著者らは、批判的に読み、原稿を編集するための博士Cliona M.マクヘイルに感謝します。この作品は、環境健康科学研究所、Lチャンに健康補助金R01ES017452の国立研究所によって資金を供給された。

1。サンプルスライドの準備<ul><li> OctoChrome FISHは含むが、培養された末梢血細胞、幹細胞/前駆細胞、および細胞株に限らず、ヒト細胞の分裂中期スプレッドの染色体の変化を調べるために設計されています。中期の細胞を逮捕するためにコルセミド処理を採用することにより、細胞を膨潤する低張処理し、約0.5×10 6細胞懸濁液で細胞を固定するためのカルノア固定液：サンプルは分裂3,11-13を収集するための標準手順に従って準備する必要がありますmlあたり。 </li><li> 2分間純エタノールに浸漬：8平方スライド（PMP 803、Cytocell株式会社は、英国ケンブリッジ、図2 OctoChromeキットで提供され、カタログ＃）を清掃してください。 </li><li>サンプルスライドを作成する前に、細胞密度を確認するにはスライド上に細胞を少量のドロップします。細胞密度が（あまりにも多くの重複セル）が高すぎる場合は、新鮮な固定液で懸濁液を希釈してください。細胞密度（細胞数が少なすぎる低すぎる場合スライド上のs）は、新鮮な固定液の少量の細胞をスピンダウンし、再懸濁する。 2月8日 - - 5分の3〜6分の4の正方形1月7日を交互シーケンスのテンプレートのスライドの8の各分野への細胞懸濁液をピペット5〜10μlの。最初の二つの正方形は、空気乾燥させていたら、同じ方法で残りの四角形を見つける。これは、互いに干渉するからセルのスプレッドを防ぐことができます。 </li><li>乾燥したスライドは、ハイブリダイゼーションの前に5年以上のために窒素雰囲気下で-20℃で保存することができます。 </li></ul> 2。自動化システムを使用して、中期の局在<ul><li>スライドの各半分の中央に4を20μl &#39;,6-ジアミジノ-2 - フェニルインドール（0.2μg/ ml）を適用し、カバースリップでカバーしています。 </li><li> Metaferソフトウェア（メタシステム、Altlussheim、ドイツ）を用いて分裂中期細胞をローカライズするためのサンプルスライドの自動スキャンを実行します。これは、すべての分裂と今後のすべてのセル画像の再評価の位置を容易にします。 </li><li>手動でスキャン結果を表示し、非分裂アーティファクトを削除します。 </li></ul> 3。ハイブリダイゼーション<ol><li>サンプルスライドとOctoChrome装置の準備</li><ul><li> DAPIを洗い流すために30分間室温で2×SSC緩衝液のサンプルスライドを浸します。 </li><li>エタノール洗浄（2 70％分ずつ、85％および100％エタノール）の一連のサンプルスライドを脱水、乾燥、37、5分間°C、ホットプレート上に配置することができます。 </li><li> 37°Cの水浴中でChromoprobeのマルチプローブハイブリダイゼーションチャンバー（OctoChromeキットで提供）を配置し、37と同じになるように°C（+ / - 1°C）。 </li><li> 37℃にOctoChromeデバイスごとに繰り返しピペッティングし、事前暖かい25μlのアリコートでハイブリダイゼーション溶液（OctoChromeキットで提供）ミックス</li><li>ホットプレート上でデバイスのラベル面を下に配置することにより、37°C​​まで（図2を参照してくださいOctoChromeキットで提供）は、各OctoChromeデバイスプレ暖かい。 tを触れないでください彼OctoChromeデバイスのエンボス面がプローブを含んでいるので。 </li></ul><li> OctoChromeデバイス上のサンプルスライドの位置（図2） </li><ul><li>それは37℃のまま予め温めておいたOctoChromeデバイス上の8つの領域のそれぞれに予め温めておいたハイブリダイゼーション溶液2μlを加え℃に</li><li>慎重に逆さまになりました数1は、OctoChromeデバイスの右上領域の上に配置されているようなOctoChromeデバイス上のテンプレートスライドを反転させる。正方形の1を見つけやすくするために、デバイス上のその位置はオレンジでマークされています。 </li><li>テンプレートのスライドを注意深くOctoChromeデバイス上でマッチング領域に揃えて配置されていることを確認してください。ハイブリダイゼーション溶液の滴スライドに接触するように慎重にOctoChromeデバイス上のスライドを下げる。ハイブリダイゼーション溶液はOctoChromeデバイス上で発生した領域のそれぞれの端に広がっていることを確認するために穏やかな、均一な圧力を適用します。 </li><li>持ち上げるスライド/ OctoChrome、慎重にスライドガラスのフロスト端を保持し、スライドがOctoChromeデバイスの下になるように反転させる。これはプローブの交差汚染を引き起こす可能性があり、デバイスがテンプレートのスライドを越えて塗抹しないことを確認してください。 </li><li> 37場所°C（+ / - 1℃）10分間ホットプレート。 </li></ul><li>変性</li><ul><li>それはレベルを保持するために特定の世話をしてホットプレートにサンプルスライド/ OctoChromeデバイスに転送します。サンプルスライド均等に接触するホットプレートを確認します。 </li><li> 75ホットプレート上で変性℃（+ / - 1°C）で5分間。 </li></ul><li>ハイブリダイゼーション</li><ul><li> Chromoprobeのマルチプローブハイブリダイゼーションチャンバー内の場所のサンプルスライド/ OctoChrome装置。 </li><li>蓋を交換し、37室をフロート°C（+ / - 1℃）の水浴（非 - 攪拌）で一晩。ご注意：ハイブリダイゼーションチャンバーの蓋を密封しないで、水浴上で蓋を密封しないでください。でハイブリダイズしないインキュベーター。これらの手順は、湿度を制御するために重要です。 </li></ul><li>ハイブリダイゼーション後の洗浄</li><ul><li>洗浄溶液の調製 解決策1：20倍拡大SSCを含むコプリン/ Hellendahl jarファイルを準備します。 72平衡℃（+ / - 1℃）の水浴中とpHを7.0に調整します。 解決策2：2×SSC、0.05％Tween 20を含むコプリン/ Hellendahl jarファイルを準備します。室温で放置することができます。 </li><li>スライドから慎重にOctoChromeデバイスを削除し、2分間解決方法1のスライドに配置します。 </li><li> 30秒間ソリューション2にスライドを配置します。 </li></ul></ol> 4。マウントと結果の可視化<ul><li>スライドの各半分の中心にDAPI液20μlを適用し、カバースリップを適用します。 </li><li>蛍光顕微鏡で表示する前に室温で10分間、暗所でインキュベートします。 </li></ul> 5。代表的な結果ふぃぎゅ再3Aは、広場2で正常分裂中期細胞を示しています。 （1） - （3）：染色体8、21、12、赤、緑、青を描いた、テキサスレッド、FITC、それぞれDEACフィルターを通して可視化された、（4）：DAPI / FITCを介して可視化/テキサスレッド、トリプルフィルター。一般的には、青色で塗装染色体はトリプルフィルターを通して明らかになっていない、特定のDEACフィルターの下に表示する必要があります。 図3Bは、白血病に特異的な染色体転座や異数性を持つ代表的な異常細胞を示しています。 （1）：T（8; 21）、急性骨髄性白血病の一般的な染色体転座（2）：21トリソミー、21番染色体の3つのコピー、白血病の一般的な異数性。 <img alt="図1" src="/files/ftp_upload/3619/3619fig1.jpg" /> 図1。OctoChromeデバイスと期待される染色体ペインティングの結果についての染色体の組み合わせのアレンジメント。 <img alt="図2" src= &quot;/ files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; 図2。OctoChromeデバイス上のサンプルスライドの位置決め。 図3代表的な結果OctoChrome FISH（スクエア2）から得られた。 <img alt="図3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> 図3A。スクエア2に描かれた染色体8、12、21と正常細胞。 <img alt="図3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> 図3B。広場2で白血病に特異的な染色体転座や異数性を持つ代表的な異常な細胞。

