Name: high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors
Uploaded: 2011-03-22T13:00:00
Duration: 19 min 2 s
Description: Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

We thank Drs. R. Jude Samulski and Xiao Xiao for their kind gifts of recombinant AAV3 and AAV-EGFP plasmids, respectively, and Dr. Gail Tomlinson for generously providing Hep293TT cells. This research was supported in part by Public Health Service grant grants R01 HL-076901, R01 HL-097088, and P01 DK-058327 (Project 1) from the National Institutes of Health (to AS).

1. Packaging of Recombinant Adeno-associated Virus Serotype 3 (rAAV3) Vectors
<ol>
<li>Go to the website: "<a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a>" and calculate the amount of DNA.</li>
<li>Pre-mix plasmids pHelper (Adenovirus helper), pACG2c3 (AAV helper) and pdsAAV-CB-EGFP (rAAV vector) in medium without FBS and antibiotics. Total volume should be 8,750 &mu;L for ten 15 cm2 plates.</li>
<li>Add directly 1,250 &mu;L of PEI (0.1% m/v, Polysciences, Cat# 23966, pH 4.5) into DNA solution in Step 1.2. Vortex the DNA-PEI solution for a few seconds.</li>
<li>Incubate DNA-PEI solution to form a complex for 5 min at room temperature (RT).</li>
<li>Add 1 mL of DNA-PEI solution per 15 cm2 plate.</li>
<li>Remove medium after 4 hours, replacing with fresh 25 mL complete medium+10%FBS (C-DMEM).</li>
<li>Seventy two hours post-transfection, scrape cells and pour into a 250 mL conical centrifuge tube.</li>
<li>Spin at 3000 rpm, at 4&deg;C for 10 min. Pour off supernatant.</li>
<li>Re-suspend the cell pellet in 5 mL of RB TMS Buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). Transfer to a new 15 mL conical tube.</li>
<li>Freeze in dry ice-ethanol bath for 10 min and thaw at 37&deg;C for 10 min. Repeat three times.</li>
<li>Add 1 &mu;L of 4.8 M MgCl2 and 2 &mu;L of Benzonase (25 U/ &mu;L, Novagen, Cat# 70664-3). Vortex and incubate in 37&deg;C for 40 min.</li>
<li>Spin down at 4,000 rpm, at 4&deg;C for 40 min and collect the supernatant (clarified lysate).</li>
<li>In a 13 mL Quick-Seal centrifuge tube (Beckman, Cat# 342413), load iodixanol gradient (OptiPrep, Cat# 1114542) from top to bottom: 2 mL of 15%, 2 mL of 25%, 1.5 mL of 40% and 1.5 mL of 60% iodixanol. Load the supernatant (Step 1.12) on the top of iodixanol gradient. Fill the tubes with RB TMS Buffer.</li>
<li>Centrifuge at 75,000 rpm for 1 hour at 16&deg;C using Beckman 90Ti rotor.</li>
<li>Collect the 40% iodixanol fraction by inserting a syringe at 40-60% boundary.</li>
<li>Transfer the 40% iodixanol fraction in one 50 mL conical tube and make up to 40 mL with Buffer A (20 mM Tris, 15 mM NaCl, pH 8.5).</li>
<li>Assemble the filtration apparatus using HiTrap Q HP column (GE Healthcare, Cat# 17-1154-01). Wash the column with the following buffer: 25 mL of Buffer A (20 mM Tris, 15 mM NaCl, pH 8.5); 25 mL of Buffer B (10 mM Tris, 500 mM NaCl, pH 8.5); 50 mL of Buffer A; 40 mL of virus sample in Buffer A; 50 mL of Buffer A (20 mM Tris, 15 mM NaCl, pH 8.5); 20 mL of Buffer C (20 mM Tris, 175 mM NaCl, pH 8.5);</li>
<li>Collect Buffer C in a 20 mL Apollo Concentrators (Orbital Biosciences). Centrifuge at 3,000 rpm, at 4&deg;C for 10 min.</li>
<li>Discard the flow-through and add 20 mL of chilled PBS and centrifuge at 3,000 rpm, at 4&deg;C for 10 min.</li>
<li>Discard the flow-through and add 500 &mu;L of chilled PBS. Wash the vectors off from the membrane and transfer to silicone-treated Eppendorf tubes and store frozen in small aliquots at -80&deg;C.</li>
</ol>
2. Determination of rAAV3 Vector Titers
<ol>
<li>Thaw 10 &mu;L of purified virus in an Eppendorf tube on ice. Add 40 &mu;L of ddH2O.</li>
<li>Add 0.2 &mu;L of Benzonase (25 U/ &mu;L, Novagen, Cat# 70664) and incubate at 37&deg;C for 1 hour.</li>
<li>Take 50 &mu;L of plasmid standard (0.2 ng/ &mu;L) in another Eppendorf tube.</li>
<li>Add 50 &mu;L of 100 mM NaOH in each tube and incubate in 65&deg;C for 30 min.</li>
<li>Chill on ice immediately for at least 5 min.</li>
<li>Cut transfer membrane (Millipore, Cat# INYC00010) according to Bio-Dot Filter Paper (Bio-Rad, Cat# 1620161). Rinse membrane in 10x SSC with three filter papers for 20 min.</li>
<li>Set slot blot SF Apparatus (Bio-Dot, Cat# 170-6542). Connect to a vacuum flask and add 100 &mu;L of 10x SSC to slot blot apparatus.</li>
<li>Add 100 &mu;L of 20x SSC and 1 &mu;L of 6x loading dye to each of the samples in Step 2.5.</li>
<li>Load 100 &mu;L of each samples onto the membrane in the slot blot apparatus.</li>
<li>Add another 100 &mu;L of 10x SSC into each of the samples.</li>
