンバ内でスライドを配置します。胞子に蓄積されスライドと肉眼で見えるように翌朝（ 図2）。 </li><li>必要に応じて蓄積された胞子は水でスライドをオフに洗浄し、定量化することができる。 </li><li>潜在的な子嚢胞子の放電阻害剤は、アッセイの組み立て時の寒天ブロックの背面側にそれらを追加することによって測定することができる。排出を削減するいくつかのイオンチャネル阻害剤は、以前にこのテクニックを15に検定されています。 </li></ol> 3。組換え子孫の世代<ol><li> F.の2系統とニンジン寒天培地を接種することによりクロスを開始graminearum種 （1寄生虫の卵+一NIT-）、ペトリ皿の両側に接種ポイントを配置する。 </li><li>一度菌糸が空中部分をこすり、表面を充填し、上記のようにトゥイーン-60ソリューションを適用しています。菌糸が満たすペトリ皿の側面に注意することはマーカーを使用しています。 </li><li>光に文化を返し、子のう殻が開発されるまでインキュベートし、cirrhiがありました二つの文化の交点に沿って子のう殻から形成する銃。 </li><li>解剖顕微鏡下では、ビューに二つの文化の交差点に沿って子のう殻をもたらす。虫ピンを使用して、滅菌し、滅菌水に浸し、ピンの先端で軽くこの縁に沿って子嚢胞子塊をタッチします。ピンの水分子嚢胞子塊がピンに固執することができるはずです。 </li><li>胞子を洗い流すためのエッペンドルフチュー​​ブに200μlの滅菌水でその上に子嚢胞子塊のピンの先端をすすぎます。 </li><li>炎ピンは、再びそれを湿らせて、別の子嚢胞子塊を選択します。 10から15 cirrhiを選んで、水の別のチューブにそれぞれを配置します。 </li><li>サスペンドcirrhiを含む簡単にボルテックスチューブ。 </li><li> 9センチメートルペトリ皿の中MMTS媒体1上にピペットと場所で胞子懸濁液の60μlを削除します。滅菌したホッケーのスティックと広がった。残りの胞子懸濁液は最大1週間までは4℃で保存し、必要に応じてプレーティングすることができます。 </li><li>インキュベーション3〜5日間室温でTE（光は必要ありません）非常に小さなコロニーが（ 図3）が表示されるまで。コロニー間の表現型の二種類が存在します。 NIT-コロニーは、NIT +のコロニーよりスパースな菌糸を持ちます。組換え子のう殻からCirrhiはほぼ等しい割合でコロニー表現型の両方が表示されます。その上、単一の表現型のコロニーでプレートを破棄します。時にはつ以上の表現型が他の要因の分離のために生じることに注意してください。 </li><li> V8またはストレージ用のニンジン寒天培地にコロニーを移動します。正しい表現型を確認するために、ドックスのドックス寒天培地（NIT-株はツァペックのドックスに成長しません）上および塩素（NIT +株は塩素とPDAになることはありません0.5 M塩素酸カリウム）とPDA上のテストコロニー。希望する品質のためにこれらのコロニーを調べます。 </li></ol> 4。代表的な結果子のう殻を作り出す目標は、芝生を持つことであるすべての菌糸または胞子明らかせずに子のう殻。文化の表面は黒いベルベット（ 図1）に似て表示されます。 Tweenの塗布後24時間で寒天の表面には若干の光沢を持つ必要があります。菌糸再度表示されている場合、プレートは被子器の開発を誘導するために十分なスクレイプされていない可能性があります。 子嚢胞子の放電常にあなたのアッセイで、野生型株から寒天のいくつかのブロックを提供します。それらが起動しない場合は、どちらかあなたの子のう殻は若すぎるか古すぎる。彼らは古すぎる場合には、多数の菌糸はそれを白っぽい色合いを与える文化の表面上に表示されます。これはそうでない場合、彼らは若すぎるかもしれませんし、このアッセイは、24時間で再び試行すべきである。時折、文化も放電しないし、実験が繰り返されなければなりません。あなたは適切な光条件下でアッセイを行うことを確認してください。 組換え後代<p class="jove_content"&gt;組換え子のう殻は、2つの系統間の交差点に沿って子のう殻の大部分を確認する必要があります。我々は通常のクロスから10 cirrhiを処理し、それらの少なくとも4-6組換えなければなりません。ごくまれに、突然変異が交配から歪みを防ぐことができます。両親のいずれかが異常増殖表現型を持つ場合、2つ以上の表現型はMMTS上のコロニーの間で存在する可能性があります。 <img alt="図1" src="/files/ftp_upload/3895/3895fig1.jpg" /> 図1ニンジン寒天培地上に被子器開発の段階。トゥイーンを適用した後、左、72時間。赤色顔料、表面に黒い粒のように見える非常に若い子のう殻に注意してください。右、144時間後、成熟した子のう殻で形成されています。両方の文化では表面的な菌糸体のほぼ完全な欠如に注意してください。 <img alt="図2" src="/files/ftp_upload/3895/3895fig2.jpg" /> 図2放電アッセイ。オレンジ色のニンジン寒天プラグイン指向のようにthaのです​​。強制的にその表面に成熟した子のう殻から排出されるトン胞子はスライドグラスの表面に蓄積することができます。 15時間の累積時間。 <img alt="図3" src="/files/ftp_upload/3895/3895fig3.jpg" /> 図3選択MMTS媒体上に寄生虫の卵+およびNIT-コロニーの間に成長の違い。 NIT-コロニーは（矢頭）小さくなります。寄生虫の卵+コロニー（矢印）が力強い成長を示しています。写真は7日後に撮影。

