Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium f&#252;r Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (CONSERT, CLINIGENE and PERSIST). We thank Ina Kutschera for demonstrating the protocol technique in the video.

リンカーカセット1。準備（LC） <ol><li> LC1オリゴヌクレオチド（ 表1）の40μL、LC2オリゴヌクレオチド（適切な制限酵素オーバーハングを有する表1）の40μL、110μLのTris-HCl（100ミリモル、pH7.5）にし、10μlの250のMgCl 2を混ぜる。 </li><li> 5分間95℃でインキュベートし、反応を室温までゆっくりと冷ましてみましょう。 300μlのH 2 Oを加え、遠心分離フィルターにdsLinker-DNAを集中。 0.2 PCRチューブで準備リンカーカセットの溶出液を、アリコート10μlに80μlのH 2 Oを追加します。 </li></ol>ベクターゲノム接合の2。プレ増幅<ol><li>分析される各サンプルを50μlのPCR反応物を調製するため。 <ol><li> DNAサンプルの濃度を決定する。 0.2ミリリットルのPCRチューブにDNAをピペットXμL（LAM/100-1のため1〜1,000 ngの、nrLAM 000 NG）。 DNAの量は、各サンプルにし、RANに等しくなるはずである0.5から25μLのGE。 </li><li> 表2に記載したようにPCRマスターミックスを調製ミックス（50 - x）を0.2ミリリットルのPCRチューブ中の各DNAサンプルとマスターミックス液。 </li></ol></li><li> 表2に例示したPCR条件を用いて、プリアンプするベクターゲノム接合部。PCRの完了後、各PCRチューブおよびPCR再放送番組に0.5μlのTaqポリメラーゼを加える。 PCR産物は、-20℃で最大4日間または長期間、4℃で保存することができる</li></ol> PCR産物の3。磁気分離<ol><li>磁性ビーズの作製<ol><li>ピペット1.5ミリリットルチューブにストレプトアビジン被覆磁性ビーズを20μl（200μgの）、室温で磁性粒子分離（MPS）に1分間さらすこと。上清を捨てる。 </li><li> MPSからチューブを外し、40μLPSB / 0.1％のBSA（pH7.5）中の磁気ビーズを懸濁します。 1分間のMPSに公​​開し、上澄みを捨てる。一度この手順を繰り返します。 </li><li>洗浄ビーズWI3 MのLiCl溶液20μl（3 MのLiCl、10mMトリス-HCl、1mMのEDTA）番目1分間のMPSに公​​開し、上澄みを捨てる。 6 M LiCl溶液（6M LiClと10 mMのトリス-HCl、1mMのEDTA）50μlに再懸濁ビーズ。 </li></ol></li><li>作製した磁気ビーズを50μlのステップ2.2からの全体のPCR反応を混ぜる。少なくとも室温で2時間、水平振とう器（300 rpm）の上でインキュベートする。このステップでは、コーティングされたビーズ（DNA-ビーズ複合体）をストレプトアビジンビオチン化PCR産物に結合できます。 DNAビーズ複合体を一晩振とう器上でインキュベートし、又は最大4日間4℃で保存することができる。 </li><li>室温で1分間のMPSへのDNA-ビーズ複合体を公開、上清を除去し、100μlのH 2 O中のDNA-ビーズ複合体を再懸濁すぐにLAMやnrLAMステップ5ステップ4に進みます。 </li></ol> 4。LAM-手順<ol><li>二本鎖DNA（dsDNAの）合成（LAMのみ） <ol><li> 1分間のMPSとDIS、ステップ3.3からDNA-ビーズ複合体を公開カード上清。 