<li>Repeat Steps 2.9 and 2.10 until all the sample goes through the membrane.</li>
<li>Disassemble the slot blot apparatus and remove the membrane. Put it into Inspector Linker SL-1000 UV Crosslinker. Turn on "Energy", "Optimal Crosslink" (show 1200) and push "Start".</li>
<li>Boil 1 mL of salmon sperm (SS) DNA (Fisher, Cat# NC9753983) for 5 min. Chill on ice for at least 5 min.</li>
<li>Prepare the pre-hybridization solution by mixing 27.5 mL of ddH2O, 15 mL of 20x SSC, 5 mL of 50x Denhardt's solution, 1.25 mL of 20% SDS, 0.25 mL of PolyA and 1 mL of SS DNA from Step 2.13.</li>
<li>Suspend the membrane in 25 mL of pre-hybridization solution in a capped glass cylinder and incubate at 65&deg;C in a hybridization chamber overnight.</li>
<li>Dilute 50 ng DNA templates in 10 &mu;L of ddH2O. Boil at 100&deg;C for 5 min and chill on ice immediately for at least 5 min.</li>
<li>Centrifuge at 3,000 rpm at RT for 1 min. Add 2 &mu;L of 10 mM 3 dNTPs (lacking dCTP), 2 &mu;L of Hexanucleotide Mix (Roche, Cat# 11277081001), 1 &mu;L of Klenow (Roche, Cat# 11008404001) and 5 &mu;L of &alpha;-32P-dCTP. Incubate at 37&deg;C for 10 min.</li>
<li>Centrifuge G-50 Columns (GE Healthcare, Cat# 27-5330-01) at 3,000 rpm for 2 min. load the probe in Step 2.17 into the column. Centrifuge at 3,000 rpm for 2 min and collect the flow-through.</li>
<li>Count radioactivity. Boil the probe at 100&deg;C for 5 min and chill on ice immediately for at least 5 min.</li>
<li>Add the probe (6x105 cpm/ mL) into the pre-hybridization solution with membrane.</li>
<li>Incubate overnight at 65&deg;C in a hybridization chamber.</li>
<li>Discard the hybridization solution and rinse the membrane in 2x SSC+0.1%SDS at RT for 15 min.</li>
<li>Discard the solution and rinse the membrane in 0.1x SSC+0.1%SDS at 65&deg;C for 30 min.</li>
<li>Discard the solution and rinse the membrane in 0.1x SSC at RT briefly.</li>
<li>Expose the film at -80&deg;C for 6 hours and develop the film. A typical result is shown in Figure 1.</li>
</ol>
3. rAAV3 Vector-mediated Transduction and Transgene Expression in Human Liver Cancer Cells
<ol>
<li>Seed 1x104 cells (either Huh7 or Hep293TT) in each well of 96-well plate. Incubate in a humidified CO2 incubator for at least 18 hours.</li>
<li>Remove medium from the 96-well plate.</li>
<li>Wash cells twice with 50 &mu;L of medium without FBS and antibiotics and remove.</li>
<li>Add 50 &mu;L of medium without FBS and antibiotics and rAAV vectors at 5,000 vgs/cell.</li>
<li>Incubate the plates in the CO2 incubator for 4 hours.</li>
<li>Discard the medium. Wash cells twice with 50 &mu;L of complete medium and remove.</li>
<li>Add 150 &mu;L of C-DMEM to each well and incubate the plate in CO2 incubator for 72 hours, and visualize the EGFP expression using a fluorescence microscope (DMI 4000B; Leica Microsystems).</li>
<li>Images from three wells of virus-infected cells are analyzed quantitatively by ImageJ analysis software (NIH, Bethesda, MD, USA).</li>
<li>Transgene expression is assessed as total area of green fluorescence (pixel2) per visual field. A typical result is shown in Figure 2.</li>
<li>Transgene expression can also be determined by Flow Cytometry.</li>
</ol>
4. Representative Results:
Following the protocol outlined above, one can generate AAV serotype vectors efficiently. The typical yield is ~500 &mu;L containing ~1011 vgs/ &mu;L of purified vector stock. The purity of the vector stock is determined by comparing hybridization signals on quantitative DNA slot blots, with and without Benzonase digestion. Purified rAAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines efficiently.
<img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Figure 1" /> 
Figure 1.	Quantitative DNA slot-blot analysis for determining the titers of rAAV3 vectors. Two-fold dilutions of purified viral stocks, digested with Benzonase (top row) were analyzed on quantitative DNA slot blots with 32P-labeled EGFP-specific DNA probe. The titer of AAV3 vector was determined by comparison with 1 ng (middle row) or 10 ng (bottom row) of AAV-EGFP plasmid standards loaded on the membrane. The numbers correspond to DNA copies.
<img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Figure 2" /> 
Figure 2. 	Recombinant AAV vector-mediated transgene expression in human liver cancer cells. Hep293TT cells, a recently established human hepatoblastoma cell line4, were either mock-infected (left panel), or transduced with 5,000 vgs/cell of either scAAV2-EGFP or scAAV3-EGFP vectors at 37&deg;C for 2 hours. Transgene expression was visualized 72 hours post-transduction using a fluorescence microscope.

