Name: stable and efficient genetic modification of cells in the adult mouse v-svz for the analysis of neural stem cell autonomous and non-autonomous effects
Uploaded: 2016-02-17T14:00:00
Description: Watch this Scientific Journal Video about Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects at JoVE

Riconosciamo l&#39;aiuto di MJ Palop e il supporto tecnico del SCSIE della Universidad de Valencia. Ringraziamo anche Antonia Follenzi per gli utili commenti e la discussione del manoscritto. IF è supportato dalla Fundación Botín, dal Banco Santander Santander attraverso la sua Università Divisione Global, e da sovvenzioni dal Generalitat Valenciana (Programa Prometeo, ACOMP, e ISIC) e Ministerio de Economia y Competitividad (MINECO: SAF2011-23331, CIBERNED e RETIC tercel) . Questo lavoro è stato supportato anche da BFU2010-21823 e RETIC tercel sovvenzioni dal MINECO e il Consiglio europeo della ricerca (CER) 2012-StG (260511- PD-HUMMODEL) per AC BM-P. è il destinatario di una borsa di studio FPI spagnolo del MINECO.

ETICA DICHIARAZIONE: Questo protocollo segue le linee guida per la cura degli animali dell&#39;Università di Valencia in conformità con la direttiva europea 2010/63 / UE. 1. Generazione di LV per In Vivo marcatura studi (si veda la Figura 1a) ATTENZIONE: La procedura qui descritta è livello di biosicurezza 2, eseguire quindi tutte le seguenti procedure in una cappa di rischio biologico. Assicurare che il personale di ricerca sono adeguatamente qualificato e preparato in tut...

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(39), (2010).</li><li>Ramirez-Castillejo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment+epithelium-derived+factor+is+a+niche+signal+for+neural+stem+cell+renewal.">Pigment epithelium-derived factor is a niche signal for neural stem cell renewal.</a> Nat Neurosci. 9 (3), 331-339 (2006).</li><li>Falcao, A. M., Marques, F., Novais, A., Sousa, N., Palha, J. A., Sousa, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+from+the+choroid+plexus+to+the+subventricular+zone:+go+with+the+flow!.">The path from the choroid plexus to the subventricular zone: go with the flow!.</a> Front Cell Neurosci. 6, (2012).</li><li>Delgado, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NT-3+delivered+by+vasculature+and+CSF+promotes+quiescence+of+subependymal+neural+stem+cells+through+nitric+oxide+induction.">Endothelial NT-3 delivered by vasculature and CSF promotes quiescence of subependymal neural stem cells through nitric oxide induction.</a> Neuron. 83 (3), 572-585 (2014).</li><li>Kokovay, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VCAM1+is+essential+to+maintain+the+structure+of+the+SVZ+niche+and+acts+as+an+environmental+sensor+to+regulate+SVZ+lineage+progression.">VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression.</a> Cell Stem Cell. 11 (2), 220-230 (2012).</li><li>Porlan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MT5-MMP+regulates+adult+neural+stem+cell+functional+quiescence+through+the+cleavage+of+N-cadherin.">MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin.</a> Nat Cell Biol. 16 (7), 629-638 (2014).</li><li>Ihrie, R. A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lake-front+property:+a+unique+germinal+niche+by+the+lateral+ventricles+of+the+adult+brain.">Lake-front property: a unique germinal niche by the lateral ventricles of the adult brain.</a> Neuron. 70 (4), 674-686 (2011).</li><li>Porlan, E., Perez-Villalba, A., Delgado, A. C., Ferr&#242;n, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+neural+stem+cells+in+the+subependymal+zone.">Paracrine regulation of neural stem cells in the subependymal zone.</a> Arch Biochem Biophys. 1-2 (534), 11-19 (2013).</li><li>Mamber, C., Verhaagen, J., Hol, E. 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A., Fari&#241;as, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+ex/in+vivo+assay+to+detect+effects+of+exogenously+added+factors+in+neural+stem+cells.">A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells.</a> Nat Protoc. 2 (4), 849-859 (2007).</li><li>Consiglio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+in+vivo+gene+transfer+into+adult+mammalian+neural+stem+cells+by+lentiviral+vectors.">Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors.</a> Proc Natl Acad Sci U S A. 101 (41), 14835-14840 (2004).</li><li>Dull, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Third-Generation+Lentivirus+Vector+with+a+Conditional+Packaging+System.">A Third-Generation Lentivirus Vector with a Conditional Packaging System.</a> J. 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B., Conese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+lentiviral+vector-mediated+transduction+on+the+tightness+of+a+polarized+model+of+airway+epithelium+and+effect+of+cationic+polymer+polyethylenimine.">Impact of lentiviral vector-mediated transduction on the tightness of a polarized model of airway epithelium and effect of cationic polymer polyethylenimine.</a> J Biomed Biotechnol. , (2010).</li><li>Bonazzi, M., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impenetrable+barriers+or+entry+portals?+The+role+of+cell-cell+adhesion+during+infection.">Impenetrable barriers or entry portals? The role of cell-cell adhesion during infection.</a> J Cell Biol. 195 (3), 349-358 (2011).</li><li>Padmashali, R., You, H., Karnik, N., Lei, P., Andreadis, S. 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LV offrono importanti vantaggi rispetto ad altri sistemi virali per la modificazione genetica degli adulti NSC 16,18. consegna stereotassica del lentivirus alla nicchia V-SVZ rappresenta un metodo efficace per etichettare e tracciare rado dividendo B1-NSC superare i limiti di altri metodi di uso comune come BrdU, che è diluito dopo molteplici divisioni cellulari, o retrovirus, che prendono di mira solo le cellule che proliferano al momento dell&#39;applicazione. LV, insieme con adenovirus, può infettare le ...