H 2 Oを8.25μL、1μlの10倍ヘキサバッファー、0.25μLのdNTP（10 mM）および0.5μL（2 U）ノウポリメラーゼを追加します。 1時間37℃でインキュベートする。 </li><li> H 2 Oを90μLを加え、1分間のMPSに公開。上澄み液を捨て、100μlのH 2 O中のDNA-ビーズ複合体を再懸濁</li></ol></li><li>制限消化<ol><li> 1分間のMPSへのDNA-ビーズ複合体を露出させ、上澄みを捨てる。制限酵素バッファー、制限酵素0.5μlの1μLの10倍、8.5μlのH 2 Oを加え、1時間反応をインキュベートする。ステップ4.1.2を繰り返します。 注：制限酵素のメーカーが推奨する温度で反応をインキュベートする。内に存在するか、目的のDNAで前増幅のために使用されるプライマー結合部位の下流に制限部位が存在しないことを確認してください。適当な制限酵素/制限酵素の組み合わせの選択のために9を参照してください。 </li></ol></li><li> DSリンカ（LK）の連結<ol><li> 1分間のMPSへのDNA-ビーズ複合体を露出させ、上澄みを捨てる。 5μlのH 2 O、1μLの10X FastLinkバッファー、1μlのATP（10mM）を、ステップ1.2〜2μLリンカーカセット、および1μlの高速リンクDNAリガーゼ（2単位/μl）を追加します。 5分間室温でインキュベートする。ステップ4.1.2を繰り返します。 </li></ol></li><li>合成したdsDNAの変性<ol><li> 1分間のMPSへのDNA-ビーズ複合体を露出させ、上澄みを捨てる。 5μLの0.1NのNaOHに再懸濁DNA-ビーズ複合体。水平シェーカー上で室温で5分間インキュベートする。 </li><li> 1分間のMPSへのDNA-ビーズ複合体を公開し、新しい1.5 mlチューブに上清を含む前置増幅、ベクターゲノム接合を収集します。すぐに-20℃で、ステップ6またはストア上清を進める</li></ol></li></ol> 5。nrLAM - 手順<ol><li>一本鎖リンカーのライゲーション（SSLC）（nrLAMのみ） <ol><li> MPSにステップ3.3からDNA-ビーズ複合体を公開1分間、上澄みを捨てる。 6.5μlのH 2 Oを追加し、1μlを10倍反応バッファーを、CircLigase 0.5μLのMnCl 2（50mm）で、0.5のATP（1 mM）をμL、1μlのssLinkerオリゴヌクレオチドおよび0.5μlのCircLigase（100 U /μL）。 1時間60℃でインキュベートする。 </li><li> H 2 Oを90μLを加え、1分間のMPSに公開。上澄み液を捨て、100μlのH 2 O中のDNA-ビーズ複合体を洗うこの場合も、1分間のMPSに公開上清を廃棄し、10μlのH 2 O中のDNA-ビーズ複合体を再懸濁</li></ol></li></ol> 6。指数関数的増幅I <ol><li>分析される各サンプルを50μlのPCR反応物を調製するため。 <ol><li> （ステップ4.4.2（LAM）または5.1.2（nrLAM）から）、ピペット2μlのテンプレートDNA 0.2ミリリットルのPCRチューブに。 </li><li> 表3に記載したようにPCRマスターミックスを調製する。ステップ6.1.1から各サンプルにマスターミックス48μLを追加し、PCR conditioによりベクターゲノム接合部を増幅する表3に例示したナノは、PCR産物は、-20℃で最大4日間または長期間、4℃で保存することができる。 </li></ol></li></ol> PCR産物の7。磁気分離<ol><li> 3.1.3 - ステップ3.1.1に例示したように磁気ビーズを準備します。 6 M LiCl溶液（6M LiClと10 mMのトリス-HCl、1mMのEDTA）の（nrLAM用）（LAM用）20μLまたは50μLに再懸濁ビーズ。