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No conflicts of interest declared.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus (AAV) have been developed, and are currently in use in a number of gene therapy clinical trials5. Previously, of the 10 most commonly used AAV serotypes, the transduction efficiency of AAV3 vectors in general has been reported to be particularly low, both in vitro and in vivo1. However, our recent observation that AAV3 vectors transduce human liver cancer cell lines exceedingly well, as determined quantitatively by flow cytometry2, and that AAV3 utilizes the human hepatocyte growth factor receptor (hHGFR) as a cellular co-receptor for binding and entry in these cells3, strongly suggest a selective tissue-tropism of AAV3 for human liver cells in general, and human liver cancer cells in particular. However, since AAV3 vectors also transduce normal human hepatocytes efficiently2, their potential use in cancer gene therapy application in vivo would be negatively impacted. One possible strategy to circumvent this potential problem involves transcriptional-targeting of cancer cells by identifying a gene product that is selectively produced by the tumor, but not by normal hepatocytes. For example, previous studies have shown that serum levels of &alpha;-fetoprotein (AFP) are used as a specific marker for tracking the presence, progression, and/or reoccurrence of certain types of liver cancers, since normal hepatocytes generally produce very small amounts of this protein. Indeed, we have used AAV3 vectors containing the AFP promoter to target transgene expression in liver cancer cells, but not in normal hepatocytes2, and studies are currently underway to test the efficacy of this approach in a murine xenograft model of liver cancer. In our additional studies, we have observed that the transduction efficiency of various AAV serotype vectors can be significantly augmented by site-directed mutagenesis of surface-exposed tyrosine residues in the viral capsids6-14. Since 6 of 7 surface-exposed tyrosines are also conserved in AAV3, we have performed site-directed mutagenesis of these residues and observed that the transduction efficiency of tyrosine-mutant AAV3 vectors is significantly enhanced in human liver cancer cells (unpublished data). Studies are also currently underway to evaluate the safety and efficacy of the tyrosine-mutant AAV3 vectors in mouse xenograft models for human hepatoblastoma and hepatocellular carcinoma, and if successful, the optimal tyrosine-mutant AAV3 serotype vectors may prove to be useful for targeting human liver cancers for the potential gene therapy.

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

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 number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

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 this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

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

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Medicine

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

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

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

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

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.

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

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

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

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

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;

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.

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.

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.

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.

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.

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

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

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

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

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