La zona ventricolare-subventricular murino (V-SVZ), nelle pareti del ventricolo laterale rivolta verso lo striato, è una regione germinale molto attivo in cui un processo continuo di replicazione delle cellule progenitrici e differenziazione risultati nella produzione persistente bulbo olfattivo (OB ) interneuroni e oligodendrociti corpo calloso 1. La generazione per tutta la vita di queste cellule sembra essere sostenuta dalla presenza in questa regione di cellule neurali staminali (NSC; anche chiamate cellule B1), che esprimono l&#39;antigene astrociti proteina silicea fibrillare gliale (GFAP) e gambo marcatori di cellule, come nestina, Id1 e Sox2 2. GFAP-cellule che esprimono B1 generare cellule (TAP) (cellule C), che esprimono fattori di trascrizione Dlx2 transito amplificando progenitore (distale-meno homeobox 2) e Ascl1 (mammiferi achaete-Schute omologo 1) e dividere rapidamente un paio di volte prima che danno luogo a neuroblasti migrazione (cellule a) o oligodendroblasts 3. Appena generata prolifneuroblasti migrano rative anteriormente, formando il flusso migratorio rostrale (RMS) per l&#39;OB, in cui si integrano nel granulari e strati glomerulare come interneuroni inibitori differenziati. Migrazione giovani oligodendroblasts muovono al CC, dove diventano cellule NG2-positive immature che continuano a dividere localmente o differenziarsi in oligodendrociti mielinizzanti maturi 1,4. celle B1, che derivano da cellule gliali radiali fetali, conservano la morfologia allungata e polarizzata dei loro predecessori e presentano un rapporto altamente specializzato con la loro nicchia. Essi spaziano tra il ependima che allinea il ventricolo e la rete di vasi sanguigni che irrigano la nicchia V-SVZ. Il piccolo processo apicale delle cellule B1 intercala tra ependymocytes multiciliated e termina in un singolo ciglio primario non mobili, mentre il loro processo basale si estende lunghe distanze per avvicinarsi il plesso vascolare planare che irriga questo finale nicchia nel BAsal lamina dei capillari del plesso 2,5-8. Il modo più affidabile per distinguere B1-NSC da astrociti non neurogena, che sono anche GFAP +, nella nicchia V-SVZ intatto si basa sulla preparazione della parete laterale del ventricolo e la loro analisi del 3-D microscopia confocale tutto il montaggio dopo immunocolorazione per GFAP per etichettare il sottile processo apicale B1-NSC, β-catenina a delineare le membrane cellulari, e sia γ-tubulina come marcatore di corpi basali cilial o acetilata α-tubulina di etichettare l&#39;entità di ogni ciglio 5,8. Le osservazioni di questi interi-monti dalla superficie ventricolare hanno indicato che le cellule B1 e ependimali sono disposti in &quot;girandole&quot; 5, in cui i processi apicali uniciliated di uno o più GFAP + B1 cellule sono circondate da una rosetta di cellule ependimali multiciliated. La morfologia caratteristica delle cellule B1 correla con l&#39;evidenza sperimentale indicating che i vasi sanguigni / cellule endoteliali e ventricolare liquido cerebrospinale (CSF) costituiscono fonti regolamentati di segnali solubili che agiscono su NSC 2,6,9-11. In superficie ventricolare, omotipica e le interazioni apico-laterale eterotipica che coinvolgono cellule ependimali e B1 comprendono giunzioni strette e giunzioni aderenti 5,12. Inoltre, molecole di adesione coinvolte nei complessi giunzionali tra cellule B1 e ependimali, come N-caderina e V-CAM, hanno dimostrato di regolare non solo il posizionamento altamente organizzato di B1 nella nicchia V-SVZ, ma anche la loro quiescenza 12 , 13. Il monostrato di cellule ependimali-B1 sembra agire come barriera di diffusione che consente il flusso regolamentato di acqua e piccole molecole dal CSF, ma limitando il passaggio intercellulare grandi proteine ​​10,11. Evidenze sperimentali indicano che la posizione unica ciglio apicale delle cellule B1 potrebbe svolgere un ruolo di sensore di segnalazione polipeptidi presenti nel liquor 2,5-7. Cellule ependimali sono, di per sé, anche una sorgente di segnali solubili e di membrana con un ruolo nella regolazione del comportamento NSC 14,15. Nucleosidi tracciabili, come bromo-deossiuridina (BrdU), o retrovirus sono stati ampiamente utilizzati per marcare le cellule progenitrici, tra NSC, in vivo. Tuttavia, questi metodi non sono ottimali per il destino tracciamento a lungo termine, perché i segnali BrdU diluito attraverso divisioni cellulari ripetuti e retrovirus sembrano indirizzare preferenzialmente transitoriamente amplificare le cellule a causa della loro esigenza di proliferazione delle cellule per la trasduzione 16,17. Per esaminare NSC fisiologia in vivo, comprese le interazioni con i componenti di nicchia, è fondamentale stabilire un metodo per etichettare e tracciare le cellule raramente che dividono, come B1-NSC sono in gran parte a riposo e le loro cellule ependimali vicini non si dividono in condizioni fisiologiche 3. Qui, dimostriamo che vettori lentivirali (LV) consentono di marchio gene ad alta efficienzazione e modifica a lungo termine di NSC adulti e non dividendo le cellule ependimali, causa più ragionevolmente alla loro capacità di trasdurre e di integrarsi nel genoma delle cellule bersaglio in modo indipendente ciclo cellulare. Inoltre, si mostra come il percorso di consegna e virali aiuto titolo di trasdurre specificamente cellule ependimali, ma non le cellule B1 consentendo in tal modo l&#39;analisi di nicchia-dipendente, effetti ependimali su NSC.

