著者は、原稿の校正にジャクリーン · ローゼンバーグを認めると思います。この作品は DeClerck 屋の付与と米国保健社会福祉省/国立衛生研究によって支えられた (5R01 CA 129377 付与) と TJ マーテル財団。MH クバラはサバン研究所子供のロサンゼルスの病院での研究のキャリア開発奨励賞の受信者です。

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	<li>Ryding, A. D., Sharp, M. G., Mullins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+transgenic+technologies.">Conditional transgenic technologies.</a> J Endocrinol. 171 (1), 1-14 (2001).</li><li>Mantovani, A., Marchesi, F., Malesci, A., Laghi, L., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-associated+macrophages+as+treatment+targets+in+oncology.">Tumour-associated macrophages as treatment targets in oncology.</a> Nat Rev Clin Oncol. , (2017).</li><li>Franklin, R. 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+cellular+and+molecular+origin+of+tumor-associated+macrophages.">The cellular and molecular origin of tumor-associated macrophages.</a> Science. 344 (6186), 921-925 (2014).</li><li>Ruffell, B., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+therapeutic+resistance+in+cancer.">Macrophages and therapeutic resistance in cancer.</a> Cancer Cell. 27 (4), 462-472 (2015).</li><li>Sharma, P., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+immune+checkpoint+therapy.">The future of immune checkpoint therapy.</a> Science. 348 (6230), 56-61 (2015).</li><li>Mizuguchi, H., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+adenovirus-mediated+tetracycline-controllable+expression+system.">Characteristics of adenovirus-mediated tetracycline-controllable expression system.</a> Biochim Biophys Acta. 1568 (1), 21-29 (2001).</li><li>Meyer-Ficca, M. 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=Comparative+analysis+of+inducible+expression+systems+in+transient+transfection+studies.">Comparative analysis of inducible expression systems in transient transfection studies.</a> Anal Biochem. 334 (1), 9-19 (2004).</li><li>Wiederschain, 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=Single-vector+inducible+lentiviral+RNAi+system+for+oncology+target+validation.">Single-vector inducible lentiviral RNAi system for oncology target validation.</a> Cell Cycle. 8 (3), 498-504 (2009).</li><li>Wee, 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=PTEN-deficient+cancers+depend+on+PIK3CB.">PTEN-deficient cancers depend on PIK3CB.</a> Proc Natl Acad Sci U S A. 105 (35), 13057-13062 (2008).</li><li>Chen, Y., Stamatoyannopoulos, G., Song, C. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+CXCR4+by+inducible+small+interfering+RNA+inhibits+breast+cancer+cell+invasion+in+vitro.">Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro.</a> Cancer Res. 63 (16), 4801-4804 (2003).</li><li>Wiznerowicz, M., Trono, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+suppression+of+cellular+genes:+lentivirus+vector-mediated+drug-inducible+RNA+interference.">Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.</a> J Virol. 77 (16), 8957-8961 (2003).</li><li>Solari, V., 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=MYCN-dependent+expression+of+sulfatase-2+regulates+neuroblastoma+cell+survival.">MYCN-dependent expression of sulfatase-2 regulates neuroblastoma cell survival.</a> Cancer Res. 74 (21), 5999-6009 (2014).</li><li>Liao, Y., Tang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+RNAi+system+and+its+application+in+novel+therapeutics.">Inducible RNAi system and its application in novel therapeutics.</a> Crit Rev Biotechnol. 36 (4), 630-638 (2016).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods Mol Biol. 294, 15-22 (2005).</li><li>Jungi, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+chemotaxis+by+a+reversible+Boyden+chamber+eliminating+cell+detachment.">Assay of chemotaxis by a reversible Boyden chamber eliminating cell detachment.</a> Int Arch Allergy Appl Immunol. 48 (3), 341-352 (1975).</li><li>Jung, H. 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=Monoclonal+antibodies+against+autocrine+motility+factor+suppress+gastric+cancer.">Monoclonal antibodies against autocrine motility factor suppress gastric cancer.</a> Oncol Lett. 13 (6), 4925-4932 (2017).</li><li>Park, 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=Effect+of+Sorbus+commixta+on+the+invasion+and+migration+of+human+hepatocellular+carcinoma+Hep3B+cells.">Effect of Sorbus commixta on the invasion and migration of human hepatocellular carcinoma Hep3B cells.</a> Int J Mol Med. , (2017).</li><li>Schildberger, A., Rossmanith, E., Eichhorn, T., Strassl, K., Weber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes,+peripheral+blood+mononuclear+cells,+and+THP-1+cells+exhibit+different+cytokine+expression+patterns+following+stimulation+with+lipopolysaccharide.">Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide.</a> Mediators Inflamm. 2013, 697972 (2013).</li><li>Heil, T. L., Volkmann, K. R., Wataha, J. C., Lockwood, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+peripheral+blood+monocytes+versus+THP-1+monocytes+for+in+vitro+biocompatibility+testing+of+dental+material+components.">Human peripheral blood monocytes versus THP-1 monocytes for in vitro biocompatibility testing of dental material components.</a> J Oral Rehabil. 29 (5), 401-407 (2002).