Name: detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice
Uploaded: 2017-01-11T15:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

この研究は、国立航空宇宙局を通じてアーカンソースペースグラントコンソーシアムと国立宇宙生物医学研究所によってサポートされていました、付与NNX15AK32A（RP）とRE03701（MH-J）、およびP20のGM109005（MH-J）、および米国退役軍人局（ MH-J）。私たちは、原稿の準備で編集の支援のために、クリストファーフェテス、アーカンソー医科大学環境学科や産業衛生プログラム・コーディネーターに感謝します。

すべての動物実験は、国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って実施しました。動物プロトコルは、アーカンソー医科大学の施設内動物管理使用委員会によって承認されました。新鮮な空気と、標準的なげっ歯類の餌と水に自由にアクセスの15時間ごとのサイクル - すべての動物は、10と20±2℃で、標準的なエアコン付きの動物施設で飼育されました。到着すると、マウスを1週間検疫で開催されたと認定齧歯動物食を提供しました。 1.Radiation曝露と中期細胞のインビボ逮捕で <ol><li>照射室で2 Gyの全身照射に非麻酔したマウスを公開します。照射中、彼らは自由に移動し、均一な線量を受けていないことを確認する風通しのよい保持室にマウスを置きます。 </li><li> 0.05％コルヒチン溶液100μLを注入（C腹腔内に1mlの使い捨て注射器を取り付けた25 G針を使用して、カルシウム及びマグネシウムを含まないPBSに溶解olchicine粉末）。任意の器官に直接注入は避けてください。 </li><li>骨髄細胞の収穫前に2時間ケージに動物を残します。痛みや苦痛の徴候のために動物を観察します。 </li></ol> 2.骨髄細胞収穫と密度勾配遠心分離により骨髄単核細胞の単離<ol><li> CO 2窒息によりマウスを安楽死させます。 </li><li>背と腹側の70％エタノールをスプレーします。 </li><li>鋭いハサミで腹部皮膚上の2cmの切開を行います。鈍ピンセットで切開部の両側に皮膚をつかみ、ゆっくりと腹筋を開き引き出します。 </li><li>ハサミで後ろ足の両方を切断し、すぐに4％のFBSを予め冷却PBSでそれらを置きます。 </li><li>慎重に鋭いカミソリの刃と綿ガーゼ包帯で後肢に接続されているすべての筋肉を清掃してください。 </li><li>大腿骨から脛骨を区切ります。カミソリの刃でそれぞれの骨の両方の先端をトリミング。 </li><li> 3 mLシリンジに取り付けた23 Gの針を用いて（4％FBSを含む）3 mLのPBSで骨髄の内容をフラッシュし、15 mL遠心チューブ内のコンテンツを収集します。単一細胞懸濁液を作製するために針を通して細胞懸濁液を少なくとも10回通過します。 </li><li>細胞懸濁液と、リンパ球分離媒体との間の界面を乱すことなく、リンパ球分離媒体（3 mL）を等量の細胞懸濁液を慎重に重なります。室温で30分間、400×gで遠心分離します。 </li><li>新しい15 mL遠心管に他の層と転送を乱すことなく、慎重にバフィーコートを収集します。 </li></ol>中期細胞スプレッドの調製<ol><li>バフィーコートを含むチューブに10 mLのPBSを追加します。 </li><li>室温で5分間、400×gで遠心分離します。 </li><li>上清を注意深く取り除きます。その後、トンを破ります彼は穏やかなタッピングでペレットをセルと10 mLのPBSを追加します。 </li><li>室温で5分間、400×gで遠心分離します。 </li><li> 、細胞ペレットを乱すことなく上清を除去し、ペレットを分割し、予め温めた低張0.075 M塩化カリウム溶液の4 mLを加え。穏やか一定に振盪しながら降下により低張液滴を追加します。 </li><li>水浴中で37℃で20分間細胞をインキュベートします。 </li><li> （：氷酢酸、3：メタノール1 v / v）を低張処理した後、固定液の等量（4 ml）を追加し15 mLチューブにおいて、チューブを反転させることにより穏やかに混合します。 </li><li>室温で5分間、400×gで遠心分離し、上清を捨てます。 </li><li>数秒間穏やかにボルテックス続いタッピングして細胞ペレットを破砕し、一定に振盪しながら降下により固定液滴の3ミリリットルを追加します。 </li><li>室温で30分間インキュベートし、次いで5分間400×gで遠心します。 </li><li> 、上清を除去ゲントで細胞ペレットを破りますルは、タッピング、および3 mLの新鮮な固定液を追加します。 </li><li>室温で5分間、400×gで遠心分離し、上清を除去します。穏やかなタッピングで細胞ペレットを破壊し、そして新鮮な3 mLの固定液を追加します。 </li><li>手順を繰り返し3.12倍以上。 </li><li>室温で5分間、400×gで遠心分離し、細胞ペレットを乱すことなく、上清を除去します。そして、400〜600μLの固定液を追加し、ピペッティングでよく混ぜます。 </li><li> 45°の角度で傾斜予備洗浄とウェットスライド上に30μLの固定細胞を削除し、完全に一晩空気乾燥さスライドを可能にします。 </li></ol> mFISH 4.マウス染色体の絵画<ol><li>染色体変性<ol><li>室温で2分間、2×SSC中で中期細胞のスプレッドを含むスライドを置きます。 </li><li> 70％、80％、100％で2分間、連続エタノール洗浄によってスライドを脱水。 65℃で60分間インキュベートします。 </li><li>予熱40 mLの変性溶液（70％ホルムアミド/ 2×SSC; 30分間ガラスコプリンジャー中で70℃（±2℃）にpHが7.0）。 1.5分 - 1のための予熱した変性溶液中でスライドをインキュベートすることによって染色体を変性させます。 </li><li>変性処理を停止するとともに、再アニーリングから変性染色体を防ぐために2分間氷冷70％エタノール中で直ちにクエンチスライド。次に2分間80％エタノールで洗浄することにより脱水します。 2分間100％エタノールでエタノール洗浄を繰り返します。完全に室温でスライドを乾燥させます。 </li></ol></li><li>プローブ混合物の変性およびハイブリダイゼーション<ol><li>簡単に言うと、製造業者によって供給されたプローブの混合物を遠心分離し、7分間水浴中で80℃（±2℃）でインキュベートすることによりキャップ遠心管をスナップ500μLにプローブ混合物の10μLを転送し、変性。 </li><li> 10分間37℃の水浴中でプローブ混合物を用いた遠心分離管を置きます。 </li><li>変性chromosoでスライド上に変性したプローブ混合物を適用しますMES。 </li><li>慎重に18ミリメートルX 18ミリメートルガラスカバースリップでエリアをカバーし、非常に静かにスライドさせるカバースリップを押すことにより、目に見える気泡を除去します。ゴムセメントでカバースリップのすべての4つの側面をシールし、ハイブリダイゼーションのための加湿チャンバー内で37℃、暗所で16時間〜12時間インキュベートします。 </li></ol></li><li>ポストハイブリダイゼーション洗浄および検出<ol><li>慎重にゴムセメントとカバースリップを削除します。 </li><li> 5分間74℃（±2℃）で予熱した0.4×SSCに入れスライド。 </li><li> 2分間の洗浄溶液II（4×SSC / 0.1％のTween-20）で洗浄スライド。 </li><li> DAPIの対比（1.5μgの/ mL）で抗退色封入剤の20μLを置き、ガラスカバースリップでカバーしています。気泡や過剰取り付け溶液を除去するためのラボの組織とカバースリップ上でそっと押し。マニキュアでカバースリップの端をシールします。 </li><li>適切なフィルターを備えた蛍光顕微鏡を使用して表示スライド。 </li></ol></l私&gt; </ol>マウス染色体の5スペクトル核型分析（SKY） <ol><li>染色体およびプローブ変性<ol><li>振盪せず、2分間、室温で2×SSC中でスライドを平衡化します。 </li><li>室温で2分間、エタノールシリーズ（70％、80％、および100％エタノール）中でスライドを脱水します。エタノールを完全に除去するために、スライドを空気乾燥させます。 </li><li>ウォーム40 mLの変性溶液（70％ホルムアミド/ 2×SSC; pH7.0）で30分間ガラスコプリンジャーです。 </li><li> 1〜1.5分間予熱した変性溶液中でスライドをインキュベートします。 </li><li>直ちに変性プロセスを停止するとともに、再アニーリングから変性染色体を防ぐために2分間氷冷70％エタノールにスライドを浸します。 </li><li> 2分間80％エタノール中に置き、次いで室温で2分間、100％エタノールにすることによってスライドを脱水。 </li><li> 80℃（±2℃）に設定された水浴中でSKYプローブ（メーカーによって供給されたバイアル＃1）変性7分間、その後すぐに10分間37℃に設定し、異なる水浴中に配置します。 </li></ol></li><li>プローブのハイブリダイゼーション<ol><li>変性した染色体上に変性SKYプローブの10μLを追加します。 </li><li>空気の泡がカバースリップの下に閉じ込めされないように、慎重にSKYプローブ上に18ミリメートル×18ミリメートルのガラスカバースリップを配置。 </li><li>ゴムセメントでカバースリップの端をシールします。 </li><li>加湿遮光チャンバー内でスライドを置き、36時間〜24時間、37℃の暗所でインキュベートします。 </li></ol></li><li>ハイブリダイゼーション後の洗浄及び蛍光検出<ol><li>カバースリップを乱すことなく、非常に慎重にゴムセメントを削除します。 </li><li>振盪しながら5分間72℃（±2℃）で予熱した迅速な洗浄溶液（0.4×のSSC）にスライドを置きます。洗浄液III（4×SSC / 0.1％のTween 20）にスライドを浸し、振とうしながら1分間インキュベートします。 </li><li>オプション：ブロッキング試薬の80μLを追加します。ハイブリダイゼーションの領域上に（製造業者によって供給されたバイアル＃2）の2​​4mm×60ミリメートルのプラスチックカバースリップを置き、加湿チャンバー内で37℃で30分間、暗所でインキュベートします。 </li><li>静かにプラスチックカバースリップを削除し、5分間予熱した（45ºC）洗浄液IIIとスライドを洗います。 </li><li> 80μLのCy5の染色試薬（メーカーによって供給されたバイアル＃3）を適用し、24ミリメートル×60ミリメートルのプラスチック製カバースリップを置き、加湿チャンバー内で40分間、暗所で37℃でインキュベートします。 </li><li> （4×SSC / 0.1％Tween 20）で予熱した（45ºC）洗浄液IIIを含有するガラスコプリンジャーにスライドを浸漬し、振盪しながら2分間、水浴中で45℃でインキュベートします。この洗浄工程を3回繰り返します。 </li><li> 80μLのCy5.5の染色試薬（メーカーによって供給されたバイアル＃4）を適用し、24ミリメートル×60ミリメートルのプラスチック製カバースリップを置き、加湿チャンバー内で40分間、暗所で37℃でインキュベートします。 </li><li> （予め温めてスライドを3回洗浄45ºC）洗浄液III。 </li><li>余分な水分を排出するためにペーパータオルに対する傾斜位置でスライドを保持します。 20μL抗フェードDAPI試薬（メーカーによって供給されたバイアル＃5）を追加し、慎重に気泡を導入することなく、24ミリメートル×60ミリメートルのガラスカバースリップを配置。マニキュアでカバースリップの端をシールし、SKY画像を取り込むために装備落射蛍光顕微鏡で観察します。 </li></ol></li></ol>

