Biz Dr teşekkür ederim. R. Jude Samulski cömertçe Hep293TT hücreleri sağlamak için rekombinant AAV3 ve AAV-EGFP plazmid, sırasıyla, ve Dr. Gail Tomlinson tür hediyeler ve Xiao Xiao. Bu araştırma Ulusal Sağlık Enstitüleri Kamu Sağlık Hizmetleri hibe hibe R01 HL-076.901, R01 HL-097.088, P01 DK-058.327 (Proje 1) (AS) tarafından kısmen desteklenmiştir.

1. Rekombinant adeno-ilişkili virüs serotip 3 (rAAV3) Vektörler Ambalaj <ol><li> Web sitesine gidin: &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;ve DNA miktarını hesaplamak. </li><li> Ön-mix FBS ve antibiyotik olmadan orta plazmid pHelper (Adenovirüs yardımcı), pACG2c3 (AAV yardımcı) ve pdsAAV-CB-EGFP (rAAV vektör). Toplam hacmi on 15 cm 2 tabak için 8750 mcL olmalıdır. </li><li> Adım 1.2 DNA çözüm PEI doğrudan mcL 1.250 (% 0.1 m / v, Polysciences, Kedi # 23966, pH = 4.5) ekleyin. Vortex birkaç saniye için DNA-PEI çözüm. </li><li> 5 dakika oda sıcaklığında (RT) için bir kompleks oluşturacak şekilde inkübe DNA PEI çözüm. </li><li> DNA PEI çözüm 15 cm 2 plaka başına 1 ml ekleyin. </li><li> Taze 25 ml tam orta yerine, 4 saat sonra orta çıkarın +% 10 FBS (C-DMEM). </li><li> Yetmiş iki saat sonrası transfeksiyon, kazıma hücreleri ve 250 ml konik santrifüj tüpüne dökün. </li><li> 3000 rpm&#39;de 4 Spin, ° C 10 dakika. Süpernatant kapalı dökün. </li><li> Yeniden askıya alma, RB TMS Tampon (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) 5 ml hücre pelletini. Yeni bir 15 ml konik tüp transfer. </li><li> 37, 10 dakika ve çözülme için kuru buz-etanol banyo Dondurulmuş ° C 10 dak. Üç kez tekrarlayın. </li><li> 4.8 M MgCl 2 1 mcL ve 2 mcL Benzonase (25 U / ml, Novagen, Kedi # 70.664-3) ekleyin. Vortex ve inkübe 37 ° C de 40 dk. </li><li> Dakika süreyle 40 ° 4, 4,000 rpm&#39;de C Spin ve süpernatantı toplamak (lizat açıklık). </li><li> , 2 ml,% 15% 25 2 ml, 1.5 ml% 40 ve bir 13 ml Hızlı-Seal santrifüj tüpüne (Beckman, Kedi # 342.413), yukarıdan aşağıya doğru yük iodixanol degrade (OptiPrep, Kedi # 1.114.542) 1.5 mL% 60 iodixanol. Iodixanol degrade üstüne süpernatant (Adım 1.12) yükleyin. RB TMS Buffer tüpler doldurun. </li><li> 16 1 saat süreyle 75.000 rpm Santrifüj ° C Beckman 90Ti rotor. </li><li> % 40 iodixanol fraksiyonu% 40-60 sınırında bir enjektör takarak toplayın. </li><li> % 40 iodixanol fraksiyonu, biri 50 ml konik tüp transferi ve Tampon ile 40 ml makyaj (20 mM Tris, 15 mM NaCl, pH 8.5). </li><li> Filtrasyon aparatı HiTrap S HP kolon (GE Healthcare, Kedi # 17-1154-01) kullanarak birleştirin. Aşağıdaki tamponu ile sütun yıkayın: 25 ml Buffer (20 mM Tris, 15 mM NaCl, pH 8.5); Tampon B (10 mM Tris, 500 mM NaCl, pH 8.5) 25 ml, 50 ml Tampon bir; Tampon olarak virüs örnek 40 ml, 50 ml Buffer (20 mM Tris, 15 mM NaCl, pH 8.5); 20 ml Tampon C (20 mM Tris, 175 mM NaCl, pH 8.5); </li><li> 20 ml Apollo Konsantratörleri (Orbital Biosciences) Tampon C toplayın. 3.000 rpm&#39;de santrifüj, 4 ° C 10 dk. </li><li> Flow-through atın ve 4, 3,000 rpm&#39;de 20 ml soğutulmuş PBS ve santrifüj eklemek ° C&#39;de 10 dk. </li><li> Flow-through atın ve soğutulmuş PBS 500 mcL ekleyin. -80 Küçük alikotları donmuş silikon tedavi Eppendorf tüpleri ve mağaza membran ve transfer ° C vektörleri yıkayın. </li></ol> 2. RAAV3 Vektör titreleri Belirlenmesi <ol><li> Buz üzerinde bir Eppendorf tüp virüs saflaştırılmış 10 mcL çözülme. GKD 2 O. 40 mcL ekle </li><li> 0.2 mcL Benzonase (25 U / ml, Novagen, Kedi # 70.664) ekleyin ve 37 ° C&#39;de 1 saat süreyle. </li><li> Başka bir Eppendorf tüp plazmid standart (0.2 ng / ml) 50 mcL alın. </li><li> Her tüp ve 65 ° C&#39;de 30 dakika için 100 mM NaOH 50 mcL ekleyin. </li><li> Hemen en az 5 dakika buz üzerinde soğutun. </li><li> Bio-Dot Filtre Kağıdı (Bio-Rad, Kedi # 1.620.161) göre transfer membran (Millipore, Kedi # INYC00010) kesin. 