</li><li>Tsuchiya, 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=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>de Almeida, M. C., Silva, A. C., Barral, A., Barral Netto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+human+peripheral+blood+monocyte+isolation.">A simple method for human peripheral blood monocyte isolation.</a> Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).</li><li>Flo, R. W., 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=Negative+selection+of+human+monocytes+using+magnetic+particles+covered+by+anti-lymphocyte+antibodies.">Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies.</a> J Immunol Methods. 137 (1), 89-94 (1991).</li><li>Grondahl-Hansen, 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=Plasminogen+activator+inhibitor+type+1+in+cytosolic+tumor+extracts+predicts+prognosis+in+low-risk+breast+cancer+patients.">Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients.</a> Clin Cancer Res. 3 (2), 233-239 (1997).</li><li>Borgfeldt, C., Hansson, S. R., Gustavsson, B., Masback, A., Casslen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiation+of+serous+ovarian+cancer+from+cystic+to+solid+tumors+is+associated+with+increased+expression+of+mRNA+for+urokinase+plasminogen+activator+(uPA),+its+receptor+(uPAR)+and+its+inhibitor+(PAI-1).">Dedifferentiation of serous ovarian cancer from cystic to solid tumors is associated with increased expression of mRNA for urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1).</a> Int J Cancer. 92 (4), 497-502 (2001).</li><li>Devy, 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=The+pro-+or+antiangiogenic+effect+of+plasminogen+activator+inhibitor+1+is+dose+dependent.">The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent.</a> FASEB J. 16 (2), 147-154 (2002).</li><li>Bajou, 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=Plasminogen+activator+inhibitor-1+protects+endothelial+cells+from+FasL-mediated+apoptosis.">Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis.</a> Cancer Cell. 14 (4), 324-334 (2008).</li><li>Xu, X., Wang, H., Wang, Z., Xiao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+promotes+inflammatory+process+induced+by+cigarette+smoke+extraction+or+lipopolysaccharides+in+alveolar+epithelial+cells.">Plasminogen activator inhibitor-1 promotes inflammatory process induced by cigarette smoke extraction or lipopolysaccharides in alveolar epithelial cells.</a> Exp Lung Res. 35 (9), 795-805 (2009).</li><li>Ji, 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=Pharmacological+Targeting+of+Plasminogen+Activator+Inhibitor-1+Decreases+Vascular+Smooth+Muscle+Cell+Migration+and+Neointima+Formation.">Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.</a> Arterioscler Thromb Vasc Biol. 36 (11), 2167-2175 (2016).</li><li>Carmeliet, 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=Inhibitory+role+of+plasminogen+activator+inhibitor-1+in+arterial+wound+healing+and+neointima+formation:+a+gene+targeting+and+gene+transfer+study+in+mice.">Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice.</a> Circulation. 96 (9), 3180-3191 (1997).</li><li>Cao, 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=Endocytic+receptor+LRP+together+with+tPA+and+PAI-1+coordinates+Mac-1-dependent+macrophage+migration.">Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration.</a> EMBO J. 25 (9), 1860-1870 (2006).</li><li>Thapa, B., Koo, B. H., Kim, Y. H., Kwon, H. J., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+regulates+infiltration+of+macrophages+into+melanoma+via+phosphorylation+of+FAK-Tyr(9)(2)(5).">Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr(9)(2)(5).</a> Biochem Biophys Res Commun. 450 (4), 1696-1701 (2014).</li><li>Kortlever, R. M., Higgins, P. J., Bernards, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+is+a+critical+downstream+target+of+p53+in+the+induction+of+replicative+senescence.">Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.</a> Nat Cell Biol. 8 (8), 877-884 (2006).</li><li>Fang, H., Placencio, V. R., DeClerck, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protumorigenic+activity+of+plasminogen+activator+inhibitor-1+through+an+antiapoptotic+function.">Protumorigenic activity of plasminogen activator inhibitor-1 through an antiapoptotic function.</a> J Natl Cancer Inst. 104 (19), 1470-1484 (2012).</li><li>Bennett, S., Breit, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+in+the+isolation+and+culture+of+human+monocytes+that+are+of+particular+relevance+to+studies+of+HIV.">Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV.</a> J Leukoc Biol. 56 (3), 236-240 (1994).</li><li>Geissmann, F., 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+monocytes,+macrophages,+and+dendritic+cells.">Development of monocytes, macrophages, and dendritic cells.</a> Science. 327 (5966), 656-661 (2010).</li><li>Bauer, M., Goldstein, M., Heylmann, D., Kaina, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocytes+undergo+excessive+apoptosis+following+temozolomide+activating+the+ATM/ATR+pathway+while+dendritic+cells+and+macrophages+are+resistant.">Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.</a> PLoS One. 7 (6), e39956 (2012).</li></ol>