<ol>
	<li>Kodama, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+chromosome+aberrations+in+atomic+bomb+survivors:+results+from+25+years+of+investigation.">Stable chromosome aberrations in atomic bomb survivors: results from 25 years of investigation.</a> Radiat Res. 156, 337-346 (2001).</li><li>Lucas, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+signature+for+ionizing+radiation.">Cytogenetic signature for ionizing radiation.</a> Int J Radiat Biol. 73, 15-20 (1998).</li><li>Zaccaria, A., Barbieri, E., Mantovani, W., Tura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+radiation-induced+aberrations+in+patients+with+Hodgkin's+disease.+Possible+correlation+with+second+malignancy?.">Chromosome radiation-induced aberrations in patients with Hodgkin's disease. Possible correlation with second malignancy?.</a> Boll. Ist. Sieroter. Milan. 57, 76-83 (1978).</li><li>Fisher, N. L., Starr, E. D., Greene, T., Hoehn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+and+limitations+of+chromosome+banding+in+pre-+and+postnatal+service+cytogenetics.">Utility and limitations of chromosome banding in pre- and postnatal service cytogenetics.</a> Am J Med Genet. 5, 285-294 (1980).</li><li>Hande, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+chromosome+aberrations+persist+in+individuals+many+years+after+occupational+exposure+to+densely+ionizing+radiation:+an+mFISH+study.">Complex chromosome aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: an mFISH study.</a> Genes Chromosomes. Cancer. 44, 1-9 (2005).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+detection+of+RNA-DNA+hybrid+molecules+in+cytological+preparations.">Formation and detection of RNA-DNA hybrid molecules in cytological preparations.</a> Proc Natl Acad Sci U S A. 63, 378-383 (1969).</li><li>Bauman, J. G., Wiegant, J., Borst, P., van, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochromelabelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA.</a> Exp Cell Res. 128, 485-490 (1980).</li><li>Hopman, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-color+detection+of+two+target+DNAs+by+non-radioactive+in+situ+hybridization.">Bi-color detection of two target DNAs by non-radioactive in situ hybridization.</a> Histochemistry. 85, 1-4 (1986).</li><li>Nederlof, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-color+fluorescence+in+situ+hybridization+for+the+simultaneous+detection+of+multiple+nucleic+acid+sequences.">Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences.</a> Cytometry. 10, 20-27 (1989).</li><li>Nederlof, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fluorescence+in+situ+hybridization.">Multiple fluorescence in situ hybridization.</a> Cytometry. 11, 126-131 (1990).</li><li>Szeles, A., Joussineau, S., Lewensohn, R., Lagercrantz, S., Larsson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+spectral+karyotyping+(SKY)+in+biodosimetry+for+the+triage+situation+following+gamma+irradiation.">Evaluation of spectral karyotyping (SKY) in biodosimetry for the triage situation following gamma irradiation.</a> Int J Radiat Biol. 82, 87-96 (2006).</li><li>Camparoto, M. L., Ramalho, A. T., Natarajan, A. T., Curado, M. P., Sakamoto-Hojo, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+analysis+by+the+FISH-painting+method+for+retrospective+dose+reconstruction+in+individuals+exposed+to+ionizing+radiation+10+years+after+exposure.">Translocation analysis by the FISH-painting method for retrospective dose reconstruction in individuals exposed to ionizing radiation 10 years after exposure.</a> Mutat. Res. 530, 1-7 (2003).</li><li>Edwards, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critique+of+'Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">A critique of 'Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 78, 867-871 (2002).</li><li>Bauchinger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+exercise+on+the+use+of+FISH+chromosome+painting+for+retrospective+biodosimetry+of+Mayak+nuclear-industrial+personnel.">Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.</a> Int J Radiat Biol. 77, 259-267 (2001).</li><li>Hieber, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+rearrangements+in+post-Chernobyl+papillary+thyroid+carcinomas:+evaluation+by+spectral+karyotyping+and+automated+interphase+FISH.">Chromosomal rearrangements in post-Chernobyl papillary thyroid carcinomas: evaluation by spectral karyotyping and automated interphase FISH.</a> J Biomed. Biotechnol. 2011, 693691 (2011).</li><li>Cohen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=detection+of+chromosome+rearrangements+in+two+cases+of+tMDS+with+a+complex+karyotype.">detection of chromosome rearrangements in two cases of tMDS with a complex karyotype.