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我々は、開示することは何もありません。

この小説OctoChrome-FISHアッセイは1つが同時に複数のスライド上の単一のハイブリダイゼーションに全24ヒト染色体を調べることができます。 8マスに染色体の組み合わせの配置は、最も一般的な白血病およびリンパ腫の非ランダムな染色体再配列の識別を容易にするために設計されています。したがって、このアッセイは、同時に数値（異数性）だけでなく、構造（転座）染色体の変化を検出?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td><tr><tr><td> OctoChromeキット</td><td> Cytocell株式会社は、英国ケンブリッジ、 </td><td> PMP 803 </td><td></td></tr><tr><td>フィトヘマグルチニン（PHA） </td><td>インビトロジェン株式会社</td><td> 10576-015 </td><td></td></tr><tr><td>コルセミド</td><td>インビトロジェン株式会社</td><td> 15212-012 </td><td></td></tr><tr><td>カルノア固定液</td><td></td><td></td><td>メタノール：氷酢酸= 3：1 </td></tr><tr><td>蛍光マイルcroscope </td><td></td><td></td><td>同時に異なる色を表示するには、DAPI、テキサスレッド、FITC、およびクマリンスペクトル個別DAPI / FITC /テキサスレッドトリプルフィルタを表示するには、フィルタを装備</td></tr><tr><td> Metaferソフトウェア</td><td>メタシステム、Altlussheim、ドイツ</td><td></td><td>異常をすべて分裂を見つけるために、再評価することが容易に</td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