作製した磁気ビーズ（ステップ7.1）でステップ6.1.2から20μL（LAM）またはPCR反応液50μl（nrLAM）を混合し、室温で2時間、水平振盪（300回転）上でインキュベートする。 DNAビーズ複合体を一晩振とう器上でインキュベートし、又は最大4日間4℃で保存することができる。 </li><li> 1分間のMPSへのDNA-ビーズ複合体を公開、上清を除去し、100μlのH 2 O中のDNA-ビーズ複合体を再懸濁1分間のMPSへのDNA-ビーズ複合体を露出させ、上澄みを捨てる。 </li><li> 20μL（LAM）または5μL（nrLAM）0.1NのNaOHに再懸濁DNA-ビーズ複合体。 Incu水平シェーカー上で室温で10分間BATE、1分間のMPSに公​​開し、新しい1.5 mlチューブに上清を含む増幅されたDNAを収集します。すぐに-20℃でステップ8.1またはストア上清を進める</li></ol> 8。指数関数的増幅II <ol><li>分析される各サンプルを50μlのPCR反応物を調製するため。 <ol><li> 0.2ミリリットルのPCRチューブに（ステップ7.3から）ピペット2μlのテンプレートDNA。 </li><li> 表4に記載したようにPCRマスターミックスを調製する。ステップ8.1.1からの各サンプルにマスターミックス48μlを添加し、表4に例示したPCR条件によってベクターゲノム接合を増幅する。PCR産物は、最大4 4℃で保存することができる-20℃での日または長期</li></ol></li><li>可視化するために（NR）LAM-PCR産物を2％アガロースゲル上で、ステップ8.1.2からのPCR産物の10μlを負荷。バンドが可視である場合、高解像度のゲル上でLAM-PCR産物を10μlを分析する。注意：のEthidium臭化変異原性である。非常に慎重に作業し、常に適切な手袋を着用してください。 </li></ol>ハイスループット配列決定のための9。準備<ol><li> （NR）LAM-PCR産物の精製<ol><li>室温AMPure XP磁性ビーズ44μlのステップ8.1.2から40μlのPCR産物を混合します。室温で5分間インキュベートし、さらに2分間、MPSに公​​開。 </li><li>上澄み液を捨て、MPSに200μlの70％エタノールで二度洗い。 </li><li>上澄み液を捨て、30μlのH 2 O中のDNA-ビーズ複合体を再懸濁1分間インキュベートし、新鮮な0.2ミリリットルチューブに上清を移す。精製したDNAの濃度を測定する。 </li></ol></li><li> Fusionprimer PCRは、特定のアダプターの配列を決定する追加します。 <ol><li>分析される各サンプルを50μlのPCR反応物を調製するため。 </li><li> 0.2ミリリットルのPCRチューブにDNAをピペットXμL（40 NG）。 DNAの量は、各試料中および0.5〜25μLの範囲で同じにする必要があります。 </li><li>表5に記載したようにPCRマスターミックスを調製入れる。（50 - x）が、ステップ9.2.2からの各サンプルにマスターミックス液を、表5に例示したPCR条件によって（NR）LAM-PCR産物をシーケンシングアダプターを導入するPCR産物-20℃で最大4日間または長期間、4℃で保存することができる。 </li><li> Fusionprimer-PCR産物を可視化するためには、2％アガロースゲル上で、ステップ9.2.3からのPCR産物10μlのロード。 9.1.3 - 9.1.1の手順に記載されるように、残りのPCR産物を精製する。正確Fusionprimer-PCR産物の濃度およびフラグメントサイズを定量化するための自​​動化された高分解能電気泳動デバイス上の工程9.4から1μlの精製PCR産物を分析する。 </li></ol></li></ol>