<table>
	<tbody>
		<tr>
			<td colspan="4">Part 1: Generation of LV for in vivo delivery.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima XL-100K</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>SW-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman Coulter</td>
			<td>358126</td>
			<td>25X89 mm</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman Coulter</td>
			<td>326819</td>
			<td>13X51 mm</td>
		</tr>
		<tr>
			<td>Ultracentrifuge adapters</td>
			<td>Beckman Coulter</td>
			<td>358156</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>SPL</td>
			<td>PLC-30006</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>SPL</td>
			<td>PLC-30024</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>SPL</td>
			<td>PLC-20101</td>
			<td>100x20 style</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Afora</td>
			<td>DE400800</td>
			<td>12x75 mm, 5 ml</td>
		</tr>
		<tr>
			<td>Cup sterile FACS filter</td>
			<td>BD</td>
			<td>340626</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>Nitrocellulose filter</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22 &#956;m&#160;</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>LSR Fortessa</td>
			<td>Blue laser 488 nm</td>
		</tr>
		<tr>
			<td>Steritop filter</td>
			<td>Biofil</td>
			<td>FPE-204-500&#160;</td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12251</td>
			<td>Core packaging plasmid</td>
		</tr>
		<tr>
			<td>pRSV.REV plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12253</td>
			<td>Core packaging plasmid</td>
		</tr>
		<tr>
			<td>pMD2G plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td>Envelope plasmid</td>
		</tr>
		<tr>
			<td>pRRL-SIN-PPT.PGK.EGFP.Wpre plasmid&#160;</td>
			<td>Addgene</td>
			<td>#12252</td>
			<td>Transfer vector plasmid</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td>Biowest</td>
			<td>L0101-500</td>
			<td>For HeLa cell culture</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td>Life technologies</td>
			<td>12440-053</td>
			<td>For 293T cell culture</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE)</td>
			<td></td>
			<td></td>
			<td>Tris-HCl (sigma, T5941), 0.1 mM EDTA (sigma, E5134), pH 7.6,&#160; DNAse/RNAse-free, 0.2 &#181;m sterile-filtered</td>
		</tr>
		<tr>
			<td>2X HBS</td>
			<td></td>
			<td></td>
			<td>0.28 M NaCl (Sigma, S7653), 0.05 M HEPES (Sigma, H7523), 1.5 mM anhydrous Na2HPO4 (Sigma, S7907) in dH2O (preferably not MilliQ). Adjust pH to 7.0 with NaOH solution (Calbiochem, 567530).</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biowest</td>
			<td>S181B-500</td>
			<td>Stock solution at 100X, used to prepare HeLa and 293T culture medium at a final concentration of 10X.</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513-100</td>
			<td>Stock solution at 200 mM, used to prepare HeLa and 293T culture medium at a final concentration of 6 mM.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life technologies</td>
			<td>11360-039</td>
			<td>Stock solution at 100 mM, used to prepare HeLa and 293T culture medium at a final concentration of 1 mM.</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Life technologies</td>
			<td>35050-061</td>
			<td>Used to prepare 293T culture medium at a final concentration of 1%.</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458</td>
			<td>Stock solution contains 5,000 units/ml penicillin and 5 mg/ml streptomycin. Used to prepare HeLa and 293T culture medium at a final concentration of 1%.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td>With phenol red, contains 2.5 g porcine trypsin and 0.2 g EDTA 4Na/L HBSS.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160; (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>Without calcium chloride and magnesium chloride, 10X, liquid, sterile-filtered, suitable for cell culture. Stock solution used to prepare 1X PBS in cell culture grade water.</td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>Powder. Prepare a 1000X stock solution at 8 mg/ml in dH2O</td>
		</tr>
		<tr>
			<td>Paraformaldehyde EM grade 16%</td>
			<td>EM Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 2: Sterotaxic injection of LV into the SEZ proper or the lateral ventricle.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td><a href="http://www.leicabiosystems.com/">Vernier stereotaxic instrument</a></td>
			<td>NeuroLab, Leica</td>
			<td>39463001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cunningham mouse and neonatal rat adaptor</td>
			<td>NeuroLab, Leica</td>
			<td>39462950</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe holder</td>
			<td>KD Scientific</td>
			<td>KDS-311-CE</td>
			<td></td>
		</tr>
		<tr>
			<td>33-gauge syringe</td>
			<td>Hamilton</td>
			<td>P/N&#160;&#160;&#160; 84851/00</td>
			<td>#85RN</td>
		</tr>
		<tr>
