Agradecemos a drs. R. Jude Samulski e Xiao Xiao para os presentes o seu tipo de AAV3 recombinante e AAV-EGFP plasmídeos, respectivamente, e Gail Dr. Tomlinson para generosamente fornecimento de células Hep293TT. Esta pesquisa foi suportada em parte pelo Serviço de Saúde Pública conceder bolsas R01 HL-076901, R01 HL-097088, e P01 DK-058327 (Projeto 1) do National Institutes of Health (para AS).

1. Embalagem de adeno-associados recombinantes do vírus sorotipo 3 (rAAV3) Vetores <ol><li> Acesse o site: &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;e calcular a quantidade de DNA. </li><li> Pré-mix plasmídeos pHelper (helper Adenovirus), pACG2c3 (AAV helper) e pdsAAV-CB-EGFP (rAAV vector) em meio sem SFB e antibióticos. Volume total deve ser 8.750 mL por dez placas de 15 cm 2. </li><li> Adicionar diretamente mL de 1250 PEI (0,1% m / v, Polysciences, Cat # 23966, pH 4.5) na solução de DNA no Passo 1.2. Vortex solução do DNA-PEI por alguns segundos. </li><li> Incubar solução DNA-PEI para formar um complexo de 5 min à temperatura ambiente (RT). </li><li> Adicionar 1 mL de DNA-PEI solução por 15 cm 2 chapa. </li><li> Remova o meio após 4 horas, substituindo com fresca média de 25 mL completa 10% FBS (C-DMEM). </li><li> Setenta e duas horas após a transfecção, as células raspar e despeje em um tubo de centrífuga de 250 mL cônico. </li><li> Giram a 3000 rpm, a 4 ° C por 10 min. Deitar fora o sobrenadante. </li><li> Re-suspender o pellet celular em 5 mL de RB TMS Buffer (pH 50 mM Tris-HCl, 150 mM NaCl, 8,0). Transferência para um novo tubo de 15 mL cônico. </li><li> Congelamento em banho de gelo-seco de etanol por 10 min e descongelamento a 37 ° C por 10 min. Repita três vezes. </li><li> Adicionar 1 mL de 4,8 M MgCl 2 e 2 mL de Benzonase (25 U / mL, Novagen, Cat # 70664-3). Vortex e incubar em 37 ° C por 40 min. </li><li> Girar a 4.000 rpm, a 4 ° C por 40 min e coletar o sobrenadante (lisado clarificado). </li><li> Em um tubo de centrífuga de 13 mL Seal-rápida (Beckman, Cat # 342413), o gradiente de carga iodixanol (OptiPrep, Cat # 1114542) de cima para baixo: 2 mL de 15%, 2 mL de 25%, 1,5 mL de 40% e 1,5 mL de iodixanol 60%. Carregar o sobrenadante (Passo 1.12) na parte superior do gradiente iodixanol. Encher os tubos com RB buffer TMS. </li><li> Centrifugar a 75.000 rpm por 1 hora a 16 ° C utilizando rotor Beckman 90TI. </li><li> Recolher a fração iodixanol 40% através da inserção de uma seringa no limite de 40-60%. </li><li> Alienar a fração iodixanol 40% em um tubo de 50 mL cônica e fazer até 40 mL com tampão A. (20 mM Tris, 15 mM NaCl, pH 8,5) </li><li> Montar o aparelho de filtração utilizando HiTrap Q coluna HP (GE Healthcare, Cat # 17-1154-01). Lavar a coluna com o tampão seguinte: 25 mL de tampão A (20 mM Tris, 15 mM NaCl, pH 8,5);; 25 mL de tampão B (10 mM Tris, 500 mM NaCl, pH 8,5), 50 mL de tampão A 40 mL de amostra de vírus em tampão A; 50 mL de tampão A (20 mM Tris, 15 mM NaCl, pH 8,5);; 20 mL de tampão C (20 mM Tris, 175 mM NaCl, pH 8,5) </li><li> Colete C em um tampão de 20 mL Apollo Concentradores (Orbital Biosciences). Centrifugar a 3.000 rpm, a 4 ° C por 10 min. </li><li> Descartar o fluxo