Ve James Neiswender; Kristen Kwan ve bu çalışmada kullanılan plazmid hediyeler için geç Chi-Bin Chien;; antikor boyama ile ilgili yardım için James Lister ve David Raible Biz kimin laboratuar bu teknikleri başlangıçta geliştirilen Dr Leonard I. Zon ederim mikroenjeksiyon video. Bu çalışma CJC için NIH hibe R00AR056899-03 tarafından finanse edilmiştir

1. Melanom Onset Değiştiricileri için Tarama <ol><li> PCR ilgi genlerinin tüm uzunlukta açık okuma çerçevesi (GOI) yükselterek ve BP klonaz II (Invitrogen) kullanılarak pDONR 221 içine yeniden birleştirilmesini ile orta ağ geçidi giriş klonlar oluşturur. MiniCoopR vektörü (Şekil 1A) mitfa organizatörü altında ilgi genleri yerleştirmek rekombinasyona p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 ve miniCoopR 5 Multisite Geçidi teknolojisi (Invitrogen) kullanın. </li><li> Önceden 7 açıklandığı gibi mitfa (lf) triply homozigot zebrafish embriyolar; p53 (lf): tek hücreli Tg (BRAF V600E mitfa) içine 25 pikogram Tol2 transposase mRNA ile birlikte her klon 25 pikogram enjekte edilir. 28.5 enjekte edilen embriyolar inkübe ° C. 24 hpf herhangi ölü embriyolar çıkarın. </li><li> Diseksiyon mikroskop üzerine enjekte embriyoları içeren Petri kabı koyarak 72 hpf at kurtarıldı melanosit ile transgenik hayvanlar seçinbeyaz bir arka plan (Şekil 1A) karşı gelen ışığı altında e. </li><li> Önceden 8 açıklandığı gibi 4 dpf at zebrafish tesisin kreş 3 L tanklarına hayvanlar aktarın. </li><li> 2 ay sonra, 4 mm 2 (Şekil 1A) &#39;den daha büyük bir melanosit kurtarma en az bir alana sahip hayvanlar seçin. 72 hpf de melanosit kurtarma derecesi ve 2 ay melanosit kurtarma derecesi arasında güçlü bir ilinti vardır. Melanosit kurtarma ile embriyolar 72 hpf azından seçilmiş olduğunda, bunların kısmı (~% 80) 2 ay azından 4 mm 2 &#39;den daha büyük bir melanosit kurtarma en az bir alan vardır. </li><li> Ekran tümörlerin varlığı (Şekil 1B) için haftalık seçilen hayvanlar. Histopatolojik ve morfolojik değişiklikler arasında güçlü bir ilişki vardır. Benign ve malign Geçiş lezyonların hayvanın yüzeyden yükseltilmiş haline bir morfolojik değişim olarak kabul edilmektedir. Tümörlü anima Isolateçalışma için ls. </li><li> Ordinat üzerindeki apsis ve yüzde melanom sağkalım üzerine hafta içinde yaşla birlikte melanom sağkalım eğrileri çizin. Ilgi bir gen ifade melanosit Hayvanlar, kontrol hayvanlarına kıyasla olduğunu melanosit ekspres EGFP (gelişmiş yeşil flüoresan protein) (Şekil 1C). MiniCoopR-EGFP enjekte hayvanlar için medyan tümör başlangıcı yaklaşık 18 haftadır. Iki eğri bir log rank testi kullanılarak istatistiksel olarak farklı olup olmadığını belirleyin. </li></ol> 2. Tümör Invasion Assay <ol><li> Arka beyin ve sırt yüzgeci (Şekil 2A) anterior sınır posterior sınırı arasındaki bir bölgede, dorsal tümörler geliştirmek izole zebrafish seçin. </li><li> Melanom başlangıçlı Massachusetts Tıp Fakültesi İnş Üniversitesi tarafından Ötenazi tarihinde Amerikan Veteriner Hekimleri Birliği Paneli tarafından belirlenen ve onaylanan yönergelere göre balık ötenazi İki hafta sonratitutional Hayvan Bakımı ve Kullanımı Komitesi (IACUC). Solungaç hareket durana kadar Spesifik olarak, bir balık, 0.6 mg / ml &#39;tricaine ile bir çanak içine yerleştirilmiştir. </li><li> Bir neşter ile balık periton boşluğunda bir kesi yapın ve sonra fiksasyonu için 4 ° C gecede% 4 paraformaldehid balık (PFA) yerleştirin. </li><li> 4 gece boyunca 0.5 M EDTA ile tedavi ° C kirecini için. </li><li> Gecede, 60 az 2 saat her biri için 2 kez ° C&#39;de 1 saat boyunca ksilen 3 kez ile açık, 1 saat boyunca kurutmak ve daha sonra parafin ile tedavi kaset formalin ile tespit </li><li> Bir kalıp içinde parafin içinde doku gömmek ve yaklaşık 30 dakika, 1 saat için katılaşmaya izin. </li><li> 5 mikron bölümleri, bir her 50 mikron olun. Bölümler enine ve derin istila noktasına tespit edilebilir, böylece tüm lezyon aracılığıyla olmalıdır. Hematoksilen ve eozin boyama ile izleyin. </li><li> Tümör hücreleri kollajen tabakanın compactum dışında kalır olmadığını ölçerek tümör invazyonu değerlendirin, dorsal içine işgalkas veya omurga (Şekil 2B, C) ​​içine daha fazla işgal. Numunelerin iki takım arasında istatistiksel fark belirleyin. </li></ol> 3. Ölçek Melanositler Antikor Boyama <ol><li> 80 ml ​​0.1 M Na 2 HPO 4 ve 20 ml 0.1M NaH 2 PO 4 ile PO4 tamponu Makyaj. PH 7,3 olduğundan emin olun. 8.0 g sakkaroz, 0,15 ml 0,2 M CaCl2 ve 90 ml 0,2 M PO 4 tamponu, pH 