Diese Arbeit wurde von der slowenischen Research Agency (Projekt J2-9770, infrastrukturelle Zentrum IP-0510 und das Programm P2-0249) unterstützt. Dieses Video stellt ergänzendes Material für die &quot;Elektroporation basierenden Technologien und Behandlungsmethoden&quot; wissenschaftlichen Workshop und Aufbaustudium, von der Fakultät für Elektrotechnik an der University of Ljubljana, Slowenien organisiert. Autoren danken auch Duša Hodzic für die freundliche Bereitstellung Plasmid-DNA.

1. Zellkultur, Plasmid-und Puffer-Vorbereitung für das Experiment <ol><li> In diesem Experiment chinesischen Hamsters (CHO-K1) eingesetzt werden. Die Zellen werden in ein Nährstoff Mischung HAM-F12 (PAA) mit 2 mM L-Glutamin, 10% fötalem Rinderserum, 400 ul / l Gentamycin (alle von Sigma-Aldrich Chemie GmbH, Deisenhofen, Deutschland), und 1 ergänzt gewachsen ml / l crystacilin (Pliva, Zagreb, Kroatien). Die Zellen werden bei 37 ° C in einer befeuchteten 5% CO 2-Atmosphäre in den Inkubator für 24 Stunden gehalten. </li><li> Amplify Plasmid pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) Kodierung grün fluoreszierende Protein (GFP) in DH5a Stamm von Escherichia coli und isolieren mit HiSpeed ​​Plasmid Maxi Kit (Qiagen, Hilden, Deutschland). Plasmid-DNA-Konzentration (in TE-Puffer aufgelöst Plasmid) sollte spektrophotometrisch bei 260 nm bestimmt und bestätigt werden durch Gelelektrophorese. </li><li> Bereiten isoosmolaren Natriumphosphat-Puffer (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 1 mM MgCl 2, 250 mM Saccharose, pH 7,4). </li><li> Am Tag der Vorbereitung des Experiments Zellsuspension durch Trypsinierung mit 0,25% Trypsin / EDTA-Lösung (Sigma-Aldrich Chemie GmbH, Deisenhofen, Deutschland). Centrifuge Zellen für 5 min bei 1000 rpm (180 xg) bei 4 ° C (Sigma, Deutschland) und resuspendieren Zellpellet in isoosmolaren Natriumphosphatpuffer auf eine Zelldichte von 5 × 10 6 Zellen / ml. </li></ol> 2. Hardware-Ausstattung <ol><li> Zellen sind elektrische Feld in Pipettenspitze (Abbildung 1) mit integrierten Elektroden verbunden mit einem Hochspannungs-Prototyp-Generator ausgesetzt. Die Spitze und Elektrode Geometrie ermöglicht den Einsatz von relativ homogenen elektrischen Feld und der Generator ermöglicht Lieferung von elektrischen Impulsen in verschiedene Richtungen. Die Spitze und der Generator wurden im Labor von Biokybernetik, Fakultät für Elektrotechnik, Universität Ljubljana 1 entwickelt. </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Vertikale und horizontale (a) Querschnitt und Foto (b) der Pipettenspitze mit integrierten Elektroden. In den Querschnitt graue Farbe wird für die Kunststoff-Gehäuse und schwarz für die Elektroden verwendet. Die Pipettenspitze mit integrierten Elektroden besteht aus vier zylinderförmigen Stab-Elektroden. Die Elektroden sind aus Edelstahl gefertigt, deren Durchmesser 1,4 mm, benachbarten Elektroden sind 1 mm auseinander und gegenüberliegenden Elektroden sind 2 mm voneinander entfernt. Die Elektroden werden in die Kunststoff-Spitze parallel verleimt und gegebenenfalls deren Länge beträgt 30 mm 2. 