<ol>
	<li>Schmidt, 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=Detection+and+direct+genomic+sequencing+of+multiple+rare+unknown+flanking+DNA+in+highly+complex+samples.">Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.</a> Hum Gene Ther. 12, 743-749 (2001).</li><li>Schmidt, 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=High-resolution+insertion-site+analysis+by+linear+amplification-mediated+PCR+(LAM-PCR).">High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR).</a> Nat Methods. 4, 1051-1057 (2007).</li><li>Schroeder, A. 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=HIV-1+integration+in+the+human+genome+favors+active+genes+and+local+hotspots.">HIV-1 integration in the human genome favors active genes and local hotspots.</a> Cell. 110, 521-529 (2002).</li><li>Wu, X., Li, Y., Crise, B., Burgess, 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=Transcription+start+regions+in+the+human+genome+are+favored+targets+for+MLV+integration.">Transcription start regions in the human genome are favored targets for MLV integration.</a> Science. 300, 1749-1751 (2003).</li><li>Vigdal, T. J., Kaufman, C. D., Izsvak, Z., Voytas, D. F., Ivics, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+physical+properties+of+DNA+affecting+target+site+selection+of+sleeping+beauty+and+other+Tc1/mariner+transposable+elements.">Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements.</a> J Mol Biol. 323, 441-452 (2002).</li><li>Lewinski, M. K., 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=Retroviral+DNA+integration:+viral+and+cellular+determinants+of+target-site+selection.">Retroviral DNA integration: viral and cellular determinants of target-site selection.</a> PLoS Pathog. 2, (2006).</li><li>Mueller, P. R., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+footprinting+of+a+muscle+specific+enhancer+by+ligation+mediated+PCR.">In vivo footprinting of a muscle specific enhancer by ligation mediated PCR.</a> Science. 246, 780-786 (1989).</li><li>Silver, J., Keerikatte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+of+polymerase+chain+reaction+to+amplify+cellular+DNA+adjacent+to+an+integrated+provirus.">Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.</a> Journal of virology. 63, 1924-1928 (1989).</li><li>Gabriel, 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=Comprehensive+genomic+access+to+vector+integration+in+clinical+gene+therapy.">Comprehensive genomic access to vector integration in clinical gene therapy.</a> Nature medicine. 15, 1431-1436 (2009).</li><li>Harkey, M. 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=Multiarm+high-throughput+integration+site+detection:+limitations+of+LAM-PCR+technology+and+optimization+for+clonal+analysis.">Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis.</a> Stem Cells Dev. 16, 381-392 (2007).</li><li>Wang, G. 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=DNA+bar+coding+and+pyrosequencing+to+analyze+adverse+events+in+therapeutic+gene+transfer.">DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.</a> Nucleic acids research. 36, (2008).</li><li>Paruzynski, 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=Genome-wide+high-throughput+integrome+analyses+by+nrLAM-PCR+and+next-generation+sequencing.">Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.</a> Nat. Protocols. 5, 1379-1395 (2010).</li><li>Hacein-Bey-Abina, 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=Insertional+oncogenesis+in+4+patients+after+retrovirus-mediated+gene+therapy+of+SCID-X1.">Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.</a> J Clin Invest. , 3132-3142 (2008).</li><li>Hacein-Bey-Abina, 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=LMO2-associated+clonal+T+cell+proliferation+in+two+patients+after+gene+therapy+for+SCID-X1.">LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.</a> Science. 302, 415-419 (2003).</li><li>Howe, S. J., 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=Insertional+mutagenesis+combined+with+acquired+somatic+mutations+causes+leukemogenesis+following+gene+therapy+of+SCID-X1+patients.">Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.</a> J Clin Invest. 