7.3 ihtiva 2x düzeltme tampon Makyaj. Gerekli NaOH veya HCI ile 7.3 &#39;e ayarlayın, sonra pH 4 tampon PO ile 100 mL&#39;ye tamamlanır. </li><li> 300 mg katı PFA kadar tartılır ve bir mikrosantrifüj tüpüne ekleyin. PFA 4.5 x kütle, mg olarak (örn., 450 ul 5 mM NaOH ile 100 mg PFA) eşit bir hacim ile 5 mM NaOH ekleyin. Isı 60-70 ° C, PFA çözününceye kadar sallama sıra ile. Kalan partiküller aşağı spin ve 20% PFA süpernatant kurtarabilirsiniz. Taze her zaman olun. </li><li> 1x fi içeren fiksasyon çözeltisi hazırlayınx tampon ve% 4 PFA. </li><li> 0.17 mg / ml tricaine içinde balık anestetize. </li><li> Keskin ince uçlu forseps kullanarak sırt pulları, koparmak bir diseksiyon mikroskobu altında olay ışık kullanarak ölçek melanosit içeren yarısı (Şekil 3A) zarar vermemeye özen. Direkt olarak 1 ml tespit çözeltisi ile oda sıcaklığında inkübe edin, içine forseps Sıra terazi açarken, ≥ 2 saat için. Balık suya balık aktarın ve başarılı kurtarma onaylamak için balık izlemek. </li><li> Ağartıcı pigmentli melanosit oda sıcaklığında 5 dakika için PBST (1 x PBS, pH 7.4,% 0.1 Triton X-100) içinde 2 kez sabit terazi yıkamak için. DH 2 O ile 5 ml içinde 0.4 ml% 10 KOH ve 0.15 ml,% 30 H2O 2, içeren 1 ml taze yapılmış ağartıcı çözelti Tüplerinin açık patlama gaz önlemek için parafilm veya kapak kilitleri giy. Pigment (Şekil 3B, C) ​​(10 dk tipik) gidene kadar beyazlatma devam edin. Oda sıcaklığında 5 dakika için PBST dört kez yıkayınerature. </li><li> 1 x PBS, pH 7.4,% 0,2 Triton X-100, 2 mg / ml BSA,% 1 DMSO,% 0.02 NaN3,% 2 kuzu serumu oluşan blok çözelti içinde, en azından 30 dakika için Blok. HRP reaksiyon ile gelişmekte olmadığını NaN 3 dışarı bırakın. </li><li> Gece boyunca oda sıcaklığında blok çözelti içinde birincil antikor ile inkübe edin. Bunları batık tutmak için numune üzerine çözelti en az 400 ul olması gerekir. </li><li> Oda sıcaklığında PBST içinde 30 dakika için terazi 3 kez yıka. </li><li> 2 saat oda sıcaklığında blok çözelti gece boyunca sekonder antikor ile inkübe edin. </li><li> PBST + 0.1 uM DAPI ile 10 dakika boyayın. </li><li> PBST ile 5 dk için 3 kere yıkayın. </li><li> Ölçekler konkav slayt aşağı doğru bakacak şekilde vectashield bir damla bir cam slayt üzerine ölçekler monte ediniz ve onlar üzerinde bir cam kapak kayma yerleştirin. Net oje ile slayt kenarları mühür ve bir floresan mikroskop (Şekil 3B, E, F, G altında slaytlar gözlemlemek </strong&gt;). </li></ol> 4. Transplantasyon Assay <ol><li> Transplantasyonu 10 öncesi gama ışınlaması 25 Gy ile bir gün aydınlatarak tarafından alıcıya 2-3 aylık casper 9 balık hazırlayın. Balık balık suda kurtarmak için izin ver. </li><li> Daha önce açıklandığı gibi, bir tümör taşıyan balık (Bölüm 2.2) Euthanize. </li><li> Tümör kes ve% 5 FBS ile 0.9x PBS sterilize yaklaşık 5 ml filtre ile bir Petri kabı koyun. Bir jilet çözüm ise tümörün zar. </li><li> Oda sıcaklığında çalışmak, tek hücreler almak için P1000 pipet ile çiğnemek. % 5 FBS ile filtre sterilize 0.9x PBS ile hacmi 25 ml&#39;ye kadar olun. </li><li> 40 uM gözenekli filtre süzülür. </li><li> 10 dk için 1500 rpm (453 rcf) bir Eppendorf 5810R tezgah üstü santrifüj içinde Spin. </li><li> Süpernatantı ve kabaca istenen konsantrasyonu (100 mm 3 tümör için 10 7 hücre varsayalım) hücre süspansiyonu olun. </li><li> T Hesaplao tam hücre sayısı, bir hemositometre kullanılarak ve nihai enjeksiyon hacmi yaklaşık 5 ul olacak şekilde hücre süspansiyonu seyreltin. 50.000 hücre enjekte edilmesi durumunda, örneğin, bir hücre süspansiyonu 10,000 hücre / ul nihai konsantrasyon ile seyreltilmelidir. Hücre süspansiyonu hücre kümelenmesi önlemek için her bir kaç dakika içeren tüp hafifçe vurun. </li><li> 2-3 kez% 100 etanol ve 0.9x PBS ile bir 26s ölçer (konik ucu) 701 N 10 ul Hamilton şırınga yıkayın. Hücre süspansiyonu 5 ul ile şırınga yerleştirin. </li><li> 0.17 mg / ml tricaine olarak ışınlanmış alıcı balık anestetize. Nemli Kimwipe üzerinde yan Yeri balık (Şekil 4A). </li><li> Tek elle balık stabilize ve yaklaşık yarım arka beyin ve dorsal yüzgeç anterior sınır posterior sınırı arasındaki periton boşluğuna yukarıdaki balık kanadı içine 45 ° açıyla yukarı bakacak fasetli iğne takın. Yavaşça piston (Şekil 4B) basınız. &lt;/ Li&gt; <li> Balık taze balık suda kurtarma ve tümör engraftman (Şekil 4C) için günlük balık gözlemlemek için izin ver. Engraftman meydana gelmişse, sürekli büyüme ve hastalık gelişimi de görülebilir. </li></ol>