著者が明らかに何もありません。

さまざまな種類の細胞から成る、TME は癌の開発のために重要です。最適な成長条件を確認するためには、がん細胞は単球走化性因子の分泌を通じてを引き付けます。ここは、腫瘍細胞の in vitro単球採用のメカニズムを研究するためのプロトコルを提案する.このため、誘引可能な遺伝子発現系、プライマリのひと単球の遊走アッセイによる、ボイデン室アッセイの例として浄化に組み?...

促進することによって腫瘍の開発に貢献する腫瘍関連マクロファージ (Tam) 腫瘍の増殖、転移、および2免疫応答を調節します。がん細胞の腫瘍に潜入し、pro 幹 Tam3に分化するリクルートの炎症性単球。Tam と腫瘍の浸潤臨床予後と相関してマクロファージ4、5の免疫抑制の役割にリンクされています。しかし、腫瘍に大食細胞の募集のメカニズムはよく検討しないと関与する経路を理解フィールドと有望な治療の更なる向上のために重要です。腫瘍細胞と腫瘍微小環境 (TME) における正常細胞間の相互作用を勉強する際の課題の 1 つはメカニズムの解明や細胞、クロストークの解剖を許可する生体外でアプローチを必要とする複雑です。がん細胞由来のパラクライン効果、体外の大食細胞のような他の細胞型の移行の分泌タンパク質の研究に適用できる汎用性の高い方法論をご紹介します。単球の募集に関与するがん細胞由来タンパク質に対して shRNA 発現がテトラサイクリン誘導性プロモーターの制御下では、システムを使用して、分泌タンパク質の単球のパラクライン効果は量的に表わされます。このプロトコルでは規制のベクトルを提示テトラサイクリンに shRNA 配列のクローニングは続いて安定した癌細胞の世代。さらに、プライマリのひと単球・ ボイデン室アッセイの浄化は、単球の移行にがん細胞に由来するタンパク質のパラクライン効果を分析に使用されます。
蛋白質コーディングの遺伝子のダウンレギュレーション、一般的 siRNA と shRNA のテクニックの使用に制限はないがなくて適用されます。長期的なノックダウン (KD) 遺伝子の実験結果を妨げる細胞の二次的適応反応を引き出すことがあります。遺伝子発現の時空間制御の欠如では、時間の経過や細胞の生存に重要なタンパク質の役割蛋白質の動的役割を研究する困難になります。この問題は、腫瘍が確立された後にのみ腫瘍の開発そして進行中のタンパク質の役割が興味の蛋白質の発現のダウンレギュレーションを必要があります設定体内で特に重要です。条件付き KD 早い致死効果 KD の細胞と無条件の KD は腫瘍の開発の欠如につながる間、腫瘍の成長のさまざまな段階で蛋白質の役割の解析を有効にするを防止する利点があります。
条件付きの KD システムの数は、安定した KD の制限に対処する開発されましたが。条件遺伝子発現系には、エクダイソン誘導発現システム、チトクローム P-450 誘導システム1テトラサイクリン調節遺伝子発現系があります。テトラサイクリン調節遺伝子発現システムは、抗生物質テトラサイクリン (またはより安定したアナログ - ドキシサイクリン) の添加によって shRNA 発現を制御できます。テトラサイクリン/ドキシサイクリン KD、遺伝子発現の結果存在下でテトのシステム、shRNA 発現が誘導される shRNA 発現は遺伝子発現、テトラサイクリン存在下で抑制システム テト オフ中。テトラサイクリン誘導システムの欠点は、ドキシサイクリン - いわゆる水漏れしたため6、7の不在で shRNA 発現以前に報告された低レベルです。テトのシステムで説明した、テトラサイクリンの投与時に関心の shRNA のプロモーター領域が H1、テト応答要素 (TRE) シーケンスに恒常発現テトラサイクリン リプレッサー (TetR) 蛋白質の結合抑制されます。ShRNA 発現とテトラサイクリン依存的8,9の興味の蛋白質の翻訳の抑制でこの結果します。