118, 3143-3150 (2008).</li><li>Cartier, 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=Hematopoietic+stem+cell+gene+therapy+with+a+lentiviral+vector+in+X-linked+adrenoleukodystrophy.">Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.</a> Science. 326, 818-823 (2009).</li><li>Matrai, J., 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=Hepatocyte-targeted+expression+by+integrase-defective+lentiviral+vectors+induces+antigen-specific+tolerance+in+mice+with+low+genotoxic+risk.">Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.</a> Hepatology. 53, 1696-1707 (2011).</li><li>Penaud-Budloo, 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=Adeno-associated+virus+vector+genomes+persist+as+episomal+chromatin+in+primate+muscle.">Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle.</a> Journal of virology. 82, 7875-7885 (2008).</li><li>Kaeppel, 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=A+largely+random+AAV+integration+profile+after+LPLD+gene+therapy.">A largely random AAV integration profile after LPLD gene therapy.</a> Nature medicine. 19, 889-891 (2013).</li><li>Voigt, K., 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=Retargeting+sleeping+beauty+transposon+insertions+by+engineered+zinc+finger+DNA-binding+domains.">Retargeting sleeping beauty transposon insertions by engineered zinc finger DNA-binding domains.</a> Mol Ther. 20, 1852-1862 (2012).</li><li>Yanez-Munoz, R. J., 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=Effective+gene+therapy+with+nonintegrating+lentiviral+vectors.">Effective gene therapy with nonintegrating lentiviral vectors.</a> Nature medicine. 12, 348-353 (2006).</li><li>Boztug, K., 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=Stem-Cell+Gene+Therapy+for+the+Wiskott-Aldrich+Syndrome.">Stem-Cell Gene Therapy for the Wiskott-Aldrich Syndrome.</a> N Engl J Med. 363, 1918-1927 (2010).</li><li>Deichmann, 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=Vector+integration+is+nonrandom+and+clustered+and+influences+the+fate+of+lymphopoiesis+in+SCID-X1+gene+therapy.">Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.</a> J Clin Invest. 117, 2225-2232 (2007).</li><li>Schwarzwaelder, K., 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=Gammaretrovirus-mediated+correction+of+SCID-X1+is+associated+with+skewed+vector+integration+site+distribution+in+vivo.">Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.</a> J Clin Invest. 117, 2241-2249 (2007).</li><li>Gabriel, 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=An+unbiased+genome-wide+analysis+of+zinc-finger+nuclease+specificity.">An unbiased genome-wide analysis of zinc-finger nuclease specificity.</a> Nat Biotechnol. 29, 816-823 (2011).</li><li>Dieter, S. 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=Distinct+types+of+tumor-initiating+cells+form+human+colon+cancer+tumors+and+metastases.">Distinct types of tumor-initiating cells form human colon cancer tumors and metastases.</a> Cell Stem Cell. 9, 357-365 (2011).</li><li>Montini, E., 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=Hematopoietic+stem+cell+gene+transfer+in+a+tumor-prone+mouse+model+uncovers+low+genotoxicity+of+lentiviral+vector+integration.">Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.</a> Nature biotechnology. 24, 687-696 (2006).</li><li>Zavidij, O., 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+long-term+blood+formation+by+stem+cells+in+murine+steady-state+hematopoiesis.">Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.</a> Stem Cells. 30, 1961-1970 (2012).</li><li>Martins, V. 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=Thymus-autonomous+T+cell+development+in+the+absence+of+progenitor+import.">Thymus-autonomous T cell development in the absence of progenitor import.</a> J Exp Med. 209, 1409-1417 (2012).</li><li>Arens, 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=Bioinformatic+clonality+analysis+of+next-generation+sequencing-derived+viral+vector+integration+sites.">Bioinformatic clonality analysis of next-generation sequencing-derived viral vector integration sites.</a> Hum Gene Ther Methods. 23, 111-118 (2012).</li></ol>