através de e adicionar 20 mL de PBS refrigerado e centrifugar a 3.000 rpm, a 4 ° C por 10 min. </li><li> Descartar o fluxo através de e adicionar 500 mL de PBS gelado. Lavar os vetores fora da membrana e transferir para tubos de silicone tratados com Eppendorf e armazenar congelada em pequenas alíquotas a -80 ° C. </li></ol> 2. Determinação de rAAV3 Títulos Vector <ol><li> Thaw 10 mL de vírus purificado em um tubo Eppendorf no gelo. Adicionar 40 mL de DDH 2 O. </li><li> Adicionar 0,2 mL de Benzonase (25 U / mL, Novagen, Cat # 70664) e incubar a 37 ° C por 1 hora. </li><li> Tomar 50 mL de padrão plasmídeo (0,2 ng / mL) em outro tubo Eppendorf. </li><li> Adicionar 50 mL de NaOH 100 mM em cada tubo e incubar em 65 ° C por 30 min. </li><li> Frio no gelo imediatamente para pelo menos 5 min. </li><li> Corte de membrana de transferência (Millipore, Cat # INYC00010) de acordo com papel de filtro Bio-Dot (Bio-Rad, Cat # 1620161). Enxágüe membrana em SSC 10x com três papéis de filtro por 20 min. </li><li> Set Slot blot SF Aparelho (Bio-Dot, Cat # 170-6542). Conectar a um balão de vácuo e adicionar 100 mL de SSC 10x para slot blot aparelho. </li><li> Adicionar 100 mL de 20x SSC e 1 mL de corante carregamento 6x de cada uma das amostras no Passo 2.5. </li><li> Carga de 100 mL de cada amostra para a membrana no aparelho blot slot. </li><li> Adicionar outro mL 100 de SSC 10x em cada uma das amostras. </li><li> Repita os passos 2.9 e 2.10 até que todas as amostras o atravessa a membrana. </li><li> Desmontar o aparelho blot slot e remover a membrana. Colocá-lo em Linker Inspector SL-1000 Agente de crosslinking UV. Transformar em &quot;Energia&quot;, &quot;Crosslink Optimal&quot; (show 1200) e empurre &quot;Start&quot;. </li><li> Ferva 1 mL de esperma de salmão (SS) DNA (Fisher, Cat # NC9753983) por 5 min. Frio no gelo por pelo menos 5 min. </li><li> Prepare a solução de pré-hibridação através da mistura de 27,5 mL de DDH 2 O, 15 mL de 20x SSC, 5 mL de solução 50x Denhardt é, 1,25 mL de SDS 20%, 0,25 mL de Polya e 1 mL de SS DNA a partir da Etapa 2.13. </li><li> Suspender a membrana em 25 mL de solução de hibridização pré-nos um cilindro de vidro tampado e incubar a 65 ° C em uma câmara de hibridização durante a noite. </li><li> Diluir 50 modelos DNA ng em 10 mL de DDH 2 O. Ferver a 100 ° C por 5 min e frio no gelo imediatamente para pelo menos 5 min. </li><li> Centrifugar a 3000 rpm em temperatura ambiente por 1 min. Adicione 2 mL de 10 mM dNTPs 3 (falta dCTP), 2 mL da mistura de Hexanucleotide (Roche, Cat # 11277081001), 1 mL de Klenow (Roche, Cat # 11008404001) e 5 mL de α -32 P-dCTP. Incubar a 37 ° C por 10 min. </li><li> Centrífuga G-50 Colunas (GE Healthcare, Cat # 27-5330-01) a 3.000 rpm por 2 min. carga a sonda em Passo 2,17 na coluna. Centrifugar a 3.000 rpm por 2 min e recolher o escoamento. </li><li> Contagem de radioatividade. Ferva a sonda a 100 ° C por 5 min e frio no gelo imediatamente para pelo menos 5 min. </li><li> Adicionar a sonda (6x10 5 cpm / mL) na solução de pré-hibridação com membrana. </li><li> Incubar durante a 65 ° C em uma câmara de hibridação. </li><li> Descartar a solução de hibridação e enxaguar