Name: stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord
Uploaded: 2013-03-18T13:00:00
Description: Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Vi takker Bakhos A. Tannous, Ph.D., direktør for Vector Udvikling og Produktion i Neuroscience Center i Massachusetts General Hospital, Charlestown, Massachusetts, for at give os med rAAV-EGFP vektor, og John Whang for teknisk assistance. Dette arbejde blev støttet af tilskud R01 NS050408 (til JS) fra National Institute of Neurologiske Lidelser.

Den kirurgiske beskrevne procedure er godkendt af Institutional Animal Care og Use Committee (IACUC) fra Columbia University. 1. Fremstilling af udstyr og virus partikelsuspension <ol><li> Rengøre og desinficere udstyret, sterilisere kirurgiske instrumenter og V-notch spikes der vil blive anvendt til at fastsætte ryghvirvel L1. </li><li> Træk og smig glaspipetter. Vi anvender pipetter, der har en spids diameter på 40 um og er affaset i en vinkel på 20 °. Sterilisere glaspipetter. </l...

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Stereotaktisk vektor injektion tillader målretning rygmarvsneuroner til applikationer såsom neurale netværk kortlægning baseret på transsynaptisk virusset spredes 6,7 eller optogenetic dissektion 8, axon vejledning under regenerering fra skade 9,10 eller genterapi til forebyggelse eller behandling af neurodegeneration 11, 12. Virale vektorer er blevet anvendt til genmanipulation i rygmarven for at undersøge somatosensoriske, motoriske og autonom veje 9,10,13-15.</su...

Avancerede teknologier betinget genmanipulation i musen så mangesidede tilgange til udforskning af synaptiske veje og funktionelle forbindelser i centralnervesystemet. Transgener kan reguleres af små molekyler effektorer, såsom doxycyklin, der virker på et tetracyclin-reguleret transaktivator, som kan udformes til at fungere som en repressor eller aktivator af gentranskription eller tamoxifen erkender et muteret ligand-bindende domæne af østrogenreceptoren en . Irreversibel transgen modifikation er almindeligvis opnås ved deoxyribonukleinsyre (DNA) rekombinaser. Cre (årsager rekombination) og Flp (flippase rekombination enzym) katalysere excision, inversion eller translokation af DNA-fragmenter, der er flankeret af loxP (locus af krydsning x over, P1) eller Frt (flippase anerkendelse mål) sites, henholdsvis 1. Applikationerne omfatter genaktivering eller nedregulering og inducerbar ribonukleinsyre (RNA) interferens <sup&gt; 2. Betinget ekspression af fluorescerende eller enzymatiske reportere, såsom β-galactosidase eller alkalisk phosphatase kan anvendes til at mærke neuroner og undersøge deres topisk organisation og konnektivitet 3. Storstilede mutagenese projekter i Nordamerika ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) og Europa ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) producerer biblioteker af muse embryonale stamceller kloner med betingede genmål og fælder, der i sidste ende vil dække hele musegenomet. Mus genereres fra disse kloner kan krydses med et voksende antal muselinjer, som udtrykker DNA-rekombinaser under promotorer eller loci er specifikke for en bestemt population af neuroner til selektiv genmanipulation ( <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . php</a> ). <p class= &quot;Jove_content&quot;&gt; Imidlertid kan begrænse genmanipulation til forskellige populationer af neuroner eller særlige regioner af interesse ikke opnås ved genetisk målretning alene, hvis en promotor specifik for neuron populationen af ​​interesse ikke kendes eller ikke udtrykkes af alle neuroner i regionen af interesse. I rygmarven, kan eksperimentelle design kræver rumlig begrænsning af genmanipulation til et eller to craniocaudal segmenter. Stereotaktisk injektion af en viral vektor, der udtrykker Cre eller FLP mulighed for at begrænse genrekombination til regioner i rygmarven af ​​mus, hvor DNA-fragmenter er flankeret af loxP eller FRT sites, såkaldte floxet eller flrted alleler. I modsætning til konstitutiv DNA-omlejring, som vil medføre fra krydsning dyrene med rekombinasegenkendelsessites udtrykker mus, denne strategi giver også tidsmæssig kontrol over gen-aktivering eller nedregulering. Virale vektorer, der koder floxet eller flirtede transgener har en omvendt mulighed for genmanipulation i mus der udtrykker corresponde rekombinase nedstrøms for en neuron-specifik promotor. Adskillige rekombinante vektorer med affinitet til neuroner er tilgængeligt 4. Høj kapacitet (rygradsløse) adenovirus, adenoassocieret virus, herpes simplex virus og lentivirus er almindeligt anvendt neurotrofiske vektorer. Valg af passende virus til et forsknings-spørgsmål er en afgørende del af det eksperimentelle design. Størrelsen af transgenet, tilførselsvej, specificitet af infektionen til neuroner i modsætning til gliaceller, infektion effektivitet, inflammatoriske og toksiske bivirkninger skal overvejes 4. Her beskrives stereotaktisk injektion af en viral vektor i det dorsale horn i rygmarven, en teknik, som vi anvender for betinget genregulering i vores forskning på den neurobiologiske smerte. Den dorsale horn modtager afferent input fra primære somatosensoriske neuroner, herunder nociceptive neuroner. Lokale interneuroner behandler oplysningerne før projektions neuroner overbringe det fradet dorsale horn til hjernen 5. Vi viser infektionen af ​​baghornsneuroner på spinal segmental niveau L4 med en neurotropisk rekombinant adenoassocieret virus (rAAV), som udtrykker forbedret grønt fluorescerende protein (EGFP) under en grundlæggende aktiv cytomegaloviruspromotor.