			<td>Electric drill</td>
			<td>Fine Science Tool</td>
			<td>98096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal blanket</td>
			<td>Ufesa</td>
			<td>AL5512/01</td>
			<td>230-240 V, 100-110 W, type C_AL01</td>
		</tr>
		<tr>
			<td>Shaver&#160;</td>
			<td>Jata</td>
			<td>MP373N</td>
			<td>Model: beauty, 3 V, 300 mA, type HT-03.</td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Esteve</td>
			<td>DOMTOR&#160;</td>
			<td>Comercial solution at 1 mg/ml.&#160;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td>Imalgene 500&#160;</td>
			<td>Comercial solution at 50 mg/ml</td>
		</tr>
		<tr>
			<td>Medetomidina/ketamine mixture</td>
			<td></td>
			<td></td>
			<td>Prepare a working mixture of medetomidine at a final concentration of 0.2 mg/ml dilution and ketamine at a final concentration of 15 mg/ml in saline solution. Use as anesthesia injecting a volume to get a final concentration of 0.5-1 mg medetomidina per kg body weight and 50-75 mg ketamine per kg body weight</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Pfizer</td>
			<td>Torbugesic</td>
			<td>Stock solution at 10 mg/ml. Used as analgesia at 1 mg/ml in saline solution.</td>
		</tr>
		<tr>
			<td>Atipamezole</td>
			<td>Esteve</td>
			<td>Antisedan</td>
			<td>Stock solution at 5 mg/ml, used in a final concentration of 0.5 mg/ml in saline solution to exit from anesthesia.</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Braun</td>
			<td>13465412</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>Braun</td>
			<td>1050052</td>
			<td>Topical skin adhesive</td>
		</tr>
		<tr>
			<td>HydroGel</td>
			<td>Clear H2O</td>
			<td>70-01-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34120</td>
			<td>11x21 cm</td>
		</tr>
		<tr>
			<td>Bleach/Virkon</td>
			<td>Dupont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical marker pen</td>
			<td>Staedler</td>
			<td>313-9</td>
			<td>Permanent lumocolor</td>
		</tr>
		<tr>
			<td>Ophthalmic lubricant &#160;</td>
			<td></td>
			<td>SICCAFLUID</td>
			<td>0.5 g/dosis, carbomer 974P</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Betadine</td>
			<td>694109.6</td>
			<td>10% povidone-iodine</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 3: Histological analysis.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment:</td>
		</tr>
		<tr>
			<td>Automatic peristaltic pump</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-07524-55</td>
			<td>Masterflex L/S variable-speed economy drive, 1.6-100 rpm, 230 V</td>
		</tr>
		<tr>
			<td>Pump head</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-07518-00</td>
			<td>Masterflex L/S Easy-Load pump head for precision tubing; PSF housing, CRS rotor</td>
		</tr>
		<tr>
			<td>Silicone tube</td>
			<td>Cole-Parmer Inst. Co.</td>
			<td>HV-96410-16</td>
			<td>Platinum L/S 16</td>
		</tr>
		<tr>
			<td>Scalp vein set</td>
			<td>Vygon V-green</td>
			<td>70246.05T</td>
			<td>25G, 30 cm tube length</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FluoView FV10i</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Tehtnica</td>
			<td>SHP-10</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Reagents:</td>
		</tr>
		<tr>
			<td>Phosphate buffer (PB)</td>
			<td></td>
			<td></td>
			<td>0.2 M PB: 0.2 M Na2HPO4 (Sigma, S7907) and 0.2 M NaH2PO4 (Panreac, 141965.1211) in dH2O, adjust pH to 7.2-7.4</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Panreac</td>
			<td>141451.1211</td>
			<td>Prepare fresh every time. Heat dH2O up to 55&#8211;60 &#176;C using a hot plate placed in a fume hood and pour PFA powder while stirring to obtain an 8% solution. The solution is cloudy white as PFA does not dissolve easily. Add 1N NaOH drop by drop just until the solution clears. Cool down, filter through Whatman paper and add an equivalent volume of 0.2 M PB.</td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td></td>
			<td></td>
			<td>0.9% NaCl in dH2O</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>LOCTITE</td>
			<td>767547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Panreac</td>
			<td>122712.1608</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore</td>
			<td>S30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Anti-GFP rabbit antibody</td>
			<td>ROCKLAND</td>
			<td>600-401-215</td>
			<td>Use at a 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Molecular probes</td>
			<td>A-21206</td>
			<td>Use at a 1:750 dilution</td>
		</tr>
		<tr>
			<td>6-Diamindino-2-phenylindole dihydrochloride hydrate (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Fluorescent nuclear staining. Use at 2 mg/ml in ddH2O. Keep in the dark at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>EM Sciences</td>
			<td>17984-25</td>
			<td>Mounting medium for fluorescent preparations</td>
		</tr>
	</tbody>
</table>