The authors have nothing to disclose.

LAM-PCR法は、既知のDNA領域に隣接する未知のDNA配列を同定することができる。なぜなら公知のDNA配列にハイブリダイズする特異的プライマーを用いた接合の前増幅から得られる高感度のではなく、単一細胞レベルまでさえ稀接合を増幅して検出することができる。反して、ポリクローナル状況でLAM-PCRは、単一の反応において異なる数千の接合部を増幅することができる。 <p class="jove_content"...

PCR（LAM-PCR）媒介線形増幅は、任意の起源の既知のDNAに隣接する未知の隣接DNAを同定し、特徴づけることができます。具体的には、LAM-PCRは、宿主ゲノム内に1,2（IS）ウイルスベクター組込み部位を局在化するために開発された。レトロウイルスやトランスポゾンのような遺伝的要素が（半）ランダムに3-6で宿主ゲノムにそれらのゲノムを統合。多くの場合、これらのベクターは、集積正確に位置を知ることが決定的である。 LAM-PCRは、ライゲーション媒介PCR法7およびその変種またはインバースPCR 8のような代替技術よりも優れていることが証明されています。この方法の感度およびロバスト性は、ベクターゲノム接合の初期前置増幅、増幅されたPCR産物の磁気選択から生じる。上記の代替的な方法と同様に、LAM-PCR法は、IS 9月11日の検索能力に偏りを導入し、制限酵素の使用に依存している。このように、ISレパートリー（integrome）のサブセットのみが1の反応で検出することができる。このバイアスは、制限酵素9の最適な組み合わせを使用して所与の試料の平行分析することによって最小化される。近年、非制限LAM-PCR（nrLAM-PCR）と呼ばれる技術の変形は、制限酵素の使用を回避し、単一の反応9,12試料の公平なゲノムワイドな解析を可能にする開発されている。 過去には、LAM-PCRは、原因レトロウイルスは、臨床遺伝子治療試験13-15における少数の患者における白血病を引き起こすIS同定するために使用されている。それ以来、LAM-PCRを識別するように適合されている他の組込みベクター（レンチウイルスベクター、トランスポゾン）にも受動的にアデノ随伴ベクター（AAV）またはインテグラーゼ欠損型レンチウイルスベクター（IDLV）のようなベクトルを統合する統合パターンを識別するためにからのものである16 -21。 LAM-PCR法の応用を広げている。伝統的なLY、技術が広く遺伝子治療を受けた患者における遺伝子改変細胞のクローン組成を研究するために、またはそれらの積分15,16,22-24挙動を解明することにより、新規なベクター系の生物学的安全性を評価するために使用される。最近では、LAM-PCRはIDLV捕獲アッセイ25でデザイナーのヌクレアーゼの特異性とオフターゲット活性を測定することができました。 また、LAM-PCR法は簡単に生物中で時間をかけて導入細胞の運命を追跡することができます。このことは、癌原遺伝子、ならびに腫瘍抑制遺伝子を同定するために、また、造血又は癌幹細胞生物学26-28を研究することを可能にする。少なくとも最後のではなく、LAM-PCR法は、ヒト29（および未発表データ）におけるT細胞受容体の多様性を研究するようになった。 テクノロジーの本質的なパワーは、一塩基rで未知のフランキングDNAの何百万人を特徴づけることができ、深いシーケンシング技術にメソッドをリンクすることによって強化されている全ゲノム中esolution。以下のプロトコルでは、レンチウイルスベクターを特定するために例示的に知られていない隣接DNAの増幅および識別は、ステップバイステップを説明します。プロトコルで使用したオリゴヌクレオチドを表1に示す。抽出DNA又は任意の供給源のcDNAをLAM-PCRおよびnrLAM-PCRのためのDNA鋳型として使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:527px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Taq DNA Polymerase</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3001.5000</td>
			<td>Alternative Taq Polymerases may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR Buffer</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:120px;">201203</td>
			<td>Use of this buffer is recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">dNTP-Mixture</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3015.4020</td>
			<td>or any other dNTPs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Oligonucleotides (Primers)</td>
			<td style="width:203px;">MWG Biotech</td>
			<td style="width:120px;"></td>
			<td>HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">11206D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PBS</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:120px;">14190-086</td>
			<td>0.1 % wt/vol BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6M LiCl</td>
			<td style="width:203px;">Roth</td>
			<td style="width:120px;">3739.1</td>
			<td>10 mM Tris-HCl&#160; (pH 7.5)/1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tris-HCl, pH 7.5</td>
			<td style="width:203px;">USB Corporation&#160;</td>
			<td style="width:120px;">22637</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A1103,0250</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Klenow Polymerase</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">10104523001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Hexanucleotide mixture</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11277081001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Restriction endonuclease</td>
			<td style="width:203px;">NEB</td>
			<td style="width:120px;"></td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fast-Link DNA ligation kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">LK11025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CircLigase ssDNA Ligase Kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">CL4111K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NaOH</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">72068</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose LE</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11685660001</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TBE buffer</td>
			<td style="width:203px;">Amresco</td>
			<td style="width:120px;">0658</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethidium bromide</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A2273,0005</td>
			<td>Ethidium bromide is mutagenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">100 bp DNA Ladder</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">15628-050</td>
			<td>or any other DNA ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">20 mM NaCl</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">71393-1L</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158501</td>
			<td>for use with 1.5 ml Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158696</td>
			<td>for use with 96 well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-0.5, Ultracel-30 membrane</td>
			<td style="width:203px;">Millipore</td>
			<td>UFC503096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PerfectBlue Gelsystem Midi S</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;">40-1515</td>
			<td>or other electrophoresis system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TProfessional 96</td>
			<td style="width:203px;">Biometra</td>
			<td style="width:120px;">050-551</td>
			<td>or other Thermocycler for 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Orbital shaker KS 260 basic</td>
			<td style="width:203px;">IKA</td>
			<td style="width:120px;">2980200</td>
			<td>or other horizontal shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR softtubes 0.2 ml</td>
			<td style="width:203px;">Biozym Scientific GmbH</td>
			<td style="width:120px;">711082</td>
			<td>or other 0.2 ml PCR tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">1.5 ml tubes</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">12682</td>
			<td>or other 1.5 ml tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Gel documentation system</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;"></td>
			<td>or any other gel documentation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nanodrop ND-1000 spectrophotometer</td>
			<td style="width:203px;">Thermo Scientific</td>
			<td style="width:120px;">ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Spreadex EL1200 precast gel</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">3497</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:360px;">Submerged gel electrophoresis apparatus SEA 2000&#160;</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">2001E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2100 Electrophoresis Bioanalyzer</td>
			<td style="width:203px;">Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