蛍光 in situハイブリダイゼーション （FISH）は細胞核1に中期または間期の染色体上に検出される特定のDNA配列を可能にする技術です。技術は、全体の染色体または特定の染色体領域にハイブリダイズするユニークな配列を有するDNAプローブを使用しており、古典的な細胞遺伝学への強力な補助としての役割を果たします。例えば、多くの以前の研究は、古典的な細胞遺伝学的分析2を使用して 、ベンゼン暴露、ベンゼン中毒の患者、およびベンゼンにさらされる労働者の健康との関連性白血病患者で増加した染色体異常を頻繁に検出を報告した。 FISHを用いて、白血病に特異的な染色体の変化は、ベンゼン誘発性白血病におけるcytogentic変化の重要な役割を示し、ベンゼン3月6日にさらされ、明らかに健康な労働者で上昇することが観察されている。 一般に、単一のFISHアッセイは、1つまたは少数の全体の染色体検査またはスライドごとに特定の遺伝子ので、複数のハイブリダイゼーションは、ヒト染色体のすべてをカバーするために複数のスライドに実施する必要があります。スペクトル核型分析（SKY）は、同時に全ゲノムの可視化が、特別なソフトウェアや機器の制限とその応用7の要件を可能にします。ここでは、新たなFISHアッセイ、我々はこのような染色体全体の異数性試験などの人間研究では、1つのスライド全24ヒト染色体の同時分析（CWAS）としてここで定義Chromosomicsに適用することができるOctoChrome-FISHを、記述し8。 ChromoprobeのマルチプローブシステムとしてCytocellにより市販されている方法の基礎は、3つの異なる全染色体ペイントプローブ（図1）を運び、それぞれが8マスに分割され、OctoChromeデバイスです。 3つのプローブの各々は、直接に異なる色の蛍光体、緑色（FITC）、赤（テキサスレッド）、青（クマリン）が付いています。 OctoChrom上で染色体の組み合わせの配置は、t（15; 17）は、t（8; 21;電子デバイスは、インスタンスt（22 9）は、最も一般的な白血病およびリンパ腫に見られる非ランダム染色体構造変化（転座）の識別を容易にするために設計されています）は、t（14; 18）9。また、染色体の数値の変化（異数性）を同時に検出することができます。対応するテンプレートのスライドは、中期スプレッドがバインドされているその上に8マス（図2）に分割され、OctoChromeデバイス上に配置されています。プローブとターゲットDNAは高温多湿のチャンバー内でハイブリダイズした時に変性し、全24ヒト染色体を同時に可視化することができる。 OctoChrome FISHは、白血病とリンパ腫の臨床診断のために、全ての染色体の異数性を検出するための有望な技術です。我々は、ベンゼンにさらされた中国人労働者のCWAS研究8,10のこの新しい染色体のアプローチを適用している。

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

新規蛍光<em&gt;その場で</em同時に、特に数値と染色体構造の変化、白血病とリンパ腫に関連付けられた特定の染色体転座の両方を調べて&gt;ハイブリダイゼーション（FISH）法は、一つのハイブリダイゼーションの単一のデバイス上のすべての24のヒト染色体のうち、記載されています。

Chromosomics：同時に新規OctoChrome FISHアッセイを使用した全24ヒト染色体における数値と構造変化の検出

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

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18.7K ビュー.  University of California, Berkeley. 新規蛍光その場でハイブリダイゼーション（FISH）法は、一つのハイブリダイゼーションの単一のデバイス上のすべての24のヒト染色体のうち、記載されています。

ビデオ: Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

蛍光その場で</emヒト精子の>ハイブリダイゼーション（FISH）プロトコル

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

生物学

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

使用BMP4のエフェクター-錯体のダウンストリームシグナリングの検出その場で PLA、近接ライゲーションアッセイ

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

リグノセルロース系材料のためのハイスループット糖化アッセイ

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

エクソンキャプチャと超並列配列決定することにより腫瘍標本における体細胞遺伝子変異を検出する

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

HEK 293F懸濁細胞における構造生物学のための組換えタンパク質の発現：小説とアクセアプローチ

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

蛍光標識ナノ粒子を用いたマウス、ヒトおよびブタ起源のライブ細胞の細胞内遺伝子発現の検出

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

勉強リボヌクレオチド定款: 酵母ゲノムとリボヌクレオチド誘発突然変異を測定のリボヌクレオチド鎖特異的検出

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...