a membrana em 2x SSC SDS 0,1% no RT por 15 min. </li><li> Descartar a solução e lave a membrana em 0,1 x SSC SDS 0,1% a 65 ° C por 30 min. </li><li> Descartar a solução e lave a membrana em 0,1 x SSC em breve RT. </li><li> Expor o filme a -80 ° C durante 6 horas e desenvolver o filme. Um resultado típico é mostrado na Figura 1. </li></ol> 3. rAAV3 Transdução Vector mediada e expressão do transgene em células humanas de câncer de fígado <ol><li> Semente de 1x10 4 células (ou Huh7 ou Hep293TT) em cada poço da placa de 96 poços. Incubar em umidificado incubadora de CO 2 por pelo menos 18 horas. </li><li> Remova o meio da placa de 96 poços. </li><li> Lave as células duas vezes com 50 mL de meio, sem FBS e antibióticos e remover. </li><li> Adicionar 50 mL de meio sem FBS e antibióticos e vetores rAAV menos 5.000 vgs / célula. </li><li> Incubar as placas na incubadora de CO 2 para 4 horas. </li><li> Descartar o meio. Lave as células duas vezes com 50 mL de meio completo e remover. </li><li> Adicionar 150 mL de C-DMEM a cada poço e incube a placa na incubadora de CO 2 por 72 horas, e visualizar a expressão EGFP usando um microscópio de fluorescência (DMI 4000B; Leica Microsystems). </li><li> Imagens a partir de três poços de células infectadas por vírus são analisados ​​quantitativamente através de análise de software ImageJ (NIH, Bethesda, MD, EUA). </li><li> Expressão do transgene é avaliada como a área total de fluorescência verde (pixel 2) por campo visual. Um resultado típico é mostrado na Figura 2. </li><li> Expressão do transgene também pode ser determinado por citometria de fluxo. </li></ol> 4. Resultados representativos: Seguindo o protocolo descrito acima, pode-se gerar vetores AAV sorotipo eficiente. O rendimento típico é ~ 500 mL contendo ~ 10 11 vgs / mL de ações purificadas vetor. A pureza do estoque vetor é determinada pela comparação de sinais de hibridação em blots slot de DNA quantitativo, com e sem digestão Benzonase. Purificada rAAV3 vetores transduzir câncer de fígado humano - hepatoblastoma (HB) e carcinoma hepatocelular (HCC) - linhas de células de forma eficiente. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Figura 1" /> Figura 1. Quantitativa de análises de ADN Slot blot para determinar os títulos de rAAV3 vetores. Diluições duplas de purificada stocks viral, digerido com Benzonase (linha superior) foram analisados ​​em blots ranhura quantitativa DNA com 32 P-sonda marcada DNA EGFP-específicas. A título de AAV3 vetor foi determinada pela comparação com 1 ng (linha do meio) ou 10 ng (linha inferior) de AAV-EGFP padrões plasmídeo carregado na membrana. Os números correspondem a cópias de DNA. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Figura 2" /> Figura 2. AAV recombinante vector mediada expressão do transgene em células humanas de câncer de fígado. Hep293TT células, uma recém-criada linha celular humana hepatoblastoma 4, eram (painel esquerdo) mock-infectados, ou transduzidas com 5.000 vgs / célula de qualquer scAAV2-EGFP ou scAAV3-EGFP vetores a 37 ° C por 2 horas. Expressão do transgene foi visualizado 72 horas pós-transdução usando um microscópio de fluorescência.