3. Gene Elektrotransfer Protokoll <ol><li> Add-Plasmid pEGFP-N1 eine Zellsuspension in einer Konzentration 10 pg / ml. </li><li> Inkubieren Sie die Mischung für 2-3 Minuten bei Raumtemperatur, bevor elektrische Impulse. </li><li> Aspire 100 ul Zellsuspension in die Pipettenspitze mit integrierten Elektroden. </li><li> Um beste Gen Elektrotransfer Wirksamkeit erzielen und beizubehalten Lebensfähigkeit der Zellen, sollten die optimalen Parameter der elektrischen Impulse genutzt werden. In diesem Experiment ein Zug von 8 Rechteckimpulse (jeweils mit einer Dauer von 1 ms, Amplitude 225 V bei 1 Hz Wiederholfrequenz) ist zu jeder Probe aufgetragen, mit Hochspannungs-Prototyp-Generator. Zwei verschiedene elektrische Feld Protokolle (Abbildung 2) verwendet werden: in das erste Protokoll alle Impulse werden in die gleiche Richtung geliefert, während in dem zweiten Protokoll Impulse, indem alternativ das elektrische Feld Richtung und Orientierung geliefert werden. Das zweite Protokoll kann nur mit entsprechendem Impulsgeber, die Anwendung der elektrischen Impulse in verschiedene Richtungen ermöglicht werden. </li><li> Direkt nach dem Puls-Anwendung übertragen die Zellen aus Pipettenspitze in 6-Well-Platte und stell fötalem Kälberserum (FCS-Sigma, USA) (25% des Probenvolumens). </li><li> Inkubieren Zellen für 5 min bei 37 ° C, damit Zellmembran Wiederverschließen. </li><li> 2 ml HAM-F12 zu jeder Probe in 6 gut und inkubieren Zellen für 24 Stunden bei 37 ° C in einer befeuchteten 5% CO 2-Atmosphäre in den Inkubator. </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="Abbildung 2"/> . Abbildung 2 Elektrisches Feld Protokolle: (a) alle Impulse werden in die gleiche Richtung geliefert, (b) Impulse, indem alternativ das elektrische Feld Richtung und Orientierung geliefert. 4. Bildaufnahme und Bestimmung von Gen Elektrotransfer Wirksamkeit <ol><li> Die Wirksamkeit von Gen Elektrotransfer ist als der Prozentsatz von Zellen, die GFP 24h nach dem Puls Anwendung bestimmt. </li><li> Die Zellen werden beobachtet mit einem Fluoreszenzmikroskop (in unserem Fall Zeiss 200, Axiovert, ZR Deutschland) mit Anregungslicht bei 488 nm mit einem Monochromator-System (Polychrome IV, Visitron, Deutschland) und Emission bei 507 nm detektiert generiert. Die Bilder werden aufgezeichnet mit bildgebenden Systems (MetaMorph Imaging-System, Visitrauf, Deutschland), aber auch andere ähnliche Software zur Erfassung kann ebenfalls verwendet werden. </li><li> Erwerben Sie mindestens fünf Bilder (Phasenkontrast und grüne Fluoreszenz) bei 20x Objektiv-Vergrößerung. </li><li> Zählen von Zellen in der Phase kontrastreiches Bild und Zellen, die GFP sind in grüne Fluoreszenz Bild. Bestimmen Sie den Prozentsatz der Gen Elektrotransfer Wirksamkeit, indem die Anzahl von Zellen, die GFP mit der Anzahl aller Zellen in jeder entsprechende Bild (Abbildung 3). </li></ol> 5. Repräsentative Ergebnisse: <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Der Prozentsatz an Zellen, die GFP, wenn alle Impulse in die gleiche Richtung geliefert werden und Impulse werden von wechselnden alternativ das elektrische Feld Richtung und Orientierung vorgelegt wird geliefert. Die Zellen wurden zu einem Zug von acht Impulsen mit einer Amplitude von 225 V, Dauer 1 ms und Wiederholung Frequenz von 1 Hz ausgesetzt. Die Ergebnisse wurden mittels Fluoreszenz-Mikroskopie erhalten. Jeder Wert in der Grafik stellen Mittelwerte von drei unabhängigen Experimenten ± Standardabweichung. Durch die Veränderung des elektrischen Feldes Richtung und Orientierung der Anteil der Zellen, die GFP erhöht.