</a> Cancer Genet Cytogenet. 138, 128-132 (2002).</li><li>Pathak, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vitamin+E+Analog+Gamma-Tocotrienol+(GT3)+Suppresses+Radiation-Induced+Cytogenetic+Damage.">The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage.</a> Pharm Res. , (2016).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, 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=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61, 387-393 (1996).</li><li>Deng, W., Tsao, S. W., Lucas, J. N., Leung, C. S., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+improving+metaphase+chromosome+spreading.">A new method for improving metaphase chromosome spreading.</a> Cytometry A. 51, 46-51 (2003).</li><li>Hande, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+exposure+to+densely+ionizing+radiation+leaves+a+unique+permanent+signature+in+the+genome.">Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome.</a> Am J Hum Genet. 72, 1162-1170 (2003).</li><li>Tug, E., Kayhan, G., Kan, D., Guntekin, S., Ergun, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+long-term+effects+of+ionizing+radiation+through+measurement+of+current+sister+chromatid+exchange+(SCE)+rates+in+radiology+technologists,+compared+with+previous+SCE+values.">The evaluation of long-term effects of ionizing radiation through measurement of current sister chromatid exchange (SCE) rates in radiology technologists, compared with previous SCE values.</a> Mutat. Res. 757, 28-30 (2013).</li><li>Kanda, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fluorescent+chromosome+painting+using+the+peroxidase/diaminobenzidine+(DAB)+reaction.">Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction.</a> Int J Radiat Biol. 73, 529-533 (1998).</li><li>Kakazu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+spectral+colour+banding+in+cytogenetic+analysis.">Development of spectral colour banding in cytogenetic analysis.</a> Lancet. 357, 529-530 (2001).</li><li>Rithidech, K. N., Honikel, L., Whorton, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mFISH+analysis+of+chromosomal+damage+in+bone+marrow+cells+collected+from+CBA/CaJ+mice+following+whole+body+exposure+to+heavy+ions+(56Fe+ions).">mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (56Fe ions).</a> Radiat Environ Biophys. 46, 137-145 (2007).</li><li>Abrahams, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+FISHing+of+transgenic+mice+recommended:+FISH+and+SKY+define+BAC-mediated+balanced+translocation.">Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation.</a> Genesis. 36, 134-141 (2003).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+scoring+of+FISH-&amp;#34;painted&amp;#34;+chromosome-type+exchange+aberrations.">On the scoring of FISH-&amp;#34;painted&amp;#34; chromosome-type exchange aberrations.</a> Mutat. Res. 307, 345-353 (1994).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+"painting"+patterns+resulting+from+complex+exchanges.">FISH "painting" patterns resulting from complex exchanges.</a> Mutat. Res. 312, 51-60 (1994).</li><li>Tucker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAINT:+a+proposed+nomenclature+for+structural+aberrations+detected+by+whole+chromosome+painting.">PAINT: a proposed nomenclature for structural aberrations detected by whole chromosome painting.</a> Mutat. Res. 347, 21-24 (1995).</li><li>Knehr, S., Zitzelsberger, H., Bauchinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH-based+analysis+of+radiation-induced+chromosomal+aberrations+using+different+nomenclature+systems.">FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems.</a> Int J Radiat Biol. 73, 135-141 (1998).</li><li>Lucas, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+translocation+frequency+analysis+in+humans+decades+after+exposure+to+ionizing+radiation.">Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation.</a> Int J Radiat Biol. 62, 53-63 (1992).</li><li>Braselmann, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKY+and+FISH+analysis+of+radiation-induced+chromosome+aberrations:+a+comparison+of+whole+and+partial+genome+analysis.">SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.</a> Mutat. Res. 578, 124-133 (2005).</li><li>Sokolova, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+multitarget,+multicolor+fluorescence+in+situ+hybridization+assay+for+the+detection+of+urothelial+carcinoma+in+urine.">The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.</a> J Mol Diagn. 2, 116-123 (2000).</li><li>Lindholm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodosimetry+after+accidental+radiation+exposure+by+conventional+chromosome+analysis+and+FISH.">Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH.</a> Int J Radiat Biol. 70, 647-656 (1996).</li></ol>