<ol>
	<li>Beroukhim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. 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=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. 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=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. 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=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. 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=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, 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> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

Çıkar çatışması ilan etti.

MiniCoopR yöntem zebrafish melanosit ilgi genlerinin ifadesini sağlar. Bu yaklaşım zebrafish mitfa geni hücre-özerk davrandığını aslında yararlanır. Bu nedenle, miniCoopR vektör tarafından kurtarıldı melanosit minigene ve fiziksel birleştiğinde ilgilendiren herhangi bir gen içeren kesin. Kurtarılan melanosit açıkça görülebilir ve tek hücreli embriyolar olarak enjekte edilmiştir hayvanlarda elde edilebilir. Kimerizmin Belirtilen derecesi seçilir, ve bu kesim aşarak hayvanlar tümör ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Ağ Geçidi rekombinasyon reaktifler </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Referans 5 </td></tr><tr><td> Mitfa antikor </td><td></td><td></td><td> Referans 5 </td></tr><tr><td> FITC keçi anti-tavşan IgG antikoru </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> Vectashield </td><td> Vektör Labs </td><td> H-1000 </td><td></td></tr><tr><td> casper Zebra balığı </td><td></td><td></td><td> Referans 9 </td></tr><tr><td> 701N 10 ul Şırınga </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 um bir filtreden </td><td> BD Falcon / Fisher </td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