<ol>
	<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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Effects of DNA topology on cell uptake and transfection efficiency.</a> Biophys J. 63, 1026-1031 (1992).</li><li>Rols, M., Delteil, C., Serin, G., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+electrotransfection+of+mammalian+cells.">Temperature effects on electrotransfection of mammalian cells.</a> Nucleic Acids Res. 22, 540-540 (1994).</li><li>Golzio, M., Teissie, J., Rols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization+effect+on+mammalian+cell+permeabilization+and+gene+delivery+by+electric+field.">Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.</a> Biochim Biophys Acta. 1563, 23-28 (2002).</li><li>Haberl, S., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mg+ions+on+efficiency+of+gene+electrotransfer+and+on+cell+electropermeabilization.">Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization.</a> Bioelectrochemistry. 79, 265-271 (2010).</li><li>Rols, M., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+mammalian+cells+to+macromolecules:+control+by+pulse+duration.">Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.</a> Biophys J. 75, 1415-1423 (1998).</li><li>Kanduser, M., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+gene+electrotransfer+using+high-and+low-voltage+pulses-An+in+vitro+study.">Mechanisms involved in gene electrotransfer using high-and low-voltage pulses-An in vitro study.</a> Bioelectrochemistry. 74, 265-271 (2009).</li><li>Pavlin, M., Flisar, K., Kanduser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrophoresis+in+gene+electrotransfer.">The role of electrophoresis in gene electrotransfer.</a> J Membr Biol. 236, 75-79 (2010).</li><li>Sersa, G., Cemazar, M., Semrov, D., 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=Changing+electrode+orientation+improves+the+efficacy+of+electrochemotherapy+of+solid+tumors+in+mice.">Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice.</a> Bioelectrochem Bioenerg. 39, 61-66 (1996).</li><li>Faurie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-mediated+gene+transfer+and+expression+are+controlled+by+the+life-time+of+DNA/membrane+complex+formation.">Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation.</a> J Gene Med. 12, 117-125 (2010).</li></ol>

Keine Interessenskonflikte erklärt.

Gene Elektrotransfer ist eine vielseitige Technik, Biotechnologie Transfer von DNA in Zellen ermöglicht durch Anwendung kurz-, Hochspannungs-elektrische Impulse 3 und stellt eine sichere Alternative zu viralen Vektoren aufgrund ihrer Sicherheit, Wirksamkeit und einfache Anwendung. Obwohl heute Gen Elektrotransfer weit verbreitet ist, um alle Arten von Zellen und erste klinische Phase I Studie mit dieser Methode transfizieren wurde 4 berichtet, sind die zugrunde liegenden Mechanismen noch nicht vol...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> HAM-F12 </td><td> PAA </td><td> E15-016 </td><td> Nährboden </td></tr><tr><td> L-Glutamin </td><td> Sigma-Aldrich </td><td> G7513 </td><td></td></tr><tr><td> Rinderfötenserum </td><td> PAA </td><td> A15-151 </td><td></td></tr><tr><td> Gentamicin </td><td> Sigma-Aldrich </td><td> G1397 </td><td> Antibiotikum </td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td> Antibiotikum </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></td></tr><tr><td> Na 2 HPO 4 </td><td> Merck </td><td> F640786 933 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> TKI Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl 2 </td><td> Sigma-Aldrich </td><td> M-8266 </td><td></td></tr><tr><td> Saccharose </td><td> Sigma-Aldrich </td><td> 16104 </td><td></td></tr><tr><td> Trypsin / EDTA-Lösung </td><td> Sigma-Aldrich </td><td> T4174 </td><td></td></tr><tr><td> Pipettenspitze </td><td> Custom made </td><td></td><td></td></tr><tr><td> elektrischen Impulsgeber </td><td> Custom made </td><td></td><td></td></tr><tr><td> 6-Well Platte </td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> 15 ml Zentrifugenröhrchen </td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75 cm 2 Kulturflasche </td><td> TPP </td><td> 90076 </td><td></td></tr></tbody></table>

