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

13.1K 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

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 ...

Tıp

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  ...

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.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

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 ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

içinde Recombinase aracılı Kaset Borsası tarafından Rekombinant insan pluripotent kök hücreleri hızlı ve verimli Nesil<em&gt; AAVS1</em&gt; Odağı

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

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 ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Küçük Hayvan Modellerinde Adeno İlişkili Virüs Vektörlerinin Subkonjonktival Uygulaması

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Çift İodiksanol Yoğunluk Gradyan Santrifüjleme ile AAV İzolasyonu

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Yazar Spotlight: Klinik Öncesi Uygulamalar için Verimli Adeno İlişkili Virüs İzolasyonu

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.