linear amplification mediated pcr &#8211; localization of genetic elements and characterization of unknown flanking dna

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

LAM-PCRは、各接合のために定義された断片サイズを有するベクターゲノム接合部の増幅をもたらす。個々のPCR断片のサイズは、ゲノムDNA中の既知の位置と最も近い制限酵素認識部位の間の距離に依存する。これは、例えば 、ゲル電気泳動により分析試料中の増幅された接合部の多様性を可視化することができ、単一の（モノクローナル）、（オリゴクローナ）は、いくつか、又は複数?...

Watch this Scientific Journal Video about Linear Amplification Mediated PCR â€“ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

（LAM）-PCR媒介線形増幅は、ゲノムにウイルスベクターを統合の正確な位置を特定するために開発された方法である。技術などの遺伝子治療患者におけるクローン性のダイナミクスを研究するための優れた方法、新規ベクター技術のバイオセーフティ、T細胞の多様性、癌幹細胞モデルであるように進化している

線形増幅媒介のPCR  - 不明隣接するDNAの遺伝的要素と特性の局在

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

linear-amplification-mediated-pcr-localization-genetic-elements

Research

JoVE Journal

Biology

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

29.9K ビュー.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  （LAM）-PCR媒介線形増幅は、ゲノムにウイルスベクターを統合の正確な位置を特定するために開発された方法である。技術などの遺伝子治療患者におけるクローン性のダイナミクスを研究するための優れた方法、新規ベクター技術のバイオセーフティ、T細胞の多様性、癌幹細胞モデルであるように進化している

ビデオ: Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

培養一次マウス胚線維芽細胞におけるシグナル伝達分子のアデノウイルス媒介遺伝子除去

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

で遺伝子発現プロファイルを決定 C.エレガンスマイクロアレイとリアルタイムPCRを用いて

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

タンパク質-DNA相互作用の研究のための単一分子マニピュレーションとイメージングを組み合わせる

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNAエレクトロポレーション、筋線維の単離およびイメージング

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

ラットの肝臓におけるDNA鎖切断および酸化的DNA損傷を測定するためのインビボアルカリコメットアッセイおよび酵素修飾されたアルカリコメットアッセイで

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.