Questo lavoro è stato sostenuto dall&#39;Agenzia di ricerca sloveno (progetto J2-9770, IP-0510 centro infrastrutturali e programma P2-0249). Questo video rappresenta materiale supplementare per il &quot;Elettroporazione basato Tecnologie e Trattamenti&quot; laboratorio scientifico e corso post-laurea, organizzato dalla Facoltà di Ingegneria Elettrica presso l&#39;Università di Lubiana, Slovenia. Autori ringraziano anche Dusa Hodzic per la gentile fornitura del DNA plasmidico.

1. Coltura cellulare, il plasmide e la preparazione del buffer per l&#39;esperimento <ol><li> In questo esperimento le cellule ovariche di criceto cinese (CHO-K1) sono utilizzati. Le cellule sono coltivate in una miscela di nutrienti HAM-F12 (PAA) integrato con 2 mM di L-glutamina, 10% di siero fetale bovino, 400 microlitri / l gentamicina (tutti da Sigma-Aldrich Chemie GmbH, Deisenhofen, Germania), e 1 ml / l crystacilin (Pliva, Zagabria, Croazia). Le cellule sono conservate a 37 ° C in un umidificata al 5% CO 2 nell&#39;atmosfera nell&#39;incubatrice per 24 ore. </li><li> Amplificare plasmide pEGFP-N1 (Clontech Laboratories Inc., Mountain View, CA, USA) che codifica per la proteina fluorescente verde (GFP) in DH5α ceppo di Escherichia coli e isolare con HiSpeed ​​plasmidi Maxi Kit (Qiagen, Hilden, Germania). Concentrazione di DNA plasmide (plasmide disciolti in tampone TE) dovrebbe essere determinata spettrofotometricamente a 260 nm e confermato mediante elettroforesi su gel. </li><li> Preparare isoosmolar tampone fosfato di sodio (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 1 mM MgCl2, 250 mM saccarosio, pH 7,4). </li><li> Il giorno di esperimento preparare sospensione cellulare da tripsinizzazione con il 0,25% tripsina / EDTA (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germania). Centrifugare le cellule per 5 min a 1000 rpm (180 xg) a 4 ° C (Sigma, Germania) e risospendere pellet di cellule in tampone isoosmolar fosfato di sodio per una densità cellulare di 5 × 10 6 cellule / ml. </li></ol> 2. Dotazione hardware <ol><li> Le cellule sono esposte a campo elettrico in puntale (Figura 1) con elettrodi integrata collegata ad un generatore ad alta tensione prototipo. La punta e la geometria di elettrodi permette l&#39;applicazione della relativa omogeneità di campo elettrico e il generatore permette la consegna di impulsi elettrici in direzioni diverse. La punta e il generatore sono stati sviluppati presso il Laboratorio di Biocybernetics, Facoltà di Ingegneria Elettrica, Università di Lubiana 1. </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="Figura 1"/> Figura 1. Verticale e orizzontale (a), sezione fotografia e (b), della punta della pipetta con elettrodi integrati. Nella sezione colore grigio è utilizzato per la custodia in plastica e nero per gli elettrodi. La punta della pipetta con elettrodi integrato è costituito da quattro elettrodi barra cilindrica. Gli elettrodi sono in acciaio inox, il loro diametro è di 1,4 mm, elettrodi adiacenti sono 1 mm l&#39;una dall&#39;altra, e gli elettrodi opposti sono 2 mm. Gli elettrodi sono incollate in punta di plastica in parallelo e la loro lunghezza applicabile è di 30 mm 2. 