その他の使用可能なテトのシステムは、シーケンス TATA ボックスと近位シーケンス要素 (PSE) 間および TATA ボックスと転写開始ちゃんらによって開発された重音テトの同時ノックに含めます。10このシステムは、非誘導の状態の下で shRNA 表現を規制するテトラサイクリンの有毒な線量低い shRNA のレベル未満、表されます必要があります。基 Krüppel 関連付けられているボックス (ボブ) テトのシステム11 KRAB、被験者の遺伝子は転写抑制 KRAB 結合部位の 3 kb の範囲内にある亜鉛指蛋白質が含まれます。キメラ蛋白質 tTRKRAB は、重音テトにバインドでき、大きい - DNA 制御可能な容量の範囲のため重音テト必要ないトランスクリプション間制限するサイトやプロモーターを開始しプロモーターの活性に低影響があります。ShRNA11,12; の漏らされた式の下位レベルを表示する非誘起状態の下でこの制御の RNA 干渉システムが報告されました。ただし、連続した 2 つのベクター複製のアプローチが必要です。エクダイソン誘導発現システムやチトクロム P-450 誘導システムなど開発済み条件 KD システムと比較してテトラサイクリン調節システム、堅牢性と、可逆性という利点があります、したがって最も日常的にシステムの13を使用しました。このプロトコルで使用されるシステムは、デュアル システム ノックで重音テトと KRAB テト - システムの利点それはまっすぐ進む、1 つのベクトルのクローニングは、複数のクローンの生成を許可する必要がありで水漏れしたための非常に低いレベルを発揮、ドキシサイクリンの不在。
TME は癌の開発のために重要です。腫瘍の成長を促進するには、癌細胞は走化性蛋白質の分泌によって炎症性単球を採用します。採用単球は腫瘍に潜入し、腫瘍の増殖と転移に貢献するプロ幹 Tam に分化します。免疫細胞の動員のin vitro研究利用移行アッセイ、boyden 教授区域の広く使われている試金使用14,15,16,17。この分析では、走化性因子蛋白質源、たとえば、癌細胞、または浄化された蛋白質は、下部室に配置されます。免疫細胞は、下部コンパートメントから多孔質膜で区切られた上部室に配置されます。走化性因子、および膜の下側に見られる増加勾配向かって移行セルを染色して顕微鏡下でカウントします。ここで我々 はドキシサイクリン添加による pai-1 の表現の規制、誘導性、安定した癌細胞を生成することによって単球のプラスミノーゲン活性化因子阻害剤 1 (PAI-1) の走化性因子の機能をテストします。単球の移行における pai-1 の役割を評価するのにボイデン室移行試験で人間の主要な球を使用します。人間の主な単球と THP1 のような広く使用されている単球性細胞株の違いが報告されているなど異なるサイトカイン発現パターン18;たとえば、THP1 細胞の TNF α 発現レベルは 5-10 倍に増加に比べてひと単球19。THP1 細胞白血病単球細胞から派生して、維持し、倍加時間 19-50 h20の平均で増殖し易い。逆に、成長因子の不在で寿命が短いひと単球が特徴です。単球は、個人間や精製方法によって変動のかなりの量が発生します、ドナーの血液から精製したので他のセルタイプで汚染が発生します。それにもかかわらず、主な単球は関連する、使用か生物学研究19の主な球を使用して単球性細胞株により得られた結果を確認してください推奨されています。ここで末梢血から主な単球人間の浄化のためのプロトコルについて述べる。密度勾配と接着プロトコルと Ficoll 後腹 Percoll グラデーション21続いての単一勾配と 2 つのステップ プロシージャ単球浄化の方法が含まれます。70-90% の間これらのメソッドの範囲によって得られる単球の人口の純度。負の免疫淘汰22続いて密度勾配を使用し、彼らの偶発的な活性化を回避し、結果それにより抗体と直接接触することがなくひと単球の浄化を有効にここで説明する方法を >単球の純粋な人口は 95%。
ひと癌細胞株における遺伝子発現 KD テトのシステム機能を設定するためここで提示されたプロトコルを使用して、癌由来分泌タンパク質のパイ 1 単球走化性因子の影響を検討します。パイ 1 さまざまな腫瘍で過剰発現し、その発現の臨床予後23,24逆説的に関与。Pai-1 のプロ性の役割はそのプロ血管新生の結果、抗アポトーシス機能25,26。パイ 1 は、マクロファージの炎症27サイトの募集を促進することによって炎症に寄与する示されています。パイ 1 は、Mac 1 依存マクロファージ移行30に参加する平滑筋細胞28,29を促進するために示された.パイ 1 過剰発現は、B16F10 メラノーマ腫瘍31に未加工 264.7 の大食細胞の募集を大幅強化にも示されています。ただし、タムの移行における pai-1 の役割は、詳しく解明されていません。記述されていたプロトコルを使用して、パイ 1 ががん細胞を単球を集めているかどうかの質問に答えます。この方法論は、癌細胞の分泌蛋白質を黙らせると、TME の成分を分析により腫瘍と TME の間クロストークの郭清。