20 dakika için üç filtre kağıtları ile 10x SSC membran durulayın. </li><li> Yuvası leke SF Apparatus (Bio-Nokta, Kedi # 170-6542) ayarlayın. Bir termos bağlayın ve 10x DGM&#39;nin yuvası leke aparatı 100 mcL ekleyin. </li><li> Adım 2.5 örneklerinin her 20x SSC 100 mcL ve 6x yükleme boya 1 mcL ekleyin. </li><li> Membran üzerine yuvası leke aparatı her örneklerinin 100 mcL yükleyin. </li><li> 10x SSC başka örneklerin her birine 100 mcL ekleyin. </li><li> 2.9 ve 2.10 adımları örnek zarından girene kadar tekrarlayın. </li><li> Slot leke aparatı sökün ve membran çıkarın. Müfettiş Linker SL-1000 UV Çapraz içine koyun. &quot;Enerji&quot;, &quot;Turn Optimal çapraz bağ&quot; (Show 1200) ve &quot;Başlat&quot; itin. </li><li> DNA (Fisher, Kedi # NC9753983) 5 dakika boyunca kaynatın 1 somon sperm mL (SS). En az 5 dakika buz üzerinde soğutun. </li><li> 27.5 ml, 15 ml GKD 2 O karıştırma öncesi hibridizasyon solüsyonu hazırlayın 20x 50x Denhardt çözümü SSC, 5 ml, 1.25 ml% 20 SDS, Polya 0.25 ml ve 2.13 Adım SS DNA 1 ml. </li><li> 25 ml, 65 ° C hibridizasyon odasında gece bir kapaklı cam silindir ve inkübe-hibridizasyon öncesi çözüm membran askıya alınması. </li><li> GKD 2 O 10 mcL 50 ng DNA şablonları sulandırınız. Kaynatın, 100 ° C en az 5 dakika boyunca buz üzerinde hemen 5 dakika ve soğuk için. </li><li> 1 dakika 3.000 rpm&#39;de RT santrifüjleyin. 10 mM 3 dNTP 2 mcL (dCTP eksik), (2) Hexanucleotide Mix mcL (Roche, Kedi # 11277081001), 1 mcL Klenow (Roche, Kedi # 11008404001) ve -32 α P-dCTP 5 mcL ekleyin. 37 ° C&#39;de 10 dakika inkübe edin. </li><li> 2 dakika süreyle 3000 rpm&#39;de G-50 Sütunlar (GE Healthcare, Kedi # 27-5330-01) santrifüjleyin. Adım 2.17 sütuna prob yükleyin. 2 dakika süreyle 3000 rpm&#39;de santrifüj ve flow-through toplamak. </li><li> Radyoaktivite Kont. Prob için 100 ° C 5 dakika ve soğuk &#39;hemen buz üzerinde en az 5 dakika kaynatın. </li><li> Membran ile ön hibridizasyon çözüm prob (6x10 5 kopya / ml) ekleyin. </li><li> Hibridizasyon odasında 65 ° C&#39;de bir gece inkübe edin. </li><li> Hibridizasyon çözüm atın ve 15 dakika süreyle RT 2x SSC +0.1% SDS membran durulayın. </li><li> Çözüm atın ve 30 dakika süreyle 65 ° C 0.1x SSC +0.1% SDS membran durulama. </li><li> Çözüm atın ve RT kısaca 0.1x SSC membran durulayın. </li><li> -80 ° C&#39;de 6 saat film film ortaya çıkarmak ve geliştirmek. Tipik bir sonuç Şekil 1&#39;de gösterilmiştir. </li></ol> 3. rAAV3 Vektör aracılı İletimi ve İnsan Karaciğer Kanseri Hücrelerinde Transgen İfade <ol><li> 96 plaka her iyi Tohum 1x10 4 hücreleri (HuH7 veya Hep293TT ya). Nemlendirilmiş bir CO 2 inkübatör içinde en az 18 saat süreyle inkübe edin. </li><li> 96-plaka orta çıkarın. </li><li> FBS ve antibiyotik olmadan orta 50 mcL hücreler iki kez yıkayın ve kaldırmak. </li><li> Orta 5,000 VGS / hücre FBS ve antibiyotik ve rAAV vektörlerin olmadan 50 mcL ekleyin. </li><li> CO 2 inkübatör plakalar 4 saat süreyle inkübe edin. </li><li> Orta atın. 50 mcL tam orta hücreler iki kez yıkayın ve kaldırmak. </li><li> C-DMEM her bir kuyunun 150 mcL ekleyin ve 72 saat için CO 2 inkübatör plaka inkübe ve floresan mikroskop kullanılarak EGFP ifade görselleştirmek (DMİ 4000B; Leica Microsystems). </li><li> Virüsle enfekte olan hücreleri üç kuyu Görüntüler ImageJ analiz yazılımı (NIH, Bethesda, MD, ABD) tarafından kantitatif analiz edilmektedir. </li><li> Transgen ifade görme alanı başına düşen yeşil floresan toplam alanı (piksel 2) olarak değerlendirilir . Tipik bir sonucu, Şekil 2&#39;de gösterilmiştir. </li><li> Transgen ifade Sitometrisi ile de tespit edilebilir. </li></ol> 4. Temsilcisi Sonuçlar: Yukarıda belirtilen protokolün ardından, verimli AAV serotip vektör üretebilirsiniz. Tipik verim ~~ 500 mcL ~ 10 11 VGS içeren saflaştırılmış vektör stok / mcL. Vektör stokunun saflık ve Benzonase sindirim olmadan, kantitatif DNA yuvası blot hibridizasyon sinyalleri karşılaştırarak tarafından belirlenir. Saf hücre hatları - hepatoblastom (HB) ve hepatosellüler karsinom (HSK) - rAAV3 vektörler transduce insan karaciğer kanseri verimli. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Şekil 1" /> Şekil 1 rAAV3 vektörlerin titreleri belirlemek için nicel DNA slot-blot analizi. Benzonase (üst satır) ile sindirilir viral hisse senetleri, saflaştırılmış iki kat dilüsyonları 32 P-etiketli EGFP-spesifik DNA probu ile kantitatif DNA yuvası leke incelendi. 1 ng (orta sıra) veya 10 ng (alt sıra) membran yüklenen AAV-EGFP plazmid standartları ile karşılaştırıldığında AAV3 vektör titresi tespit edildi. Numaraları DNA kopyası karşılık. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Şekil 2" /> Şekil 2 Rekombinant AAV insan karaciğer kanser hücrelerini vektör aracılı transgen ifadesi. Hep293TT hücreleri, kısa bir süre önce kurulan insan hepatoblastom hücre hattında 4, ya sahte enfekte (sol panel), ya da 2 saat boyunca 37 ° C&#39;de scAAV2-EGFP veya scAAV3 EGFP vektörler 5000 VGS / hücre transduced . Transgen ekspresyonu 72 saat sonrası transdüksiyon floresan mikroskop kullanılarak görüntülendi.

