装置<ul><li>組織培養インキュベーター、37℃、5％CO 2 </li><li>遠心</li><li> micropipettorsのセット（100、100μL） </li><li>水浴37℃ </li><li>水浴90℃の蒸気の水の源になるために</li><li>位相差顕微鏡</li></ul> FIRST STEP コルセミドで細胞を処理するこの手順では、20％ノックアウト血清代替（KSR、ギブコ社）と8ng/mLのを補充マイトマイシンC（シグマ）で不活化とDMEM/F12（Invitrogen）中で維持マウス胚線維芽細胞（MEF）上に培養したヒトES細胞のラインH9を使用線維芽細胞増殖因子（FGF - 2、R＆D）。 分裂中期スプレッドの多数を得るために、それを多くの分裂細胞が存在するで高い分裂インデックスを持つ文化を使用することをお勧めします。 <ol><li>インキュベーターから培養フラスコを取ると、層流で、0,1μg/ mLの最終濃度でコルセミド追加。 </li><li>インキュベーターにフラスコを返し、阻害剤は、染色体がうまく凝縮されており、顕微鏡下で容易に可視化できる中期、上に有糸分裂停止を引き起こす可能性があるので、3時間を待つ。 </li></ol>細胞を固定<ol start="3"><li>培地を取り出し、細胞の表面をカバーするのに十分トリプシン/ EDTA、0.05％追加。細胞は室温で5分間インキュベートし、血清を含む培養培地を追加したり、単に細胞に血清添加することによりどちらかの酵素を不活性化することができます。 注：単一の細胞懸濁液は、良好な分裂中期スプレッドを取得するために達成されていることを確認することが重要です。 </li><li> 15 ml遠心チューブと5分は122 gで遠心して細胞を移す。 </li><li>約200 mLを残し、上清を捨て、そして、ビデオに示すようにチューブを、タップして穏やかにペレットを再懸濁します。細胞がよく解離されるので、ソリューション内の任意の塊を視覚化できないことを確認してください。 </li><li>次に、低張液5 mL（塩化カリウム75mmの）37に予め温めておいた° C、ゆっくりと、以下の説明のように管壁によって。を追加する必要があります。最初だけでその後低張液で細胞を混ぜ、残りの2 mlを加え37℃で保温℃で15分間に水平位置にチューブを反転、3 mlを加える。 注：この時点で、低張液は細胞のボリューム上の増加を引き起こし、染色体を解くのに役立ちます。インキュベーションの時間は、良好な染色体のスプレッドを獲得することが重要です。タイミングが長すぎる場合、細胞膜には早すぎると染色体が失われて破裂することがあります。短すぎる場合、細胞膜が中断されないことがありますので、それは染色体のスプレッドを得ることが困難な場合があります。 </li><li>固定液（メタノール/氷酢酸3:1）の3滴を追加、休憩なしで5分間、122 グラムの細胞と遠心機で固定溶液を混合して軽くチューブを反転させる。 注：固定溶液をたててください。 注：材料の損失を防止する、細胞の減少を避けるために、休憩なしで遠心分離を使用することが重要です。 </li><li>約200 mLを残し、上清を捨て、そしてチューブの底を軽くたたくことによって静かにペレットを再懸濁します。 注記：ここでも、細胞がよく解離されるので、ソリューション内の任意の塊を視覚化できないことを確認してください。 </li><li>もしチューブを回転しながら、管壁が徐々に、固定液の最初の3 mLを加え。単に細胞を混合し、細胞をダウンさせるために管壁で固定溶液中より2 mLを追加するには、水平位置にチューブを反転。 4℃で一晩株式。 注：固定された細胞は4℃ヶ月間固定液に保存することができます℃のその膨潤状態で細胞を保護する以外に、固定液は脂質と変性蛋白質を削除します。これらのイベントは、結果として、染色体は容易に拡散させる、細胞膜が壊れやすいようにと。 </li></ol>スライドガラスのクリーニングあなたの染色体スプレッドを作り始める前にスライドが適切に洗浄されているかあなたには、いくつかの核とも中期を失う可能性があることを確認してください。あなたがどんなハイブリダイゼーション技術9試してみたい場合は、このステップでは、同様に重要です。 <ol><li>場所は、室温で少なくとも3時間のために6M塩酸を入れたビーカーにスライドします。 </li><li>この時間の後、10分間水道水を流してのスライドを洗う。 </li><li>スライドを蒸留水ですすぎ、彼らは、室温で乾燥させます。 </li><li>今スライドを96％エタノール中に保存され、使用の直前に、糸くずを乾燥させることができる。 </li></ol>注：in situハイブリダイゼーション法では、スライドを96％エタノールに2日以上保存することができます。 SECOND STEP 細胞を洗浄<ol start="10"><li>次の日に、休憩なしで5分間、122 グラムで細胞を遠心分離。 </li><li> D約200mlを残して上清をiscard。軽くチューブを回転させながら、チューブは、チューブの壁が徐々に、新鮮な固定液を5ml（メタノール/氷酢酸3:1）を追加するフリックでペレットを再懸濁します。この洗浄は3回の洗浄の合計を2回以上繰り返します。 注：固定溶液をたててください。 </li></ol>スライドの準備<ol start="12"><li>最後の遠心分離工程において（図1参照）細胞ペレットを均一化しスライドに細胞の十分な密度を得るために解決策の十分な量を残す。 </li><li>非常に近いと表面に平行にピペットを移動することで、清潔で乾燥したスライド上に細胞懸濁液の20〜30μLを加える。固定液が蒸発するように、スライドの表面が粗くなります。 </li><li>すぐに、細胞が爆破させるために30秒間お湯の蒸気（90 °）に、表向き、スライドを公開します。 注：この時点では、一緒に水で拡散染色体を刺激し増加酢酸濃​​度、結果として固定溶液が蒸発するからメタノール、。 </li><li>濃度だけでなく、細胞の分布は（図1参照）良いかどうかだけをチェックする位相差顕微鏡で見てください。 </li><li>現在のスライドは、G -バンディング（ギムザ）、FISH、SKYやCGHなどの細胞遺伝学的アプローチによる染色体のセットをチェックするために使用する準備が整いました。 </li></ol>重要なヒント分析するに十分な中期の方法を選択ラウンドと分離された分裂中期（図2D）を選択するには、2つまたはそれ以上の広がり紛れ込まない起動染色体によって生成された染色体の消失によって生成された染色体のゲインとして偽aneuploidiesを考慮して回避することができます。ので、いくつかの染色体は、それら（図2Aおよび2B）により隠れている可能性がありますので、原子核の近くに分裂中期の選択を避ける。ほかに、ラウンド分裂中期を探して、任意の染色体が分裂中期（図2C）から分離されていることを確認してください。 フィーダー細胞とヒトES細胞の核型 G -バンディングとSKYの分析のために、ヒトES細胞培養におけるフィーダー細胞の存在は、フィーダー細胞が分裂状態下にないため、結果を妨げることはありません（マイトマイシンCまたは照射によって不活性化）と中期の人口の一部にはなりません。しかし、間期核のFISH法の応用のために、それはFISH解析のために、トリプシン処理する前に手動でコロニーを収穫によってフィーダー細胞からヒトES細胞の集団を分離することが好ましい。 <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="図1" /> 図1：どのようにスライドの適切な細胞密度を知っている。 10倍の目標の位相差顕微鏡の画像。 （A）細胞密度が高すぎる。矢印は核の近すぎる広がる中期を指している。 （B）良好な細胞密度とスライド。円は、核または他の分裂中期から分離分裂中期スプレッドを示しています。してください<a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="図2" /> 図2：どのように分析するに十分な中期を選択する。染色体はDAPIと100xの目的での蛍光顕微鏡によって得られた画像で染色されています。 （A）（B）核の近くに分裂中期（矢印で示される）（C）を避け、現在の染色体は、（丸）で区切られたことが丸くなっていない分裂中期。 （D）ラウンド中期分析する良い中期です。してください<a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。