3. Gene electrotransfer protocollo <ol><li> Aggiungi plasmide pEGFP-N1 ad una sospensione di cellule in concentrazione di 10 mcg / ml. </li><li> Incubare la miscela per 2-3 minuti a temperatura ambiente, prima di applicare gli impulsi elettrici. </li><li> Aspire 100 l di sospensione cellulare in la punta della pipetta con elettrodi integrati. </li><li> Per ottenere il miglior efficacia electrotransfer genica e mantenere la vitalità cellulare, i parametri ottimali di impulsi elettrici devono essere utilizzati. In questo esperimento un treno di 8 impulsi rettangolari (ciascuno con durata di 1 ms, ampiezza 225 V a 1 Hz, frequenza di ripetizione) è applicato a ciascun campione, utilizzando ad alta tensione del generatore prototipo. Due differenti protocolli di campo elettrico (Figura 2) sono utilizzati: in primo protocollo tutti gli impulsi sono consegnati nella stessa direzione, mentre nel secondo protocollo impulsi vengono consegnati cambiando alternativamente la direzione del campo elettrico e l&#39;orientamento. Il secondo protocollo può essere utilizzato solo con generatore di impulsi appropriati, che consente l&#39;applicazione di impulsi elettrici in direzioni diverse. </li><li> Immediatamente dopo l&#39;applicazione di impulsi trasferimento alle cellule di puntale in 6 pozzetti e aggiungere siero fetale bovino (FCS-Sigma, USA) (25% del volume del campione). </li><li> Incubare le cellule per 5 minuti a 37 ° C per consentire una nuova chiusura della membrana cellulare. </li><li> Aggiungere 2 ml di HAM-F12 per ogni campione in 6 bene e incubare le cellule per 24 ore a 37 ° C in un umidificata al 5% CO 2 nell&#39;atmosfera nell&#39;incubatrice. </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="Figura 2"/> . Figura 2 protocolli di campo elettrico: (a) tutti gli impulsi sono consegnati nella stessa direzione, (b) gli impulsi vengono consegnati alternativamente cambiando la direzione del campo elettrico e l&#39;orientamento. 4. Acquisizione di immagini e la determinazione di efficacia gene electrotransfer <ol><li> L&#39;efficacia del gene electrotransfer è determinato come percentuale di cellule che esprimono GFP 24 ore dopo l&#39;applicazione di impulsi. </li><li> Le cellule sono osservati utilizzando un microscopio a fluorescenza (nel nostro caso Zeiss 200, Axiovert, ZR Germania) con luce di eccitazione a 488 nm generata con un sistema monocromatore (policromia IV, Visitron, Germania) e di emissione è rilevata a 507 nm. Le immagini vengono registrate con sistema di imaging (MetaMorph sistema di imaging, Visitrsu, Germania), ma altri software di acquisizione simili possono anche essere utilizzati. </li><li> Acquisire almeno cinque immagini (contrasto di fase e fluorescenza verde) a 20x obiettivo. </li><li> Contare le celle in immagini a contrasto di fase e le cellule che esprimono GFP in verde immagine fluorescenza. Determinare la percentuale di efficacia gene electrotransfer dividendo il numero di cellule che esprimono GFP con il numero di tutte le cellule in ogni immagine corrispondente (Figura 3). </li></ol> 5. Rappresentante dei risultati: <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="Figura 3"/> Figura 3. La percentuale di cellule che esprimono GFP, quando tutti gli impulsi sono consegnati nella stessa direzione e quando gli impulsi vengono consegnati alternativamente cambiando la direzione del campo elettrico e l&#39;orientamento è presentato. Cellule sono state esposte ad un treno di otto impulsi di ampiezza 225 V, durata 1 ms e frequenza di ripetizione di 1 Hz. Risultati sono stati ottenuti mediante microscopia a fluorescenza. Ogni valore nel grafico rappresentano media di tre esperimenti indipendenti ± deviazione standard. Cambiando la direzione del campo elettrico e l&#39;orientamento della percentuale di cellule che esprimono GFP aumenta.