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R., Ling, C., Zolotukhin, I., Ma, W., Zolotukhin, S., Srivastava, A., Zhong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+and+correction+of+murine+hemophilia+B+using+AAV2+vectors+devoid+of+multiple+surface-exposed+tyrosines.">High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.</a> Mol Ther. 18, 2048-2056 (2010).</li><li>Ojano-Dirain, C., Glushakova, L. G., Zhong, L., Zolotukhin, S., Muzyczka, N., Srivastava, A., Stacpoole, P. 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Patojen olmayan bir insan parvovirus, adeno-ilişkili virüs (AAV) dayanan rekombinant vektörler geliştirilen ve şu anda bir dizi gen tedavisi klinik çalışmalarda 5 edilmiştir . Önceleri en yaygın olarak kullanılan 10 AAV serotipleri, genel olarak AAV3 vektörlerin transdüksiyon etkinliğini in vitro ve in vivo 1 hem de, özellikle düşük olduğu bildirilmiştir. Ancak, son gözlem AAV3 flow sitometri 2 ile kantitatif tespit son derece iyi vektörler transduce insan karaciğer kanser hücresi hatlarında, ve bu AAV3 bu bağlayıcı ve giriş için ortak bir hücresel reseptör olarak insan hepatosit büyüme faktörü reseptörü (hHGFR) kullanır hücreleri 3, özellikle de güçlü AAV3 genel olarak insan karaciğer hücreleri için selektif doku-tropizm ve insan karaciğer kanseri hücrelerinin öneririz. Ancak, AAV3 vektörler aynı zamanda normal insan hepatositleri verimli 2 transduce, in vivo kanser gen tedavisi uygulama potansiyellerini kullanabilecekleri olumsuz etkiledi olacaktır . Bu olası sorunu aşmak için olası bir strateji gerektirir transkripsiyonel hedef normal hepatositler tarafından değil seçici olarak tümör tarafından üretilen bir gen ürünü tespit ederek, kanser hücrelerinin, ancak. Örneğin, normal hepatositler genellikle çok küçük miktarlarda üretmek önceki çalışmalarda, belirli bir varlığı, ilerleme, ve / veya karaciğer kanserlerinin bazı türleri tekrarını izlemek için işaretleyici olarak kullanılan serum α-fetoprotein (AFP) düzeyleri olduğunu göstermiştir bu proteinin. Nitekim, biz, karaciğer kanser hücrelerini transgen ifade hedef AFP organizatörü içeren AAV3 vektörler kullandık, ama şu anda normal hepatositler 2 ve çalışmalar karaciğer kanseri bir fare xenograft modeli bu yaklaşımın etkinliğini test etmek için çalışmalar devam etmektedir. Ek çalışmalar, 6-14 viral capsids yüzey maruz tirozin kalıntılarının site-directed mutagenesis çeşitli AAV serotip vektörler transdüksiyon etkinliğini önemli ölçüde artar olabileceğini gözlemledim . 6 7 yüzey maruz tyrosines AAV3 korunmuş olduğundan, biz bu kalıntıların site-directed mutagenesis ve tirozin-mutant AAV3 vektörlerin transdüksiyon etkinliğini önemli ölçüde insan karaciğer kanseri hücrelerinin (yayınlanmamış veri) gelişmiş olduğu görülmektedir. Araştırmalar aynı zamanda, insan hepatoblastom ve hepatosellüler karsinom için fare xenograft modelleri tirozin-mutant AAV3 vektörler güvenlik ve etkinliğini değerlendirmek için şu anda devam etmekte olup, başarılı olursa, optimal tirozin-mutant AAV3 serotip vektörleri insan karaciğer hedeflemesi için yararlı olduğu ortaya çıkabilir potansiyel gen tedavisi için kanserleri.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-0170CM</td>
<td>&nbsp;</td>
<tr>
<td>PEI</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>23966</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>HiTrap Q HP column</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-1154-01</td>
<td>&nbsp;</td>
<tr>
<td>Salmon sperm DNA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9753983</td>
<td>&nbsp;</td>
<tr>
<td>Iodixanol gradient</td>
<td>&nbsp;</td>
<td>OptiPrep</td>
<td>1114542</td>
<td>&nbsp;</td>
<tr>
<td>G-50 Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>27-5330-01</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