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tet-pLKO-puro lentiviral vector</td>
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			<td height="21" style="height:21px;">Histopaque</td>
			<td style="width:177px;">Sigma</td>
			<td>10771-500ML</td>
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			<td height="21" style="height:21px;">EasySep Human Monocyte Enrichment Kit</td>
			<td style="width:177px;">StemCell</td>
			<td>19059</td>
			<td></td>
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			<td height="21" style="height:21px;">EasySep Magnet</td>
			<td style="width:177px;">StemCell</td>
			<td>18002</td>
			<td></td>
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			<td height="21" style="height:21px;">EcoRI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3101S</td>
			<td></td>
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			<td height="21" style="height:21px;">AgeI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3552S</td>
			<td></td>
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			<td height="21" style="height:21px;">Cut Smart buffer</td>
			<td style="width:177px;">NEB</td>
			<td>B7200S</td>
			<td></td>
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			<td height="21" style="height:21px;">EndoFree Plasmid Maxi Kit</td>
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			<td>12362</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PROMEGA Wizard SV Gel and PCR Cleanup System</td>
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			<td>A9281</td>
			<td></td>
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			<td height="21" style="height:21px;">psPAX vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
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			<td height="21" style="height:21px;">pMD2.G vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
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			<td height="28" style="height:28px;width:405px;">One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C737303</td>
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			<td height="21" style="height:21px;">Hema 3 stain set for Wright-Giemsa stain</td>
			<td style="width:177px;">Protocol</td>
			<td style="width:119px;">123-869</td>
			<td></td>
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			<td height="21" style="height:21px;">HEK293 cells</td>
			<td style="width:177px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
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			<td height="21" style="height:21px;">PBS</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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			<td height="21" style="height:21px;">XhoI restriction enzyme</td>
			<td style="width:177px;">NEB</td>
			<td>R0146S</td>
			<td></td>
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			<td height="21" style="height:21px;">Ligase</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ligase buffer</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">EDTA 0.5 M solution</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
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		<tr height="21">
			<td>Lipofectamine 2000</td>
			<td style="width:177px;">Invitrogen</td>
			<td>11668019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:405px;">The Big Easy&#34; EasySep Magnet</td>
			<td style="width:177px;">Stem Cell</td>
			<td style="width:119px;">18001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.1% Poly-L-Lysine solution</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Acetate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium butyrate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;M syringe filters</td>
			<td style="width:177px;">VWR International</td>
			<td>28145-479</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride(NaCl)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653-5 kg</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dithioerythritol (DTE)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B8255-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol (EtOH)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">792780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tryptone</td>
			<td style="width:177px;">Amresco</td>
			<td style="width:119px;">J859-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td style="width:177px;">Fluka</td>
			<td style="width:119px;">70161-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:177px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ampicillin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline hyclate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-25g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.5 M EDTA pH 8.0</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Falcon Permeable Support for 24 Well Plate with 8.0&#956;m Transparent PET Membrane</td>
			<td style="width:177px;">Becton Dickinson</td>
			<td style="width:119px;">353097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Serum Albumin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton-Tipped Applicator&#160;</td>
			<td style="width:177px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol Hema 3 STAT pack&#160;</td>
			<td style="width:177px;">Fisher Scientific Company</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resolve Immersion Oil, High Viscosity</td>
			<td style="width:177px;">Richard Allan Scientific</td>
			<td style="width:119px;">M4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Penicillin Streptomycin</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
	</tbody>
</table>

conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment

siRNA と shRNA を介したノックダウン (KD) 遺伝子発現の調整法は、遺伝子やタンパク質の機能を理解するための貴重なツールです。ただし、ケース、興味の蛋白質の KD 細胞致死効果または KD の予想される効果が時間依存性、無条件の KD メソッドを使用すると適切ではありません。条件付きシステムは、このような場合より適しているし、関心の対象とされています。エクダイソン誘導発現システム、チトクローム P-450 誘導システム1テトラサイクリン調節遺伝子発現系であります。
テトラサイクリン調整される遺伝子発現システムは、テトラサイクリン存在下で shRNA 発現誘導による蛋白質の表現の可逆制御をできます。このプロトコルでは、遺伝子発現の条件付き規制ひと癌細胞株における機能的なテトのシステムを用いた実験的なデザインを提案します。我々 は、腫瘍細胞単球の相互作用の研究ではこのシステムの使用を示します。

3 つの shRNA シーケンスのため、最も効率的な KD の PAI-1 がテストされました。このため、パイ 1 とスクランブル (表 1) shRNA シーケンス上記プロトコルを次のテト pLKO puro 発現ベクターにクローニングを行った.HT 1080 線維肉腫細胞株が生成された構造安定発現し、細胞は 3 日間のドキシサイクリンで治療されました。Pai-1 の式はウエスタンブロット(</st...

Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

このプロトコルは、単球/マクロファージにおける腫瘍微小環境の募集に腫瘍細胞由来タンパク質の役割を研究するため特に癌細胞とその後続の使用で機能するテトにシステムを設定するためのスキームとして機能します。

単球/マクロファージにおける腫瘍微小環境の採用を検討する癌細胞株における遺伝子発現の条件付きのノックダウン

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

conditional-knockdown-gene-expression-cancer-cell-lines-to-study

Research

JoVE Journal

Cancer Research

12.2K ビュー.  University of Southern California. このプロトコルは、単球/マクロファージにおける腫瘍微小環境の募集に腫瘍細胞由来タンパク質の役割を研究するため特に癌細胞とその後続の使用で機能するテトにシステムを設定するためのスキームとして機能します。

ビデオ: Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺腫瘍細胞募集試験

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

がん研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

CLIC4 ノックダウン誘起 HN4 細胞アポトーシスの in Vitro研究にミトコンドリア膜電位の検出

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

生体内でPH、 pO2、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

ウイルスの遺伝子改変マウスの前立腺に CRISPR ガイドの配信

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

抗有糸分裂治療に対するアポトーシス応答を評価するために DR3 を過剰発現細胞株の確立

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility in analyzing differential expression of groups of genes in tumor RNA-seq in a way that may be helpful for advancing precision oncology. However, CorEx produces many factors that can be challenging to analyze and connect to existing understanding. To facilitate such connections, we have built a website, CorExplorer, that allows users to interactively explore the data and answer common questions related to its analysis. We trained CorEx on RNA-seq gene expression data for four tumor types: ovarian, lung, melanoma, and colorectal. We then incorporated corresponding survival, protein-protein interactions, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, and heatmaps into the website for association with the factor graph visualization. Here we employ example protocols to illustrate use of the database for comprehending the significance of the learned tumor factors in the context of this external data.

We introduce the CorExplorer web portal, an resource for exploration of tumor RNA sequencing factors found by the machine learning algorithm CorEx (Correlation Explanation), and show how factors can be analyzed relative to survival, database annotations, protein-protein interactions, and one another to gain insight into tumor biology and therapeutic interventions.

CorExplorer Web ポータルを用した腫瘍遺伝子発現因子の解析

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

固形腫瘍細胞株における側集団の解析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

患者由来胃癌幹細胞のステム関連遺伝子を研究するための条件付きノックダウンと適応球形成アッセイを組み合わせた

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...