stable and efficient genetic modification of cells in the adult mouse v-svz for the analysis of neural stem cell autonomous and non-autonomous effects

Relativamente cellule staminali somatiche quiescenti sostenere il rinnovamento cellulare per tutta la vita nella maggior parte dei tessuti adulti. Le cellule staminali neurali nel cervello dei mammiferi adulti sono limitate a due specifiche nicchie neurogena: la zona subgranulare del giro dentato dell&#39;ippocampo e la zona ventricolare-subventricolare (V-SVZ, chiamato anche zona subependimale o SEZ) nelle pareti del laterali ventricoli. Lo sviluppo di strategie in vivo di trasferimento genico per le popolazioni di cellule staminali adulte (cioè quelli del cervello dei mammiferi) con conseguente espressione a lungo termine di transgeni desiderati nelle cellule staminali e la loro progenie derivata è uno strumento cruciale nella corrente ricerca biomedica e biotecnologica. Qui, un metodo diretto in vivo è presentato per la modificazione genetica stabile di cellule di topo adulto V-SVZ che sfrutta la cella infezioni ciclo-indipendente LV e la citoarchitettura altamente specializzato della nicchia V-SVZ. In particolare, l&#39;attuale protocollo comportas l&#39;iniezione di LV vuoti (controllo) o LV codificanti cassette specifica espressione del transgene nelle memorie V-SVZ sé, per in vivo di targeting di tutti i tipi di cellule nella nicchia, o nel lume ventricolo laterale, per l&#39;orientamento dei ependimali solo le cellule. cassette di espressione vengono poi integrati nel genoma delle cellule trasdotte e proteine ​​fluorescenti, codificate anche dal LV, permettere la rivelazione delle cellule trasdotte per l&#39;analisi di cellule, effetti autonomi e non autonomi nicchia-dipendente sulle cellule marcate e loro progenie.

