研究は、NIH 1R15GM094770によって資金を供給された。

1。コロニーの単離と固定<ol><li>指定された時間のために寒天培地に300コロニーをインキュベートする（孤立コロニーは直径に1-2 mmにしてください）​​。 </li><li>コロニー（顔のアップ）と狭いヘラを使用して、基礎となる培地を削除します。 </li><li> 2％寒天、42℃、それが固化する前に、寒天の表面に1 mLのpipetmanの先端と、すぐに場所のコロニーを用いて顕微鏡スライド上にCを数滴を置きます。 </li><li> 42 ° Cコロニーに2％の寒天を数滴を配置し、固化することができます。 </li><li>このプロトコルの残りの部分を通じてすべてのステップのための手袋を着用してください</li><li>かみそりの刃を持つブロックをトリムし、4℃で7日間、2％パラホルムアルデヒド/ 2％グルタルアルデヒド固定液を含む3.5mLのホウケイ酸塩のスクリューキャップ付きバイアルの中に置いてください</li></ol> 2。洗浄およびオスミウム処理<ol><li>約1週間後、完全に同じボリュームで5分間、その後、植民地（約1.5mL）をさらに2回をカバーするのに十分な0.15Mカコジル酸ナトリウム（pH7.2）で15分間二回インキュベートすることにより、化学煙フードの下に氷の上の寒天ブロックを洗う1X OSバッファー（100mMのKH 2 PO 4 10mMのMgCl 2、pH6.0）での。 </li><li> [2 mLのOSO 4（2％）+ 2 mLの2倍のOSのバッファ] 1％の同じ体積OSO の追加4バイアル（氷上で）に1時間化学煙フード下の寒天ブロックをカバーするために、同じ金額で2回洗浄10分ごとに1X OSバッファの。 </li><li> 4℃バイアルは一晩おきます° 1X OSのバッファでC。 </li><li>固定し、このステップですべての洗浄は、危険廃棄物として廃棄されています。 OSO 4を含むすべてのピペットは、植物油と3Xすすいだ。 </li></ol> 3。洗浄と脱水<ol><li>翌朝は、パスツールピペットを用いて、10分ごとに冷たい水の上と同じボリュームで氷の上で二回寒天ブロックを洗う。 </li><li> 25％の同じ体積で順次洗浄、10分ごとに50％、75％、95％エタノールで10分間100％エタノールで2回洗浄した。 4℃で一晩おきます〜100％エタノールでCを。 </li><li>このステップのすべての洗浄は、危険廃棄物として廃棄されています。 </li></ol> 4。スパーの準備<ol><li>次の朝は、（可能な限り早期）スパーの試薬を準備するヒュームフードの下で使い捨てのプラスチック製ビーカーに入れ、以下のソリューションを（EMサイエンス）重量を量る。この試薬を使用する後続のすべての作業では、ヒュームフードを使用し続ける。 （ERL 4221溶液5グラム） （DER 736溶液4グラム） （NSAの溶液13グラム） </li><li>これらのソリューションは、（ゆっくり）攪拌棒を使用してフードで20分間混ぜる</li><li> DMAEのソリューションを0.15グラムを追加し、上記のような20分をかき混ぜる。 </li><li>フード内の1〜2時間のためのドガ上記の混合物。 </li></ol> 5。スパーの浸透<ol><li>スパーのが上記の10分ごとに100％の室温のエタノールのと同じボリュームで寒天ブロックを5回洗浄なされるとすぐに。最後にエタノールで洗浄後に残っているエタノールは、ちょうど寒天ブロックを覆うように、パスツールピペットを用いてエタノールを除去します。 </li><li>約スパーの試薬（約0.5mL）を同量を追加し、30分間放置するし、室温で15分間ホイール上にバイアルを回転させると。 30分は完全にパスツールピペットを用いて溶液を除去した後、追加スパーは、寒天ブロック（約1.5 mL）をカバーするもの、室温で15分間ホイールにバイアルを回転させ、30分間放置する。スパーの交換、回転、およびインキュベーションさらに3回繰り返します。 </li><li>最後の30分後、スパーの試薬を除去し、ブロックをカバーするために新鮮なスパーのを追加します。寒天ブロックは4時間室温で放置する。 </li><li>スパーのを交換し、一晩回転する。 </li><li>朝にはスパーのを交換し、翌日になるまで回転しておきます。 </li><li>単に金型の底をカバーするために番号のシリコンモールドで翌朝の場所のスパーの（フィッシャーサイエンティフィック）は、その後、金型に寒天ブロックを追加します。 4時間60℃でインキュベートする</li><li>より多くのスパーのインキュベート、60℃で一晩でカビオフ上部後4時間、、。 </li><li>金型からブロックを削除した後のブロックはまだわずかに柔軟である場合、金型に戻り、追加の2日間インキュベートする</li></ol> 6。セクショニング<ol><li>ライカUltracut Sミクロトームは、光学顕微鏡では0.5μ厚の切片を0.1μ伝送EM用薄切片を切断するために使用された。 </li><li>セクションがカットされたとして、光学顕微鏡の場合は、、それらはのdH 2 Oのドロップで配置され、そして52℃のヒートブロック上で乾燥。乾燥のセクションは、1.0パーセントトルイジンブルー、5-15秒1％ホウ酸ナトリウムの滴で染色した。染色後、切片をすぐに、水の流れの下で洗浄し、乾燥し、マウントメディア（KPL）に覆われており、カバースリップは、試料上密封した。 </li></ol>酵母や他の微生物のパターン形成を測定するの広範な関連性は、以前は1日している。特に注目すべきは、無秩序な社会への相対的な組織的な微生物のコロニーの増加機能があります。 記載されている方法は大きく専門的なコロニー2を埋め込 ​​むための方法の変更であり、そして私達の変更の簡単な説明は、3を発表されている。 野生S.の植民地の中心を通ってセクションの光顕微鏡写真の例出芽酵母の酵母のコロニーを図1に示されています。 ASCI、pseudohyphaeと卵酵母がわかりやすい、基礎となる寒天を侵略植民地の地域にも明らかである。 酵母の実験室株（W303背景）の電子顕微鏡写真の例を図2に示されています。画像は、胞子形成細胞の高周波を含むコロニーの地域からです。 <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="図1" /> 図1。野生酵母のコロニーの光学顕微鏡。この酵母（YPS133）は、ツリーの滲出液4から最近単離された。コロニーは、前の切片にYNA媒体5の6日間インキュベートした。オープン矢印は、塗りつぶされた矢印は、細長い細胞のチェーン（pseudohyphae）とASCIを示す、塗りつぶされた矢印は代表的な卵形の栄養細胞を示す、代表的なASCIを示している。 <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="図2" /> 図2実験室酵母株の電子顕微鏡。 SH1020（W303背景）のコロニーが3切片の前にYNAの培地上で6日間インキュベートした。画像は、ASCIの高い周波数を持つセクションの領域を示しています。矢印は胞子壁の二層構造を示している。 <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="図3" /> 図3。コロニーにおける胞子形成細胞の分布の定量化：）その長辺に沿って積み重ね15に隣接する矩形を含むグリッドは、コロニーのセクションの画像の中央部に重畳する。その比率を一定に保ちながらグリッドは単に植民地の上部と下部をカバーするために、スケーリングされます。 （B＆C）その長方形の全細胞に対する各長方形の胞子形成細胞の割合は、4日齢（B）、8日齢（C）コロニーからの画像の目視検査によって決定されます。例えば、植民地の上部にある長方形は（&quot;1&quot;とラベルされた）のグラフの左側に対応しています。 4コロニーの平均値が表示され、エラーバーはSTDを表示します。エラー。

<ol>
	<li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

利害の衝突は宣言されません。

提示方法は、コロニーの内部構造を明らかにする。方法は、Sの範囲内のセルのタイプのパターンを決定する上で効果的ですので、別のコロニーの形態を持つ、とも近縁種S. paradoxus 5の出芽酵母株は 、この方法はまた、真菌や他の微生物の広い範囲で動作する可能性があります。 メソッドの成功のための一つの重要なステップは、コロニーの?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
<tr>
<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
<tr>
<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
</tr>
<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
</tr>
<tr>
<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19050</td>
<td>NSA</td>
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<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
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<td>Mounting media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
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<td>Rotating wheel</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
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<td>Microtome</td>
<td>&nbsp;</td>
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yeast colony embedding method

胚の異なる細胞型のパターニングは、後生動物の発生において重要なメカニズムです。このようなコロニーやバイオフィルムのような微生物のコミュニティは、また、細胞の種類のパターンが表示されます。例えば、酵母のS.出芽酵母は 、胞子形成細胞と偽菌糸細胞が一様にコロニーで配布されていません。パターニングとこれらのパターンの根底にある分子機構の機能的重要性はまだよく理解されています。 真菌コロニーの細胞型のパターンを調査に関して、1つの課題は、後生動物の組織とは異なり、コロニー内の細胞は比較的弱く、お互いに接続されていることです。特に、真菌のコロニーはほとんどの組織に見られる細胞外マトリックスの同じ豊富なレベルが含まれていません。ここでは、これらのコロニーの細胞型の内部のパターンを明らかに酵母のコロニーを埋め込むとセクショニングの方法で報告する。方法は、光学顕微鏡および透過型電子顕微鏡に適した薄切片（0.1μ）のために有用（0.5μ）厚い切片を作製するために使用することができます。これらの細胞の内部構造はEMによって可視化することができますがASCIと偽菌糸の細胞が容易に、光学顕微鏡で卵形の酵母の細胞と区別することができます。 メソッドは、寒天でコロニーを囲むスパーの培地でそれらを浸潤し、切片に基づいています。 1〜2ミリメートルの範囲の直径を持つコロニーは、このプロトコルに適しています。コロニーの内部を可視化することに加えて、メソッドは基になる寒天に侵入するコロニーの領域を可視化できる。

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

光および電子顕微鏡用切片可能に酵母のコロニーを埋め込むための方法。このプロトコルは、真菌、コミュニティ内での細胞型の組織を理解するための新しいツールを提供してコロ​​ニー内に胞子形成細胞と偽菌糸細胞の分布を決定することができます。

酵母のコロニーの埋め込み法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Research

JoVE Journal

Biology

11.4K ビュー.  University of Missouri - Kansas City. 光および電子顕微鏡用切片可能に酵母のコロニーを埋め込むための方法。このプロトコルは、真菌、コミュニティ内での細胞型の組織を理解するための新しいツールを提供してコロ​​ニー内に胞子形成細胞と偽菌糸細胞の分布を決定することができます。

ビデオ: Yeast Colony Embedding Method

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

出芽酵母の複製寿命の測定

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

生物学

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

軟寒天コロニー形成アッセイ

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面拡散と酵母染色体の免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

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 ...