This work was supported by the Slovenian Research Agency (project J2-9770, infrastructural center IP-0510 and program P2-0249). This video represents supplementary material for the "Electroporation-based Technologies and Treatments" scientific workshop and postgraduate course, organized by the Faculty of Electrical Engineering at the University of Ljubljana, Slovenia. Authors thank also Du&scaron;a Hod&#x17E;i&#x10D; for kindly providing plasmid DNA.

1. Cell culture, plasmid and buffer preparation for the experiment
<ol>
 <li>In this experiment Chinese hamster ovary cells (CHO-K1) are used. Cells are grown in a nutrient mixture HAM-F12 (PAA) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 400 &micro;l/l gentamicin (all from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany), and 1 ml/l crystacilin (Pliva, Zagreb, Croatia). Cells are kept at 37&deg;C in a humidified 5% CO2 atmosphere in the incubator for 24h.</li>
 <li>Amplify plasmid pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) encoding green fluorescent protein (GFP) in DH5&alpha; strain of Escherichia coli and isolate it with HiSpeed Plasmid Maxi Kit (Qiagen, Hilden, Germany). Plasmid DNA concentration (plasmid dissolved in TE buffer) should be spectrophotometrically determined at 260 nm and confirmed by gel electrophoresis.</li>
 <li>Prepare isoosmolar sodium phosphate buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 1 mM MgCl2, 250 mM sucrose, pH 7.4).</li>
 <li>On the day of experiment prepare cell suspension by trypsinization with 0.25% trypsin/EDTA solution (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Centrifuge cells for 5 min at 1000 rpm (180 x g) at 4&deg;C (Sigma, Germany) and resuspend cell pellet in isoosmolar sodium phosphate buffer to a cell density of 5 &times; 106 cells/ml.</li>
</ol>

2. Hardware equipment
<ol>
 <li>Cells are exposed to electric field in pipette tip (Figure 1) with integrated electrodes connected to a high-voltage prototype generator. The tip and electrode geometry allows application of relatively homogeneous electric field and the generator allows delivery of electric pulses in different directions. The tip and the generator were developed at Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana 1.</li>
</ol>

<img src="/files/ftp_upload/3309/3309fig1.jpg" alt="Figure 1"/> Figure 1. Vertical and horizontal (a) cross section and photograph (b) of pipette tip with integrated electrodes. In the cross section grey color is used for the plastic housing and black for the electrodes. The pipette tip with integrated electrodes consists of four cylindrical rod electrodes. The electrodes are made of stainless steel; their diameter is 1.4 mm, adjacent electrodes are 1 mm apart, and opposite electrodes are 2 mm apart. The electrodes are glued into the plastic tip in parallel and their applicable length is 30 mm 2.

3. Gene electrotransfer protocol
<ol>
 <li>Add plasmid pEGFP-N1 to a cell suspension in concentration 10 &mu;g/ml.</li>
 <li>Incubate the mixture for 2-3 minutes at room temperature, before applying electric pulses.</li>
 <li>Aspire 100 &micro;l of cell suspension into the pipette tip with integrated electrodes.</li>
 <li>To achieve best gene electrotransfer efficacy and maintain cell viability, optimal parameters of electric pulses should be used. In this experiment a train of 8 rectangular pulses (each with duration of 1 ms, amplitude 225 V at 1 Hz repetition frequency) is applied to each sample, using high-voltage prototype generator. Two different electric field protocols (Figure 2) are used: in the first protocol all pulses are delivered in the same direction, whereas in the second protocol pulses are delivered by changing alternatively the electric field direction and orientation. The second protocol can only be used with appropriate pulse generator, which allows application of electric pulses in different directions.</li>
 <li>Immediately after the pulse application transfer the cells from pipette tip into 6 well plate and add fetal calf serum (FCS-Sigma, USA) (25% of sample volume).</li>
 <li>Incubate cells for 5 min at 37&deg;C to allow cell membrane resealing.</li>
 <li>Add 2 ml of HAM-F12 to each sample in 6 well and incubate cells for 24h at 37&deg;C in a humidified 5% CO2 atmosphere in the incubator.</li>
</ol>

<img src="/files/ftp_upload/3309/3309fig2.jpg" alt="Figure 2"/> Figure 2.Electric field protocols: (a) all pulses are delivered in the same direction, (b) pulses are delivered by changing alternatively the electric field direction and orientation.