<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. IV. 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>

Nessun conflitto di interessi dichiarati.

Gene electrotransfer è una tecnica biotecnologica versatile che consente il trasferimento del DNA nelle cellule mediante l&#39;applicazione di brevi impulsi elettrici ad alta tensione 3 e rappresenta una alternativa più sicura di vettori virali per la sua sicurezza, efficacia e facilità di applicazione. Anche se gene electrotransfer oggi è ampiamente utilizzata per trasfettare tutti i tipi di cellule e prima fase io studio clinico con questo metodo è stato segnalato 4, i meccanismi alla base n...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> HAM-F12 </td><td> PAA </td><td> E15-016 </td><td> terreno di coltura </td></tr><tr><td> L-glutammina </td><td> Sigma-Aldrich </td><td> G7513 </td><td></td></tr><tr><td> siero fetale bovino </td><td> PAA </td><td> A15-151 </td><td></td></tr><tr><td> gentamicina </td><td> Sigma-Aldrich </td><td> G1397 </td><td> antibiotico </td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td> antibiotico </td></tr><tr><td> pEGFP-N1 </td><td> Clontech Laboratori </td><td> 6085-1 </td><td> DNA plasmidico </td></tr><tr><td> HiSpeed ​​Kit Maxi plasmidi </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> saccarosio </td><td> Sigma-Aldrich </td><td> 16104 </td><td></td></tr><tr><td> tripsina / EDTA soluzione </td><td> Sigma-Aldrich </td><td> T4174 </td><td></td></tr><tr><td> puntale </td><td> Una soluzione su misura </td><td></td><td></td></tr><tr><td> generatore di impulsi elettrici </td><td> Una soluzione su misura </td><td></td><td></td></tr><tr><td> 6 pozzetti </td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> Centrifuga da 15 ml tubo </td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75 centimetri 2 cultura fiasco </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 electrotransfer è un metodo fisico usati per fornire i geni nelle cellule mediante l&#39;applicazione di impulsi elettrici brevi e intensi, che causano destabilizzazione della membrana cellulare, rendendo permeabili a molecole piccole e permette il trasferimento di grandi molecole come il DNA. Rappresenta un&#39;alternativa ai vettori virali, grazie alla sua sicurezza, efficacia e facilità di applicazione. Per gene electrotransfer diversi protocolli di impulsi elettrici vengono utilizzati al fine di ottenere il massimo transfezione genica, uno di loro sta cambiando la direzione del campo elettrico e l&#39;orientamento durante la consegna degli impulsi. Cambio di direzione del campo elettrico e l&#39;orientamento aumentare la superficie della membrana competente per l&#39;ingresso del DNA nella cellula. In questo video, dimostriamo la differenza di efficacia gene electrotransfer quando tutti gli impulsi sono consegnati nella stessa direzione e quando gli impulsi vengono consegnati alternativamente cambiando la direzione del campo elettrico e l&#39;orientamento. A questo scopo punta con elettrodi integrati e ad alta tensione prototipo di generatore, che permette di cambiare di campo elettrico in diverse direzioni durante l&#39;applicazione di impulsi elettrici, sono stati utilizzati. Gene efficacia electrotransfer è determinato 24 ore dopo l&#39;applicazione di impulsi come il numero di cellule che esprimono la proteina fluorescente verde divisa con il numero di tutte le cellule. I risultati mostrano che la transfezione genica è aumentata quando l&#39;orientamento del campo elettrico durante la consegna degli impulsi elettrici è cambiato.

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

Trasfezione genica mediante elettroporazione è migliorato di circa due volte quando l&#39;orientamento del campo elettrico viene modificato durante l&#39;applicazione di impulsi, mentre la vitalità cellulare non è interessato. L&#39;aumento della transfezione genetica è causata dalla crescita dell&#39;area membrana che è competente per l&#39;ingresso del DNA nella cellula.

Cambiare la direzione e l&#39;orientamento del campo elettrico durante l&#39;applicazione impulsi elettrici migliora il trasferimento plasmidi Gene 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 ...

changing-direction-orientation-electric-field-during-electric-pulses

Research

JoVE Journal

Medicine

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

10.4K Views.  University of Ljubljana. Trasfezione genica mediante elettroporazione è migliorato di circa due volte quando l'orientamento del campo elettrico viene modificato durante l'applicazione di impulsi, mentre la vitalità cellulare non è interessato. L'aumento della transfezione genetica è causata dalla crescita dell'area membrana che è competente per l'ingresso del DNA nella cellula.

Video: 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.

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

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

Ricerca

Medicina

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.

Trasferimento Genico per insufficienza cardiaca ischemica in un modello preclinico

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.

L&#39;applicazione di permanente legatura dell&#39;arteria cerebrale media nel 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 ...

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.

Produzione e di rilevamento di specie reattive dell&#39;ossigeno (ROS) in Cancro

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.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di 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.

Trasferimento Gene vascolare da stent metallico superfici utilizzando Adenoviral Vettori Tethered tramite idrolizzabili Cross-linker

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; Tecnica Gene Transfer e<em&gt; In vitro</em&gt; test cellulari per lo studio della perdita ossea nei disturbi muscoloscheletrici

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.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

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.

La microscopia basato su metodi per la valutazione della migrazione delle cellule epiteliali durante In Vitro la guarigione arrotolata

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 Trasferimento del gene per l'endotelio dell'arteria carotica comune di coniglio

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 ...

Cambiare la direzione e l'orientamento del campo elettrico durante l'applicazione impulsi elettrici migliora il trasferimento plasmidi Gene In vitro

Riepilogo

Esplora Altri Video

Capitoli in questo video

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

Trasferimento Genico per insufficienza cardiaca ischemica in un modello preclinico

L'applicazione di permanente legatura dell'arteria cerebrale media nel mouse

Produzione e di rilevamento di specie reattive dell'ossigeno (ROS) in Cancro

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

Trasferimento Gene vascolare da stent metallico superfici utilizzando Adenoviral Vettori Tethered tramite idrolizzabili Cross-linker

A Novel

L'esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

La microscopia basato su metodi per la valutazione della migrazione delle cellule epiteliali durante In Vitro la guarigione arrotolata

In Vivo Trasferimento del gene per l'endotelio dell'arteria carotica comune di coniglio