Insan kanserlerini Genomik çalışmalar tümörler 1,2,3 değişmiş genler hakkında bilgi bir zenginlik vermiştir. Bu çalışmalardan kaynaklanan bir zorluk birçok genin değişmiş olduğunu ve bu dönüşüm sırasında tesadüfen ortaya çıktığını gelen tümörogenez sürdü genetik bozukluklardan ayırt etmek zor olabilir. Bu ayrım çizmek için bunu kantitatif tümörler inat ve yaymak için izin tümör gelişimini ve diğer süreçlere değişmiş bir gen etkisini ölçebilen bir test olması faydalıdır. Burada melanom başlatılması ve bakımı için gerekli adımları kapsayan bir otokton tümör modeli 4 kullanarak zebrafish aday melanom değiştiriciler çok sayıda taranması için hızlı bir araç sunuyoruz. Bu tahlilde önemli bir reaktif olan bir ağ geçidi rekombinasyon kaset için çiftler mitfa melanosit şartname faktörünün bir vahşi-tip kopya miniCoopR vektör ki, burada aday haline melanomaliler genler 5 recombined. MiniCoopR vektör promotör, açık okuma çerçevesi ve yabani-tip mitfa geninin 3&#39;-çevrilmemiş bölge içeren bir mitfa kurtarıcı minigene sahiptir. Bu bizi bir aday melanom modifiye tam boy açık okuma çerçeveleri ile yapılar yapmak için olanak sağlar. P53 (lf);; mitfa (lf) zebrafish embriyolar: Bu bireysel klonlar daha sonra tek hücre Tg (BRAF V600E mitfa) enjekte edilebilir. MiniCoopR vektör Tol2-aracılı transgenesis 6 ile entegre ve melanosit kurtarır alır. Fiziksel mitfa kurtarıcı minigene birleştiğinde Çünkü, aday genler dönüştürmek ve tümörler dönüşecek bazıları kurtarıldı melanosit olarak ifade edilmiştir. Melanom başlatılması ve melanom hücre özelliklerini bir aday genin etkisi melanom sağkalım eğrileri, invazyon deneyleri, antikor boyama ve transplantasyon yöntemleri kullanılarak ölçülebilir.

Tek hücreli Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) zebrafish embriyolar melanom onkogen mitfa promotör kontrolü altında SETDB1 5 veya EGFP, her içeren miniCoopR vektörü ile enjekte edildi. Melanosit kurtarma olan embriyolar seçilir ve olgunlaşması için izin verildi. Melanosit kurtarma ile yaş hayvanların 2 ayda 4 mm 2 den büyük seçilmiştir. Hayvanlar melanomlar için haftalık tarandı. Yetişkinler için tümör oluşumunda eğrileri S...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Bir zebrafish otokton tümör modeli kullanılarak melanoma düzenleyiciler için ekran için hızlı bir şekilde sunulmaktadır. Bu melanosit aday melanom genlerin ifadesi için izin miniCoopR vektör yararlanır. Melanom sağkalım eğrileri, bir işgal testi, ölçek melanosit antikor boyama için bir protokol ve bir melanom nakli tahlil elde bir yöntem tarif edilmiştir.

Bir Zebra balığı Otokton Tümör Modeli kullanılarak Melanom Değiştiricileri için Tarama

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

Research

JoVE Journal

Medicine

13.6K Görüntüleme.  University of Massachusetts Medical School. Bir zebrafish otokton tümör modeli kullanılarak melanoma düzenleyiciler için ekran için hızlı bir şekilde sunulmaktadır. Bu melanosit aday melanom genlerin ifadesi için izin miniCoopR vektör yararlanır. Melanom sağkalım eğrileri, bir işgal testi, ölçek melanosit antikor boyama için bir protokol ve bir melanom nakli tahlil elde bir yöntem tarif edilmiştir.

Video: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Tıp

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal Alkol Pozlama bir Zebra balığı Modelinde teratojenik değişiklikler değerlendirilmesi

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

İnsan Nöroendokrin Tümör Terapi Çalışmaları Üç Boyutlu Model İnsan nöroendokrin tümör hücre hatlarında

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes Mellitus ve Metabolik Belleğinin Zebrafish Modeli

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Yeni Bir Yüksek çözünürlüklü<em&gt; In vivo</emTümör Büyüme ve Tedavi için İntrakranial Yapıların Dinamik Tepki Eğitim&gt; Görüntüleme Tekniği

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polikistik Böbrek Hastalığı Olası Zebra balığı Modeli: demonte<em&gt; Wnt5a</em&gt; Zebra balığı Böbrekler Kistler Nedenler

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tümör-stromal Etkileşim için üç boyutlu Co-kültür Modeli

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Uveal Melanom Karaciğer Metastazlı için bir tedavi olarak izole Karaciğer Perfüzyon

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Bir deri altı tümör modelinde tümör-süzücü lökosit subsetleri Değerlendirilmesi

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...