Olmayan bir patojen insan parvovirus dayanan rekombinant vektörler, adeno-ilişkili virüs 2 (AAV2) geliştirilen ve gen tedavisi klinik çalışmalarda bir dizi şu anda kullanımda olan olmuştur. Daha yakın zamanlarda, bir dizi ek AAV serotiplerinin ayrıca çeşitli küçük ve büyük hayvan modellerinde 1 doku tropizm seçici sergilemek edildiği, izole edilmiştir. En sık kullanılan 10 AAV serotipleri, AAV3 gibi in vivo ve in vitro hücre ve dokulara geçirilmesinde kadar en az verimli . AAV3 bağlama için ortak bir hücresel reseptör olarak insan hepatosit büyüme faktörü reseptörü kullanır, çünkü son derece verimli bir hücre hatları - hepatoblastom (HB) ve hepatoselüler karsinom (HSK) - Ancak, son zamanlarda yayınlanan çalışmalarda, biz AAV3 vektörleri insan karaciğer kanseri transduce belgelediler bu hücrelerin 2,3 ve giriş. Bu makalede, biz bir muhabir geni taşıyan rekombinant AAV3 vektörleri insan karaciğer kanser hücrelerini yüksek verimlilik transdüksiyon ulaşmak için gerekli adımları açıklar. Terapötik bir geni taşıyan rekombinant AAV3 vektörlerin kullanımı sonunda, insanlarda karaciğer kanseri potansiyel gen tedavisi neden olabilir.