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

Gene Elektrotransfer ist eine physikalische Methode verwendet, um Gene in die Zellen liefern durch die Anwendung von kurzen und intensiven elektrischen Impulsen, die Destabilisierung der Zellmembran führen, wodurch es durchlässig für kleine Moleküle und ermöglicht die Übertragung von großen Molekülen wie DNA. Es stellt eine Alternative zu viralen Vektoren, wegen seiner Sicherheit, Wirksamkeit und einfache Anwendung. Für Gen Elektrotransfer verschiedenen elektrischen Impuls Protokolle verwendet werden, um eine maximale Gentransfektion zu erreichen, einer von ihnen verändert sich das elektrische Feld Richtung und Orientierung während der Impulsabgabe. Ändern elektrische Feld Richtung und Orientierung Erhöhung der Membranfläche zuständig für DNA Eintritt in die Zelle. In diesem Video zeigen wir den Unterschied in der Gen-Elektrotransfer Wirksamkeit, wenn alle Impulse in die gleiche Richtung geliefert werden und Impulse werden von wechselnden alternativ das elektrische Feld Richtung und Orientierung geliefert. Zu diesem Zweck Spitze mit integrierten Elektroden und Hochspannungs-Prototyp-Generator, der Wechsel des elektrischen Feldes in verschiedene Richtungen während der elektrische Impuls-Anwendung ermöglicht, verwendet wurden. Gene Elektrotransfer Wirksamkeit bestimmt 24 Stunden nach dem Puls-Anwendung als die Anzahl der Zellen, die grün fluoreszierendes Protein mit der Anzahl aller Zellen aufgeteilt. Die Ergebnisse zeigen, dass Gen-Transfektion erhöht wird, wenn das elektrische Feld Orientierung während der elektrische Impuls Lieferung geändert wird.

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 Transfektion durch Elektroporation etwa zwei Mal wird verbessert, wenn Ausrichtung des elektrischen Feldes während der Puls-Anwendung geändert wird, während die Lebensfähigkeit der Zellen nicht beeinträchtigt wird. Der Anstieg in Gentransfektion wird durch die Erhöhung der Membranfläche, die kompetent gemacht für die DNA-Eintritt in die Zelle verursacht wird.

Ändern der Ausrichtung und Orientierung des elektrischen Feldes während elektrische Impulse Anwendung Verbessert Plasmid Gene Transfer In-vitro-</em

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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Research

JoVE Journal

Medicine

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

10.4K Aufrufe.  University of Ljubljana. Gene Transfektion durch Elektroporation etwa zwei Mal wird verbessert, wenn Ausrichtung des elektrischen Feldes während der Puls-Anwendung geändert wird, während die Lebensfähigkeit der Zellen nicht beeinträchtigt wird. Der Anstieg in Gentransfektion wird durch die Erhöhung der Membranfläche, die kompetent gemacht für die DNA-Eintritt in die Zelle verursacht wird.

Serie: 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.

Aufbau und Verbreitung der Menschenrechte Retinoblastoma Tumore in immundefizienten Mäuse

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

Forschung

Medizin

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.

Gentransfer bei ischämischen Herzinsuffizienz in einem vorklinischen Modell

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.

Die Anwendung des dauerhaften mittleren Hirnarterie Ligation in der Maus

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.

Produktion und Nachweis von reaktiven Sauerstoffspezies (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 ...

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.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

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

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.

Gefäß Gentransfer von metallischen Stent Oberflächen mit Adenovirus-Vektoren durch Tethered Hydrolysierbare Vernetzer

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.

A Novel<em&gt; In vivo</em&gt; Gentransfer-Technik und<em&gt; In-vitro-</em&gt; Zellbasierte Assays für das Studium der Knochenverlust in Skeletterkrankungen

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

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depletion von Maus-Zellen aus menschlichem Tumorxenografte deutlich verbessert Downstream-Analyse von Zielzellen

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Mikroskopie-basierten Methoden für die Beurteilung der Epithelzelle Migration bei der In-vitro- Wundheilung

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

In Vivo Gentransfer in die Kaninchen gemeinsame Halsschlagader Endothel

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Ändern der Ausrichtung und Orientierung des elektrischen Feldes während elektrische Impulse Anwendung Verbessert Plasmid Gene Transfer In-vitro-

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