<ol>
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我々は、開示することは何もありません。

F. graminearum種は、特によく注釈付きゲノム（の在庫状況により子実体の開発研究に適応している<a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/">mips.helmholtz-muenchen.de/genre/proj/FGDB /</a> 4）、および可用性アフィメトリクス·マイクロアレイ6に基づいて。これは、子嚢胞子の放電7,11,3のために重要な遺伝子を識別する能力を促進しています。ここで紹介する方法は、研究者は、Fの子実体の遺伝的解析のための発達段階と関数の離散集合に集中することができます。 graminearum種 。方法はまた、文化のフルーツに誘導することができ、他の真菌子実体の種類の様々な開発のための標準として使用することができますに関連する菌類に容易に適応可能である。 胞子焼成表現型を評価する能力は、胞子が見て小さいと困難な種などで微妙なことができます<eM&gt; F. graminearum種。洗浄したスライド上に胞子のコレクションは、胞子がスライドをオフに洗浄し、定量することがありますので、視覚的評価、定量評価の両方を容易にします。いくつかの菌種は、その胞子を取り囲み、胞子は洗い流すことはできませんが、彼らはスライド上に残って顕微鏡でカウントする必要があり粘液を生成します。我々は、 菌核sclerotiorumの真であることを発見した。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>フザリウムgraminearum種株PH-1 </td><td>真菌の遺伝ストックセンター、カンザス州</td><td> FGSC 9075 </td></tr><tr><td>ツイーン60 </td><td> Sigma-Aldrich社</td><td> P1629 </td></tr><tr><td>ドックスのドックス寒天培地</td><td> Difco社、ベクトン·ディッキンソン</td><td> 233810 </td></tr><tr><td>塩素酸カリウム</td><td> Sigma-Aldrich社</td><td> 255572 </td></tr></tbody></table>