4. Image acquisition and determination of gene electrotransfer efficacy
<ol>
 <li>Efficacy of gene electrotransfer is determined as the percentage of cells expressing GFP 24h after the pulse application.</li>
 <li>The cells are observed using a fluorescence microscope (in our case Zeiss 200, Axiovert, ZR Germany) with excitation light at 488 nm generated with a monochromator system (PolyChrome IV, Visitron, Germany) and emission is detected at 507 nm. The images are recorded using imaging system (MetaMorph imaging system, Visitron, Germany), but other similar acquisition software can also be used.</li>
 <li>Acquire at least five images (phase contrast and green fluorescence) at 20x objective magnification.</li>
 <li>Count cells in phase contrast image and cells that are expressing GFP in green fluorescence image. Determine the percentage of gene electrotransfer efficacy by dividing the number of cells that are expressing GFP with the number of all cells in each corresponding image (Figure 3).</li>
</ol>

5. Representative results:
<img src="/files/ftp_upload/3309/3309fig3.jpg" alt="Figure 3"/> Figure 3.The percentage of cells expressing GFP when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation is presented. Cells were exposed to a train of eight pulses with amplitude 225 V, duration 1 ms and repetition frequency of 1 Hz. Results were obtained by means of fluorescence microscopy. Each value in the graph represent mean of three independent experiments &plusmn; standard deviation. By changing the electric field direction and orientation the percentage of cells expressing GFP increases.

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	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. 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No conflicts of interest declared.

Gene electrotransfer is a versatile biotechnology technique that enables transfer of DNA into cells by means of applying short, high voltage electric pulses 3 and represents a safer alternative to viral vectors due to its safety, efficacy and ease of application. Although today gene electrotransfer is widely used to transfect all types of cells and first phase I clinical trial using this method has been reported 4, the underlying mechanisms are still not completely understood. It is known, that appl...

<table border="1"><tbody><tr><td>Name of the reagent</td>
		<td>Company</td>
		<td>Catalogue number</td>
		<td>Comments</td></tr>
	<tr><td>HAM-F12</td>
		<td>PAA</td>
		<td>E15-016</td>
		<td>culture medium</td></tr>
	<tr><td>L-glutamine</td>
		<td>Sigma-Aldrich</td>
		<td>G7513</td>
		<td>&nbsp;</td></tr>
	<tr><td>fetal bovine serum</td>
		<td>PAA</td>
		<td>A15-151</td>
		<td>&nbsp;</td></tr>
	<tr><td>gentamicin</td>
		<td>Sigma-Aldrich</td>
		<td>G1397</td>
		<td>antibiotic</td></tr>
	<tr><td>crystacilin</td>
		<td>Pliva</td>
		<td>625110</td>
		<td>antibiotic</td></tr>
	<tr><td>pEGFP-N1</td>
		<td>Clontech Laboratories</td>
		<td>6085-1</td>
		<td>plasmid DNA</td></tr>
	<tr><td>HiSpeed Plasmid Maxi Kit</td>
		<td>Qiagen</td>
		<td>12662</td>
		<td>&nbsp;</td></tr>
	<tr><td>Na2HPO4</td>
		<td>Merck</td>
		<td>F640786 933</td>
		<td>&nbsp;</td></tr>
	<tr><td>NaH2PO4</td>
		<td>TKI Hrastnik</td>
		<td>0795</td>
		<td>&nbsp;</td></tr>
	<tr><td>MgCl2</td>
		<td>Sigma-Aldrich</td>
		<td>M-8266</td>
		<td>&nbsp;</td></tr>
	<tr><td>sucrose</td>
		<td>Sigma-Aldrich</td>
		<td>16104</td>
		<td>&nbsp;</td></tr>
	<tr><td>trypsin/EDTA solution</td>
		<td>Sigma-Aldrich</td>
		<td>T4174</td>
		<td>&nbsp;</td></tr>
	<tr><td>pipette tip</td>
		<td>Custom made</td>
		<td>&nbsp;</td>
		<td>&nbsp;</td></tr>
	<tr><td>electric pulse generator</td>
		<td>Custom made</td>
		<td>&nbsp;</td>
		<td>&nbsp;</td></tr>
	<tr><td>6 well plate</td>
		<td>TPP</td>
		<td>92406</td>
		<td>&nbsp;</td></tr>
	<tr><td>15 ml centrifuge tube</td>
		<td>TPP</td>
		<td>91015</td>
		<td>&nbsp;</td></tr>
	<tr><td>75 cm2 culture flask</td>
		<td>TPP</td>
		<td>90076</td>
		<td>&nbsp;</td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

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10.4K 조회수.  University of Ljubljana. 전기장의 방향이 펄스 응용 프로그램 중에 변경되면 electroporation에 의해 유전자 transfection이 약 두 배 향상이며, 동시에 세포 생존 능력은 영향을받지 않습니다. 유전자 transfection의 증가는 세포로 DNA의 항목에 대해 관할 만들어 막 면적의 증가로 인해 발생할 수 있습니다.

비디오: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...