Sistema di erogazione gene LV-mediata può essere utilizzato per il lungo termine in vivo trasduzione di cellule nel topo adulto V-SVZ, permettendo loro di inseguimento e modificazione genetica durante la proliferazione, la migrazione e la differenziazione. L&#39;infezione e l&#39;espressione sono altamente efficaci e producono numerose cellule che possono essere facilmente distinta tra le altre cellule non infette con l&#39;espressione del reporter incluso. Abbiamo finora visualizzato cellule trasdotte con i g...

Watch this Scientific Journal Video about Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects at JoVE

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Stabile ed efficiente Modificazione genetica di cellule nel topo adulto V-SVZ per l&#39;analisi di cellule staminali neurali autonoma ed effetti non autonomi

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

The overall goal of this procedure is to deliver low volumes of high titer lentiviral particles carrying specific DNA constructs to the adult mouse ventricular-subventricular zone to infect all of its cell types or to the lateral ventricle to affect ependymal cells only.This method can help answer key questions about interactions that neural stem cells of the adult ventricular-subventricular niche have with their neighboring ependymal cells.The main advantage of this technique is that lentiviral vectors integrate into the genome of target cells in a cell cycle independent way, allowing for long-term diffication of rarely dividing cells.Helping me to demonstrate the procedure will be grad student Beatriz Mart

Research

JoVE Journal

Developmental Biology

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Derivazione efficiente di cellule retiniche epitelio pigmentato da cellule staminali

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Ricerca

Biologia dello sviluppo

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Crescita dei follicoli preantrali Mouse sotto condizioni di microgravità simulata

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Bioingegneria dei microrganismi del midollo osseo umano nel mouse e la loro visualizzazione mediante imaging live

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Modello di impatto corticale controllato di lesioni cerebrali del topo con trapianto terapeutico di cellule neurali derivate da cellule staminali indotte dall'uomo

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Dissezione, cultura e analisi delle cellule staminali primarie dal mouse per lo studio della delaminazione e della migrazione delle cellule creste neurali

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Differenziazione neurale efficiente utilizzando la coltura a cella singola di cellule staminali embrionali umane

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...