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Não há conflitos de interesse declarados.

Vetores recombinantes baseados em um parvovírus não-patogênicos humanos, o vírus adeno-associado (AAV) têm sido desenvolvidas, e estão atualmente em uso em uma série de ensaios clínicos de terapia genética 5. Anteriormente, dos 10 sorotipos mais comumente usados ​​AAV, a eficiência de transdução de AAV3 vetores em geral, tem sido relatada a ser particularmente baixo, tanto in vitro como in vivo 1. No entanto, a nossa observação recente de que AAV3 vetores transdução do fígado humano linhas celulares de cancro muito bem, conforme determinado quantitativamente por citometria de fluxo 2, e que o ser humano utiliza AAV3 hepatócito receptor do fator de crescimento (hHGFR) como um co-receptor celular para a entrada obrigatória e nestes 3 células, sugerem fortemente uma seletiva do tecido tropismo de AAV3 de células do fígado humano em geral, e as células humanas de câncer de fígado em particular. No entanto, desde AAV3 vetores também transdução de hepatócitos humanos normais de forma eficiente 2, seu uso potencial no câncer de aplicação da terapia gênica in vivo seria impactado negativamente. Uma possível estratégia para contornar esse problema em potencial envolve a transcrição de metas de células de câncer através da identificação de um produto gene que é seletivamente produzida pelo tumor, mas não pelos hepatócitos normal. Por exemplo, estudos anteriores mostraram que os níveis séricos de α-fetoproteína (AFP) são usados ​​como um marcador específico para o rastreamento da presença, progressão e / ou a recorrência de certos tipos de cânceres de fígado, uma vez que os hepatócitos normais geralmente produzem quantidades muito pequenas de esta proteína. Na verdade, temos utilizado AAV3 vetores contendo o promotor AFP para atingir a expressão do transgene em células de câncer de fígado, mas não em hepatócitos normais 2, e os estudos estão em andamento para testar a eficácia desta abordagem em um modelo murino xenoenxerto de câncer de fígado. Em nossos estudos adicionais, temos observado que a eficiência de transdução de vetores sorotipo vários AAV pode ser significativamente aumentada pelo sítio-dirigida mutagênese da superfície exposta resíduos de tirosina na capsídeos virais 14/06. Desde 6 de 7 de superfície exposta tirosinas também são conservados em AAV3, temos realizado mutagênese sítio-dirigida desses resíduos e observaram que a eficiência de transdução de tirosina-mutante AAV3 vetores é significativamente maior em células de fígado humano com câncer (dados não publicados). Estudos também estão em andamento para avaliar a segurança ea eficácia da tirosina-mutante AAV3 vetores em modelos xenoenxerto mouse para hepatoblastoma humanos e carcinoma hepatocelular, e se bem sucedida, o ideal tirosina-mutante vetores sorotipo AAV3 pode vir a ser útil para a segmentação do fígado humano câncer para a terapia genética potencial.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</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>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>23966</td>
<td>&nbsp;</td>
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<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>
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<td>G-50 Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>27-5330-01</td>
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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

Vetores recombinantes baseado em um parvovirus patogênico não-humanos, o vírus adeno-associado 2 (AAV2) foram desenvolvidos e estão atualmente em uso em uma série de ensaios clínicos de terapia genética. Mais recentemente, um número de sorotipos adicionais AAV também foram isolados, que foram mostrados para expor seletiva tropismo de tecido em vários modelos animais pequenos e grandes 1. Dos 10 sorotipos mais comumente usados ​​AAV, AAV3 é de longe o menos eficiente na transdução de células e tecidos in vitro, bem como in vivo. No entanto, em nossos estudos recentemente publicados, temos documentado que AAV3 vetores transduzir câncer de fígado humano - hepatoblastoma (HB) e carcinoma hepatocelular (HCC) - linhas de células de forma extremamente eficiente porque utiliza AAV3 receptor do fator humano como um hepatócito crescimento celular co-receptor para a ligação e entrada nestas células 2,3. Neste artigo, descrevemos os passos necessários para atingir alta eficiência de transdução de células humanas de câncer de fígado por vetores recombinantes AAV3 carregando um gene repórter. O uso de vetores recombinantes AAV3 carregando um gene terapêutico pode eventualmente levar à terapia genética potencial de câncer de fígado em humanos.

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

Neste artigo, descrevemos a identificação da adeno-associated virus sorotipo 3 (AAV3) como o vetor mais eficiente para alvejar células humanas de câncer de fígado.

De Alta Eficiência Transdução de células de câncer de fígado por recombinante sorotipo de vírus adeno-associado 3 Vetores

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 visualizações.  University of Florida. Neste artigo, descrevemos a identificação da adeno-associated virus sorotipo 3 (AAV3) como o vetor mais eficiente para alvejar células humanas de câncer de fígado.

Vídeo: 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.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

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

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.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

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;

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

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.

Usando vírus adeno-associado como uma ferramenta para o estudo da retina Barreiras na Doença

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.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

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.

Desenvolvimento de um modelo de rato baseada alfa-sinucleína para a doença de Parkinson por meio de injecção estereotáxica de um vector viral recombinante adeno-associado

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.

Geração rápida e eficiente de células-tronco pluripotentes Recombinante Humana pela recombinase mediada troca cassete no<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.

Entrega de mediada por vírus adeno-associado de CRISPR para Gene cardíaco em ratos de edição

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.

Administração Subconjuntival de Vetores de Vírus Adenoassociados em Modelos de Pequenos Animais

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 de AAV por centrifugação de gradiente de densidade de iodixanol duplo

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.

Autor em destaque: Isolamento eficiente de vírus adeno-associados para aplicações pré-clínicas

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.

Produção e purificação de vírus adenoassociados em cultura de suspensão por centrifugação por gradiente de densidade de iodixanol para aplicações in vivo