The authors declare that they have no competing financial interests.

いくつかの重要なステップは、mFISHとSKYの成功を決定します。最初の最も重要なステップは、骨髄単核細胞のin vivoでの有糸分裂停止のためのコルヒチン処理を最適化することです。コルヒチン濃度と処理時間個別に、または分裂指数だけでなく、染色体凝縮-2効果的な染色体の絵画のための重要な前提条件を決定コンサートインチ高コルヒチン濃度や長い処理時間は、適切な変性およ?...

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations1, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions2. Studies by various groups have shown that stable aberrations are associated with the pathogenesis and development of a number of diseases, including cancer3. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation4. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry5. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements. 
In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes6. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.
Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic ("cryptic") rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.
The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)11-14, radiation carcinogenesis risk assessment15, as well as detection and risk estimation of radiotherapy-related secondary cancer16. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure17. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

<table><tbody><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>221198-100ML</td><td></td></tr><tr><td>SSC Buffer 20&amp;times; Concentrate</td><td>Sigma-Aldrich</td><td>S6639-1L</td><td></td></tr><tr><td>SKY Laboratory Reagent for Mouse</td><td>Applied Spectral Imaging</td><td>FPRPR0030/M40</td><td></td></tr><tr><td>CAD - Concentrated Antibody Detection Kit</td><td>Applied Spectral Imaging</td><td>FPRPR0033</td><td></td></tr><tr><td>Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua</td><td>Applied Spectral Imaging</td><td>FPRPR0182/10</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>12-545B</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>Hydrochloric acid, 37%, Acros Organics</td><td>Fisher Scientific</td><td>AC45055-0025&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Glass Staining Dishes&amp;nbsp; with Screw Cap</td><td>Fisher Scientific</td><td>08-816</td><td></td></tr><tr><td>KaryoMAX Potassium Chloride Solution&amp;nbsp;</td><td>Life Technologies</td><td>10575-090</td><td></td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Colcemid powder</td><td>Fisher Scientific</td><td>50-464-757&amp;nbsp;</td><td></td></tr><tr><td>Histopaque-1083&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10831</td><td></td></tr><tr><td>Shepherd Mark I, model 25&lt;sup&gt; 137&lt;/sup&gt;Cs irradiator</td><td>J. L. Shepherd &amp;amp; Associates</td><td>Model 484B</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD Biosciences</td><td>647911</td><td></td></tr><tr><td>Ethyl Alcohol, 200 Proof</td><td>Fisher Scientific</td><td>MEX02761</td><td></td></tr><tr><td>PBS, (1x PBS Liq.), w/o Calcium and Magnesium</td><td>Fisher Scientific</td><td>ICN1860454</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10-437-010</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454SK-4</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>AC295320010</td><td></td></tr><tr><td>Zeiss Microscope</td><td>Zeiss</td><td>AXIO Imager.Z2</td><td></td></tr></tbody></table>

detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

全身照射は、照射したマウスの骨髄細胞における多数の染色体異常を誘発します。現在のプロトコルは、放射線暴露後の骨髄細胞のin vivoでの有糸分裂停止のために最適化され、密度勾配遠心分離、中期細胞スプレッドの調製、およびそれに続くによって照射されたマウス骨髄単核細胞の単離の後脚から骨髄細胞の採取mFISHとSKY技術による放射線誘発安定した染?...

Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

複数の蛍光によるインター染色体安定型異常の検出<em&gt;その場で</em&gt;ハイブリダイゼーション（mFISH）と照射されたマウスにおけるスペクトル核型分析（SKY）

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

detection-inter-chromosomal-stable-aberrations-multiple-fluorescence

Universidad San Sebastian

Research

JoVE Journal

Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

9.8K ビュー.  University of Arkansas for Medical Sciences. The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation. 

ビデオ: Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

がん研究

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

マイクロ レーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

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

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

ヒト大腸癌細胞におけるDNA損傷および修復病巣の免疫蛍光イメージング

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

ヒト末梢血リンパ球におけるγ-H2AX Fociアッセイの自動微視的スコアリング法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

免疫・感染学

マウスの前期停止卵子におけるDNA損傷の同定

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

著者スポットライト:哺乳類の卵母細胞および着床前胚におけるDNA損傷応答の理解 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

マウス卵母細胞におけるDNA二本鎖切断の検出

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

発生生物学

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

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

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 ...

複数の蛍光によるインター染色体安定型異常の検出

要約

さらに動画を探す

この動画の章

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

マイクロレーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

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

ヒト大腸癌細胞におけるDNA損傷および修復病巣の免疫蛍光イメージング

ヒト末梢血リンパ球におけるγ-H2AX Fociアッセイの自動微視的スコアリング法

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

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

複数の蛍光によるインター染色体安定型異常の検出

要約

さらに動画を探す

この動画の章

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

マイクロ レーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

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

ヒト大腸癌細胞におけるDNA損傷および修復病巣の免疫蛍光イメージング

ヒト末梢血リンパ球におけるγ-H2AX Fociアッセイの自動微視的スコアリング法

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

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

マイクロレーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション