Ringraziamo Drs. R. Jude Samulski e Xiao Xiao per i loro doni tipo di ricombinante AAV3 e AAV-EGFP plasmidi, rispettivamente, e il dottor Tomlinson Gail generosamente per fornire cellule Hep293TT. Questa ricerca è stata sostenuta in parte dal Public Health Service concedere borse di R01 HL-076901, R01 HL-097088, e DK P01-058327 (Progetto 1) dal National Institutes of Health (AS).

1. Confezionamento di virus ricombinante adeno-associato sierotipo 3 (rAAV3) Vettori <ol><li> Vai al sito: &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;e calcolare la quantità di DNA. </li><li> Pre-mix plasmidi pHelper (helper Adenovirus), pACG2c3 (AAV helper) e pdsAAV-CB-EGFP (rAAV vettoriale) in mezzo senza FBS e antibiotici. Volume totale dovrebbe essere 8750 microlitri per una decina di 15 cm 2 piastre. </li><li> Aggiungere direttamente 1.250 ml di PEI (0,1% m / v, Polysciences, Cat # 23966, pH 4,5) in soluzione di DNA al punto 1.2. Vortex il DNA-PEI soluzione per alcuni secondi. </li><li> Incubare DNA-PEI soluzione per formare un complesso per 5 minuti a temperatura ambiente (RT). </li><li> Aggiungere 1 ml di DNA-PEI soluzione per 15 cm 2 piastre. </li><li> Rimuovere media dopo 4 ore, sostituendo con fresche 25 ml di mezzo di completa +10% FBS (C-DMEM). </li><li> Settantadue ore dopo la trasfezione, le cellule raschiare e versare in una provetta per centrifuga da 250 ml. </li><li> Rotazione a 3000 rpm, a 4 ° C per 10 min. Eliminare il surnatante. </li><li> Risospendere il pellet in 5 ml di RB TMS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8,0). Trasferimento in un nuovo tubo di 15 ml. </li><li> Congelamento in ghiaccio secco-etanolo bagno per 10 minuti e scongelare a 37 ° C per 10 min. Ripetete tre volte. </li><li> Aggiungere 1 ml di 4,8 M MgCl 2 e 2 l di benzonasi (25 U / mL, Novagen, Cat # 70664-3). Vortex e incubare a 37 ° C per 40 min. </li><li> Spin down a 4.000 giri, a 4 ° C per 40 minuti e raccogliere il surnatante (lisato chiarificato). </li><li> In una ML 13 Quick-Seal provetta da centrifuga (Beckman, Cat # 342413), carico gradiente Iodixanolo (OptiPrep, Cat # 1114542) da cima a fondo: 2 ml di 15%, 2 ml di 25%, 1,5 mL di 40% e 1,5 mL di 60% Iodixanolo. Caricare il surnatante (Passo 1.12) sulla parte superiore del gradiente Iodixanolo. Riempire i tubi con tampone RB TMS. </li><li> Centrifugare a 75.000 rpm per 1 ora a 16 ° C con rotore Beckman 90TI. </li><li> Raccogliere la frazione Iodixanolo 40% con l&#39;inserimento di una siringa al 40-60% confine. </li><li> Trasferire la frazione Iodixanolo 40% in un tubo da 50 ml e portare a 40 ml con tampone A (20 mM Tris, 15 mM NaCl, pH 8,5). </li><li> Montare l&#39;apparato di filtrazione con HITRAP Q HP colonna (GE Healthcare, Cat # 17-1154-01). Lavare la colonna con le seguenti tampone: 25 ml di tampone A (20 mM Tris, 15 mM NaCl, pH 8,5); 25 mL di tampone B (10 mM Tris, 500 mM NaCl, pH 8,5), 50 ml di tampone A; 40 ml di campione virus nel tampone A, 50 ml di tampone A (20 mM Tris, 15 mM NaCl, pH 8,5), 20 ml di tampone C (20 mM Tris, 175 mM NaCl, pH 8,5); </li><li> Raccogliere C in un buffer da 20 ml Apollo concentratori (Orbital Biosciences). Centrifugare a 3.000 giri, a 4 ° C per 10 min. </li><li> Gettare il flow-through e aggiungere 20 ml di acqua refrigerata PBS e centrifugare a 3.000 giri, a 4 ° C per 10 min. </li><li> Gettare il flow-through e aggiungere 500 ml di PBS freddo. Lavare i vettori fuori dalla membrana e trasferimento siliconata provette Eppendorf e conservare congelata in piccole aliquote a -80 ° C. </li></ol> 2. Determinazione del rAAV3 Titoli Vector <ol><li> Scongelare 10 L del virus purificato in una provetta Eppendorf sul ghiaccio. Aggiungere 40 ml di DDH 2 O. </li><li> Aggiungere 0,2 ml di benzonasi (25 U / mL, Novagen, Cat # 70664) e incubare a 37 ° C per 1 ora. </li><li> Prelevare 50 ml di standard di plasmide (0,2 ng / mL) in un&#39;altra provetta Eppendorf. </li><li> Aggiungere 50 ml di 100 mM NaOH in ogni provetta e incubare a 65 ° C per 30 min. </li><li> Freddo su ghiaccio immediatamente per almeno 5 minuti. </li><li> Tagliare a membrana di trasferimento (Millipore, Cat # INYC00010) secondo Bio-Dot filtro di carta (Bio-Rad, Cat # 1620161). Sciacquare membrana in SSC 10x con tre filtri di carta per 20 min. </li><li> Set di slot blot SF Apparatus (Bio-Dot, Cat # 170-6542). Connettersi a un fiasco vuoto e aggiungere 100 ml di SSC 10x a cancellare apparato slot. </li><li> Aggiungere 100 ml di SSC 20x e 1 ml di colorante di caricamento 6x a ciascuno dei campioni al punto 2.5. </li><li> Carico di 100 l di ciascun campione sulla membrana nell&#39;apparato macchia slot. </li><li> Aggiungere altri 100 ml di SSC 10x in ciascuno dei campioni. </li><li> Ripetere i punti 2.9 e 2.10 fino a tutto il campione passa attraverso la membrana. </li><li> Smontare l&#39;apparato macchia slot e rimuovere la membrana. Metterlo in Linker ispettore SL-1000 Crosslinker UV. Girare su &quot;Energia&quot;, &quot;Optimal Crosslink&quot; (mostra 1200) e premere &quot;Start&quot;. </li><li> Far bollire 1 ml di sperma di salmone (SS) DNA (Fisher, Cat # NC9753983) per 5 min. Freddo in ghiaccio per almeno 5 minuti. </li><li> Preparare la pre-ibridazione soluzione mescolando 27,5 mL di DDH 2 O, 15 ml di SSC 20x, 5 ml di soluzione di 50x Denhardt, 1,25 ml di SDS 20%, 0,25 ml di Polya e 1 ml di SS DNA dal punto 2.13. </li><li> Sospendere la membrana in 25 mL di soluzione pre-ibridazione in un cilindro di vetro ricoperti e incubare a 65 ° C in una camera di ibridazione durante la notte. </li><li> Diluire 50 modelli ng di DNA in 10 ml di DDH 2 O. Fate bollire a 100 ° C per 5 minuti e raffreddare immediatamente in ghiaccio per almeno 5 minuti. </li><li> Centrifugare a 3.000 rpm a temperatura ambiente per 1 min. Aggiungere 2 ml di 10 mM dNTPs 3 (manca dCTP), 2 ml di Mix Hexanucleotide (Roche, Cat # 11277081001), 1 l di Klenow (Roche, Cat # 11008404001) e 5 ml di α -32 P-dCTP. Incubare a 37 ° C per 10 min. </li><li> Centrifuga G-50 colonne (GE Healthcare, Cat # 27-5330-01) a 3.000 rpm per 2 minuti. caricare la sonda nel passo 2,17 nella colonna. Centrifugare a 3.000 rpm per 2 minuti e raccogliere i flow-through. </li><li> Conte radioattività. Lessare la sonda a 100 ° C per 5 minuti e chill su ghiaccio immediatamente per almeno 5 minuti. </li><li> Aggiungere la sonda (6x10 5 cpm / ml) nel pre-ibridazione soluzione con membrana. </li><li> Incubare per una notte a 65 ° C in una camera di ibridazione. </li><li> Gettare la soluzione di ibridazione e risciacquare la membrana in 2x SSC +0,1% SDS a temperatura ambiente per 15 minuti. </li><li> Scartare la soluzione e lavare la membrana in 0.1x SSC +0,1% SDS a 65 ° C per 30 min. </li><li> Scartare la soluzione e lavare la membrana in 0.1x SSC a breve RT. </li><li> Esporre il film a -80 ° C per 6 ore e sviluppare il film. Un risultato tipico è mostrato in Figura 1. </li></ol> 3. rAAV3 Vector-mediata trasduzione e l&#39;espressione del transgene nelle cellule umane di cancro del fegato <ol><li> Semi di 1x10 4 celle (sia Huh7 o Hep293TT) in ciascun pozzetto della piastra da 96 pozzetti. Incubare in un incubatore umidificato CO 2 per almeno 18 ore. </li><li> Togliere terreno dalla piastra a 96 pozzetti. </li><li> Lavare le cellule due volte con 50 ml di mezzo senza FBS e antibiotici e rimuovere. </li><li> Aggiungere 50 ml di mezzo senza FBS e antibiotici e vettori rAAV a 5.000 VGS / cell. </li><li> Incubare le piastre in incubatore CO 2 per 4 ore. </li><li> Gettare il mezzo. Lavare le cellule due volte con 50 ml di terreno completo e rimuovere. </li><li> Aggiungere 150 ml di DMEM-C in ciascun pozzetto e incubare la piastra di CO 2 incubatore per 72 ore, e visualizzare l&#39;espressione EGFP utilizzando un microscopio a fluorescenza (DMI 4000B, Leica Microsystems). </li><li> Immagini da tre pozzi di cellule infettate da virus sono analizzate quantitativamente mediante analisi software ImageJ (NIH, Bethesda, MD, USA). </li><li> Espressione del transgene è valutato come superficie complessiva di fluorescenza verde (pixel 2) per campo visivo. Un risultato tipico è illustrato nella figura 2. </li><li> Espressione del transgene può anche essere determinata mediante citometria di flusso. </li></ol> 4. Rappresentante dei risultati: Seguendo il protocollo di cui sopra, si può generare vettori AAV sierotipo in modo efficiente. Il rendimento tipico è ~ 500 microlitri contiene ~ 10 11 vgs / mL di purificato magazzino vettore. La purezza dello stock vettore è determinato dal confronto segnali di ibridazione su macchine di slot quantitativa del DNA, con e senza digestione benzonasi. Purificata rAAV3 vettori cancro al fegato umano trasdurre - epatoblastoma (HB) e del carcinoma epatocellulare (HCC) - linee cellulari in modo efficiente. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Figura 1" /> Figura 1. Quantitativa del DNA slot-blot analisi per la determinazione del titoli di rAAV3 vettori. Una diluizione di purificato scorte virale, digerito con benzonasi (riga in alto) sono stati analizzati su macchine di slot quantitativa del DNA con 32 P-etichettati EGFP-sonda specifica del DNA. Il titolo di AAV3 vettore è stato determinato in rapporto con 1 ng (riga centrale) o 10 ng (riga inferiore) di AAV-EGFP standard plasmide caricato sulla membrana. I numeri corrispondono alle copie del DNA. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Figura 2" /> Figura 2. Vettore ricombinante AAV-mediata espressione del transgene nelle cellule umane di cancro del fegato. Cellule Hep293TT, un recente costituzione linea cellulare umana epatoblastoma 4, erano o mock-infettate (a sinistra del pannello), oppure trasdotte con 5.000 VGS / cella di uno scAAV2-EGFP o scAAV3-EGFP vettori a 37 ° C per 2 ore. Espressione del transgene è stata visualizzata 72 ore post-trasduzione utilizzando un microscopio a fluorescenza.

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Nessun conflitto di interessi dichiarati.

Vettori ricombinanti sulla base di una non patogeno parvovirus umano, il virus adeno-associato (AAV) sono stati sviluppati e sono attualmente in uso in un certo numero di studi clinici di terapia genica 5. In precedenza, dei 10 sierotipi più comunemente utilizzati AAV, l&#39;efficienza di trasduzione del AAV3 vettori in genere è stato segnalato per essere particolarmente bassi, sia in vitro che in vivo 1. Tuttavia, la nostra recente osservazione che AAV3 vettori trasdurre fegato umano linee cellulari tumorali estremamente bene, come determinato quantitativamente mediante citometria di flusso 2 e che AAV3 utilizza il recettore umano di crescita degli epatociti (fattore hHGFR) come un cellulare co-recettore per l&#39;iscrizione vincolante e in questi cellule 3, suggeriscono fortemente una selettiva dei tessuti di AAV3 tropismo per le cellule di fegato umano in generale, e cellule umane di cancro del fegato in particolare. Tuttavia, poiché AAV3 vettori anche trasdurre normali epatociti umani in modo efficiente 2, il loro potenziale utilizzo in applicazione della terapia genica del cancro in vivo sarebbe negativamente. Una possibile strategia per aggirare questo potenziale problema coinvolge trascrizionale-targeting delle cellule tumorali attraverso l&#39;identificazione di un prodotto gene che è selettivamente prodotta dal tumore, ma non dagli epatociti normali. Per esempio, studi precedenti hanno dimostrato che i livelli sierici di α-fetoproteina (AFP) è utilizzato come marcatore specifico per il monitoraggio della presenza, la progressione, e / o la recrudescenza di alcuni tipi di tumori del fegato, dato che epatociti normali generalmente producono piccole quantità di questa proteina. Infatti, abbiamo utilizzato AAV3 vettori contenenti il promotore AFP per indirizzare l&#39;espressione del transgene nelle cellule di cancro al fegato, ma non negli epatociti normali 2, e gli studi sono attualmente in corso per testare l&#39;efficacia di questo approccio in un modello murino xenotrapianto di cancro al fegato. Nei nostri studi ulteriori, abbiamo osservato che l&#39;efficienza di trasduzione di vari vettori AAV sierotipo può essere significativamente aumentata da mutagenesi sito-diretta di superficie esposti residui di tirosina nel capsidi virale 6-14. Dal 6 di 7 superficie esposta tirosine sono inoltre conservati in AAV3, abbiamo svolto mutagenesi sito-diretta di detti residui e osservato che l&#39;efficienza di trasduzione di tirosin-mutante AAV3 vettori è significativamente migliorata nelle cellule umane di cancro del fegato (dati non pubblicati). Gli studi sono attualmente in corso per valutare la sicurezza el&#39;efficacia della tirosin-mutante AAV3 vettori nei modelli di xenotrapianto mouse per epatoblastoma umano e il carcinoma epatocellulare, e in caso di successo, l&#39;ottimale tirosina-mutante vettori sierotipo AAV3 potrebbe rivelarsi utile per indirizzare fegato umano tumori per la terapia genica potenziale.

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		<th>Catalogue Number</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-0170CM</td>
<td>&nbsp;</td>
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<td>PEI</td>
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<td>Polysciences</td>
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<td>70664-3</td>
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<td>HiTrap Q HP column</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-1154-01</td>
<td>&nbsp;</td>
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<td>Salmon sperm DNA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9753983</td>
<td>&nbsp;</td>
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<td>Iodixanol gradient</td>
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<td>1114542</td>
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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

Vettori ricombinanti sulla base di una non patogeno parvovirus umano, il virus adeno-associati 2 (AAV2) sono stati sviluppati e sono attualmente in uso in un certo numero di studi clinici di terapia genica. Più recentemente, una serie di ulteriori sierotipi AAV sono stati anche isolati, che hanno dimostrato di esporre selettivo-trofismo dei tessuti in vari modelli animali piccoli e grandi 1. Dei 10 sierotipi più comunemente utilizzati AAV, AAV3 è di gran lunga il meno efficiente in trasduzione cellule e tessuti in vitro e in vivo. Tuttavia, nei nostri studi di recente pubblicazione, abbiamo documentato che AAV3 vettori trasdurre il cancro del fegato umano - epatoblastoma (HB) e di carcinoma epatocellulare (HCC) - linee di cellule estremamente efficiente perché utilizza AAV3 umano recettore del fattore di crescita degli epatociti come un cellulare co-recettore per il legame e l&#39;ingresso in queste cellule 2,3. In questo articolo, si descrivono i passaggi necessari per ottenere alta efficienza di trasduzione di cellule umane di cancro del fegato da ricombinante AAV3 vettori che trasportano un gene reporter. L&#39;uso di vettori ricombinanti AAV3 portatrice di un gene terapeutico può portare alla terapia genica potenziale dei tumori del fegato negli esseri umani.

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

In questo articolo, si descrive l&#39;identificazione del sierotipo del virus adeno-associati 3 (AAV3) come il vettore più efficace per le cellule umane di cancro del fegato.

Ad alta efficienza di trasduzione delle cellule tumorali del fegato da adeno-associato ricombinante sierotipo Virus 3 Vettori

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 Views.  University of Florida. In questo articolo, si descrive l'identificazione del sierotipo del virus adeno-associati 3 (AAV3) come il vettore più efficace per le cellule umane di cancro del fegato.

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.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

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

Ricerca

Medicina

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.

Una piattaforma di screening Luciferase basata multiplex per interrogare il cancro-associata trasduzione del segnale in cellule in coltura

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;

Molecular Profiling del microambiente tumorale invasiva in un modello 3-Dimensional delle cellule del cancro colorettale e<em&gt; Ex vivo</em&gt; fibroblasti

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.

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

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.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

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.

Sviluppo di un Rat modello Alfa-sinucleina per la malattia di Parkinson mediante iniezione stereotassica di un ricombinante vettore virale adeno-associato

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.

Generazione rapida ed efficace delle ricombinante cellule staminali umane pluripotenti da ricombinasi mediata scambio Cassette in<em&gt; AAVS1</em&gt; Locus

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.

Adeno-associato Virus-mediate consegna di CRISPR per Gene cardiaco nei topi di Editing

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.

Somministrazione subcongiuntivale di vettori virali adeno-associati in modelli di piccoli animali

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

Isolamento AAV mediante centrifugazione in gradiente a doppia densità di iodipanolo

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.

Autore in primo piano: Isolamento efficiente dei virus adeno-associati per applicazioni precliniche

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.

Produzione e purificazione in coltura in sospensione di virus adeno-associati mediante centrifugazione a gradiente di densità di iodixanolo per applicazioni in vivo