Name: production of lentiviral vectors for transducing cells from the central nervous system
Uploaded: 2012-05-24T13:00:00
Description: Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Dette arbeidet ble støttet av NIH Neuroscience Blueprint Core-stipend (P30 NS057105, BJS) til Washington University, Program Project Grant NS032636 (BJS) og av håp Center for nevrologiske lidelser.

1. Pakking av Lentiviral Vektorer Lentiviral vektorer er produsert av cotransfection av en lentiviral overføring vektor og andre plasmider som kreves for emballasje til 293T celler ved kalsiumfosfat transfeksjon metode. Vi bruker 10 100-mm vevskulturstudier retter i denne protokollen. Det kan skaleres opp eller ned avhengig av applikasjoner. Den 293T cellelinje opprettholdes i Dulbecco modifiserte Eagles medium (DMEM) med høy glukose (4500 mg / L), supplert med 10% fosterets storfe serum (FB...

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I denne protokollen, har vi vist produksjonen av lentiviral vektorer og anvendelse av disse vektorer i neokortikale kulturer. Vi viste effektiv og celletype-spesifikk transduksjon med vektorer produsert av disse metodene. Når synapsin promoter brukes, er GFP uttrykk strengt neuron spesifikk. Når GFAP promoter brukes, er GFP uttrykk utelukkende i astrocytes. Hvis ingen celletype-spesifikke uttrykk er nødvendig, kan en allestedsnærværende promoter brukes. Vi fant både ubiqutin og phosphoglycerate kinase (PGK) arrang...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D5796 </td></tr><tr><td> MEM </td><td> Invitrogen </td><td> 11090-081 </td></tr><tr><td> Fetal bovin serum </td><td> Hyclone </td><td> SV3001403 </td></tr><tr><td> PBS </td><td> Mediatech </td><td> 21-040-CM </td></tr><tr><td> Trypsin-EDTA </td><td> Sigma-Aldrich </td><td> T3924 </td></tr><tr><td> Sodium butyrate </td><td> Sigma-Aldrich </td><td> B5887 </td></tr><tr><td> Hexadimethrine bromide (Polybrene) </td><td> Sigma-Aldrich </td><td> H9268 </td></tr><tr><td> 293T celler </td><td> ATCC </td><td> CRL-11268 </td></tr><tr><td> HT1080 celler </td><td> ATCC </td><td> CCL-121 </td></tr><tr><td> Falcon 100 x 20 mm vev kulturelte fatet </td><td> BD Biosciences </td><td> 353003 </td></tr><tr><td> 1 x 3 ½ i polyallomoer sentrifugerør </td><td> Beckman-Coulter </td><td> 326823 </td></tr><tr><td> 0,2-mikron sprøyte filter </td><td> Corning </td><td> 431219 </td></tr><tr><td> QIAamp DNA Mini Kit </td><td> Qiagen </td><td> 51304 </td></tr></tbody></table>

production of lentiviral vectors for transducing cells from the central nervous system