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Bu makalede, insan karaciğer kanser hücrelerini hedef için en verimli bir vektör olarak adeno-ilişkili virüs serotip 3 (AAV3) tanımlanması için.

Rekombinant Adeno Associated Virus serotip 3 Vektörler Karaciğer Kanseri Hücreleri Yüksek Verimlilik İletimi

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Research

JoVE Journal

Medicine

13.2K Görüntüleme.  University of Florida. Bu makalede, insan karaciğer kanser hücrelerini hedef için en verimli bir vektör olarak adeno-ilişkili virüs serotip 3 (AAV3) tanımlanması için.

Video: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Tıp

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelleme ve Görüntüleme 3-Boyutlu Toplu Hücre Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Moleküler Kolorektal Kanser Hücrelerinin bir 3-Boyutlu Modeli İnvaziv tümör mikro Profili ve<em&gt; Ex vivo</em&gt; Fibroblastlar

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Aracı Olarak Adeno-ilişkili virüs kullanarak Hastalığında Retina Engelleri Eğitim için

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Bir rekombinant adeno-ilişkili viral vektörün stereotaktik enjeksiyon yoluyla Parkinson Hastalığı için bir alfa-sinüklein Bazlı Fare Modeli Geliştirme

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Virüs-aracılı teslim CRISPR kardiyak Gene farelerde düzenleme için Adeno ilişkili

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Karaciğer Rejenerasyon Stratejisi Olarak Üç Boyutlu Kollajen Matris İskele İmplantasyonu

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...