<ol>
	<li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

染色体スプレッドの準備は、G -バンディング、およびそのようなFISH、SKYやCGHなどのより高度なテクニックとして日常的に技術による胚性幹細胞の遺伝的状態を正常に解析するための重要なステップです。 この手順は、細胞周期の長さに依存するコルセミドインキュベーション、期間を変化させることで、多くの細胞型に適用することができます。胚様体（EB）のために?...

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Techno Plastic Products</td><td>91015</td><td></td></tr><tr><td>Colcemid Karyo MAX</td><td>GIBCO, by Life Technologies</td><td>15212-012</td><td></td></tr><tr><td>EDTA</td><td>Isofar</td><td>721</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Isofar</td><td>100</td><td></td></tr><tr><td>Methanol</td><td>Isofar</td><td>208</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Merck &amp;amp; Co., Inc.</td><td>104.931.000</td><td></td></tr><tr><td>Slide</td><td>Bioslide</td><td>7105-1</td><td></td></tr><tr><td>Tripsin</td><td>Sigma-Aldrich</td><td>T4799</td><td></td></tr></tbody></table>

chromosomal spread preparation of human embryonic stem cells for karyotyping

ヒト胚性幹細胞（ヒトES細胞）安定した二倍体の核型1を提示することが示されているが、多くの研究は、培養条件に応じて、彼らはそのような全体または染色体の部品の追加などの染色体異常を取得する傾向になることを報告している。確かに、長期培養の間に、核型の変化はときに酵素的または化学的解離が観察される継代のためのコロニーのマニュアル解剖が安定核型5を保持しながら、2,3,4が使用されています。加えて、フィーダー細胞の除去など環境の変化はまた、ヒトES細胞3,6の遺伝学的完全性が損なわれるようです。一度染色体変異が細胞生理に影響を与える可能性、in vitroでのヒトES細胞の遺伝的整合性の特性は、胚発生の研究と薬物検査に不可欠なツールとして重要な考慮hESCのです。さらに、将来の治療目的のために染色体の変化が頻繁に発癌に関連付けられているように大変な問題です。 ここでは、G -バンディング、FISH、SKYやCGHの技法7,8で設定された染色体のその後の分析のために高品質の染色体のスプレッドを得るために簡単で便利な方法を示しています。我々は、転座とaneuploidiesの外観をモニターするために5継代の間隔で定期的に染色体の状態を確認することをお勧めしますプリシラブリットとラファエラサルトルは、紙にも同様に貢献した。

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

核型分析は、広く遺伝子変異を検出するために使用されるシンプルかつ有用な技術である。ここでは、文化の中で維持、これらの細胞の染色体の状態を監視するためのヒト胚性幹細胞の染色体の広がりの準備のためのステップのプロトコルバイステップで説明します。

染色体分析のためのヒト胚性幹細胞の染色体スプレッドの準備

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.6K ビュー.  Federal University of Rio De Janeiro-UFRJ. 核型分析は、広く遺伝子変異を検出するために使用されるシンプルかつ有用な技術である。ここでは、文化の中で維持、これらの細胞の染色体の状態を監視するためのヒト胚性幹細胞の染色体の広がりの準備のためのステップのプロトコルバイステップで説明します。

ビデオ: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

非統合ソームプラスミドを用いて凍結軟膜から誘導多能性幹細胞の生成

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

発生生物学

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

遺伝学

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

ゲノム編集したヒト多能性幹細胞株の樹立：分離へのターゲティングから

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

多発性嚢胞腎疾患で、ヒトとマウスの染色体異常を研究するためにスペクトル核型分析

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

医学

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

非形質転換ヒト線維芽細胞から増殖性四倍体細胞の樹立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

ヒト胚性幹細胞におけるターゲティングジンクフィンガーヌクレアーゼ遺伝子の強化

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

マウス精母細胞から減数分裂期染色体の準備をスプレッドします。

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...