Effektiv genet levering i sentralnervesystemet (CNS) er viktig i å studere gen-funksjoner, modellering nevrologiske sykdommer og utvikle terapeutiske tilnærminger. Lentiviral vektorer er attraktive verktøy i transduksjon av nerveceller og andre celletyper i CNS som de transduce både skillelinjer og ikke-delende celler, støtte vedvarende uttrykk for transgener, og har relativt stor emballasje kapasitet og lav toksisitet 1-3. Lentiviral vektorer har blitt brukt i transducing mange nevrale celletyper in vitro 4-6 og i dyr 7-10. Stor innsats er gjort for å utvikle lentiviral vektorer med forbedret biosikkerhet og effektivitet for genet levering. De nåværende tredje generasjons replikering-defekte og selv-inaktivere (SIN) lentiviral vektorer er avbildet i figur 1. De nødvendige elementene for vektor emballasje er delt inn i fire plasmider. I lentiviral transfer plasmid er U3-regionen i 5 &#39;lange terminal gjenta (LTR) erstattet med en sterk promoter fra et annet virus. Denne endringen gjør at transkripsjon av vektoren sekvensen uavhengig av HIV-1 Tat protein som normalt kreves for HIV genuttrykk 11. Emballasjen signal (Ψ) er avgjørende for encapsidation og Rev-responsive element (Børre) kreves for å produsere høy titer vektorer. Den sentrale polypurine tarmkanalen (cPPT) er viktig for kjernefysisk import av vektor-DNA, en funksjon som kreves for transducing ikke-delende celler 12. I 3 &#39;LTR, er cis-regulatoriske sekvenser fullstendig fjernet fra U3 regionen. Denne slettingen er kopiert til 5 &#39;LTR etter revers transkripsjon, som resulterer i transcriptional inaktivering av begge liter. Plasmidet pMDLg / pRRE inneholder HIV-1 gag / pol gener som gir strukturelle proteiner og revers transkriptase. pRSV-Rev koder Rev som binder seg til Børre for effektiv RNA eksport fra kjernen. pCMV-G kodervesicula stomatitt viruset glykoprotein (VSV-G) som erstatter HIV-1 konv. VSV-G utvider tropisme av vektorene og gir konsentrasjon via ultracentrifugation 13. Alle gener som koder de ekstra proteiner, deriblant Vif, VPR, VPU, og Nef er utelukket i emballasjen systemet. Produksjonen og manipulering av lentiviral vektorer skal utføres i henhold til NIH retningslinjer for forskning som involverer rekombinant DNA ( <a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a> ). En godkjenning fra individ Institusjonell biologiske og kjemiske Safety Committee kan være nødvendig før bruk lentiviral vektorer. Lentiviral vektorer er ofte produsert av cotransfection av 293T celler med lentiviral overføring plasmid og hjelperåndene plasmider som koder for proteiner som kreves for vektor emballasje. Mange lentiviral overføre plasmider og hjelpeprogrammer plasmider kan fås fra Addgene, en non-Profit plasmidet repository ( <a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/~~V</a> ). Noen stabile emballasje cellelinjer har blitt utviklet, men disse systemene gir mindre fleksibilitet og emballasje effektivitet generelt avtar over tid 14, 15. Kommersielt tilgjengelige transfeksjon kits kan støtte høy effektivitet av transfeksjon 16, men de kan være svært dyrt for storskala vektor forberedelser. Kalsium fosfat nedbør metoder gir svært effektiv transfeksjon av 293T celler og dermed gi et pålitelig og kostnadseffektiv tilnærming for lentiviral vektor produksjon. I denne protokollen, produserer vi lentiviral vektorer ved cotransfection av 293T celler med fire plasmider basert på kalsiumfosfat nedbør prinsipp, etterfulgt av rensing og konsentrasjon med ultracentrifugation gjennom en 20% sukrose pute. De vektor titere bestemmes av fluorescens-aktivert celle sortering (FACS) analysis eller ved sanntid qPCR. Produksjonen og titrering av lentiviral vektorer i denne protokollen kan være ferdig med 9 dager. Vi tilbyr et eksempel på transducing disse vektorer i murine neokortikale kulturer som inneholder både nerveceller og astrocytes. Vi viser at lentiviral vektorer støtte høy effektivitet transduksjon og celle type-spesifikk genekspresjon i primære dyrkede celler fra CNS.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

I denne protokollen beskriver vi produksjon, rensing og titrering av lentiviral vektorer. Vi tilbyr et eksempel på lentiviral vektor-mediert genet levering i primære dyrkede nerveceller og astrocytes. Våre metoder kan også gjelde for andre celletyper<em&gt; In vitro</em&gt; Og<em&gt; In vivo</em&gt;.

Produksjon av Lentiviral vektorer for Transducing Celler fra Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

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