<table border="1"><tbody><tr><td> Materialets navn </td><td> Firma </td><td> Katalognummer </td><td></td></tr><tr><td> Spinal bundplade </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Små dyr stereotaxisk instrument </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Mus gas anæstesi hoved holder </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Justerbare base-mounts </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> V notch spikes </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Små dyr temperatur kontrolsystem </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson pincet </td><td> Fin Science Tools </td><td> 11.006-12 </td><td></td></tr><tr><td> Laminektomi pincet </td><td> Fin Science Tools </td><td> 11.223-20 </td><td></td></tr><tr><td> UltraMicroPump (en) med SYS-Micro4 Controller </td><td> Verden Precision Instruments </td><td> UMP3-1 </td><td></td></tr><tr><td> Mikrosprøjte, 65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> RN kompression montering, 1 mm </td><td> Hamilton </td><td> 55.750-01 </td><td></td></tr><tr><td> Borosilikatglas kapillærer </td><td> Verden Precision Instruments </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Intraparenchymal injektion af en viral vektor muliggør betingede genmanipulation i forskellige populationer af neuroner eller særlige regioner i centralnervesystemet. Vi viser en stereotaktisk injektion teknik, der giver mulighed for målrettet genekspression eller inaktivering i det dorsale horn i muse rygmarven. Den kirurgiske procedure er kort. Det kræver laminectomy af en enkelt ryghvirvel, om hurtig genopretning af dyret og fejlfri motilitet af rygsøjlen. Styret indsprøjtning af et lille vektor suspension volumen ved lav hastighed og anvendelse af en mikrosprøjte med skrå glas kanyle minimere vævet læsion. Det lokale immunrespons til vektoren afhænger af de iboende egenskaber af det anvendte virus, i vores erfaring er det mindre og kortvarige, når en rekombinant adenoassocieret virus anvendes. Et reportergen, såsom forstærket grønt fluorescerende protein letter overvågning rumlige fordeling af vektoren og virkning og cellulære specificity af transfektion.

Vellykket transfektion giver robust genekspression i neuroner i det injicerede dorsale horn (fig. 1), besparende det dorsale horn i den kontralaterale side, den ventrale horn og den dorsale rodganglier. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figur 1" /> Figur 1. Transfektion af baghornsneuroner. (A) Ekspression af det fluorescerende reporter EGFP (grøn) i den venstre dorsale horn i L4 rygmarven, to uger efter stereotaktisk injektion af r...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Virusvektorer på målrettet genmanipulation. Vi viser en fremgangsmåde til betinget genekspression eller ablation i muse rygmarv under anvendelse af stereotaktisk injektion af en viral vektor i den dorsale horn, en fremtrædende sted for synaptisk kontakt mellem primære somatosensoriske afferenter og neuroner i centralnervesystemet.

Stereotaktisk injektion af en virusvektor til Betinget Gene Manipulation i Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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