sexual development and ascospore discharge in fusarium graminearum

フザリウムgraminearum種は糸状菌の開発と病原性の研究のためのモデル系となっています。F. graminearum種が最も簡単にんじん寒天培地上で、子のう殻と呼ばれる子実体を生成します。子のう殻は、被子器開発の特定の段階で生産、多数の組織型が含まれています。これらは、（外観の注文における）被子器のイニシャル（ascogenous菌糸を生じさせている）、外壁、副生体（ASCIを開発するまで、被子器の中心を占める滅菌菌糸体）、ASCI、及び子のう胞子の形成を含む子嚢14内。これらの組織のそれぞれの開発は約24時間で区切られ、性的発達12,8の間にトランスクリプトーム研究の基盤となっています。各ステージの写真を含む開発のより詳細な説明については、ハレンら （2007）を参照してください。ここでは、同期の生成と収穫のための方法を提示LYは、遺伝子調節、開発、および生理的過程の時間的な研究のために子のう殻の芝生を開発しています。これらの方法は、Fで使用されるように具体的に書かれていますが、 graminearum種は 、技術が他の真菌のさまざまな目的で使用することができ、結実が培養で誘導することができます提供し、開発にはいくつかの同期があります。我々は最近、Fの性的発達を研究するために、このプロトコルを適応しているverticillioides。子のう殻を開発するの芝生が誘導されなかったため、個々の子のう殻は、この種に手を選ばれる必要がありますが、プロセスが開発（Sikhakolliとトレイル、未発表）を研究するためによく働いた。 菌類の子実体の中で最も重要な機能は、胞子の飛散です。子嚢菌（子嚢は菌類を生成する）の種の多くでは、胞子を介して（とepiplasmic流体）の胞子の放出を駆動し、子嚢内の膨圧の発生により、子嚢から撮影されてい子嚢の先端2,7の細孔。強制子嚢胞子放電の我々の研究は、我々がプロセス内での変異をスクリーニングするために使用する &quot;胞子放出アッセイ&quot;の開発につながった。ここでは、このアッセイの詳細を提示します。 F. graminearum種はホモタリックであり、従って、互換性のあるパートナーの存在下で子実体を形成することができる。 homothallismの利点は、交差点では、この種の14.7の性的発達の研究を促進してきた面の特定の形質の子孫のホモ接合体を生成する必要がないことです。しかし、異体性の株が5,9を越えるために使用することができる生成されています。それは1つの菌株1のいくつかの遺伝子の変異体を取得するにはホモタリック株を横断することも可能です。これは2株で1シャーレcoinoculatingによって行われます。ミーティング·ポイントに沿って、子のう殻の大部分は、（親系統のいずれかに変異を提供阻害しない組換えになります交配）。子のう殻の年齢として、彼らは強制的にそれらを排出するのではなく、子のう胞子大挙をしみ出させる。結果胞子滲出液は、（子嚢胞子塊と呼ばれる）被子器の先端に位置し、容易に個々の胞子の回復のために削除することができます。ここでは、組換え子のう殻および組換え子孫の回復の識別を容易にするためのプロトコルを提示します。

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Sexual Development and Ascospore Discharge in Fusarium graminearum

性的な交配と組換え後代の単離は、糸状菌のための重要な研究ツールです。<em&gt;フザリウムgraminearum種</em&gt;これらのプロセスを正常に行うに必要な技術が提示されています。

の性的開発と子嚢胞子放電フザリウムgraminearum種</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

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Biology

Sexual Development and Ascospore Discharge in Fusarium graminearum

21.7K ビュー.  Michigan State University. 性的な交配と組換え後代の単離は、糸状菌のための重要な研究ツールです。フザリウムgraminearum種これらのプロセスを正常に行うに必要な技術が提示されています。

ビデオ: Sexual Development and Ascospore Discharge in Fusarium graminearum

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

マイクロアレイ実験のためのジベレラzeae子嚢胞子の生産とコレクション。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

生物学

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

IVISスペクトラムを用いた乳腺腫瘍の in vivo生物発光イメージングに

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

の心臓の鼓動の可視化ショウジョウバエ</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

から毒素誘導とタンパク質抽出フザリウム属属。</emプロテオーム研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

マウスモデルにおける乳腺の発達と機能の評価

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

組み合わせたDNA-RNA蛍光<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）差別雌マウス胚性幹細胞におけるX染色体の不活性化を研究するために、

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

分裂酵母セクシャルライフサイクルの顕微鏡観察

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...