Name: a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells
Uploaded: 2013-06-19T13:00:00
Description: Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

Forfatterne vil gerne anerkende Michael Chou, Bingbing Dai og Liang Xiao for at bidrage data til dette manuskript. Vi anerkender også Dr. John Gray for de generøse gaver af reagenser, der anvendes i denne undersøgelse. PB er understøttet af en postdoc stipendium fra National Cancer Center. Denne forskning blev støttet af tilskud fra National Institute of Health (R01AI68978, P01CA132681 og RCA170820A), et tilskud fra Bill og Melinda Gates Foundation, en translationel acceleration tilskud fra Fælles Center for Translationel Medicin og en bevilling fra California HIV / AIDS forskningsprogram.

DC-LV stabile producerende celler er konstrueret baseret på GPR pakkecellelinie 11, der indeholder de nødvendige lentiviral komponenter gagpol, rev og tet-off system. Først retroviral transduktion bruges til at generere en GPRS pakkecellelinie der koder for et tet-afhængig SVGmu glycoprotein. Derefter er concatemer matrix transfektion anvendes til at transficere GPRS cellelinien med en lentivirusvektor transgen såsom GFP. Denne stabile producent-cellelinje, betegnet som LV-MGFP, kan testes i...

<ol>
	<li>Naldini, L., 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=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272, 263-267 (1996).</li><li>Kootstra, N. A., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+with+viral+vectors.">Gene therapy with viral vectors.</a> Annu. Rev. Pharmacol. Toxicol. 43, 413-439 (2003).</li><li>Montini, 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=Hematopoietic+stem+cell+gene+transfer+in+a+tumor-prone+mouse+model+uncovers+low+genotoxicity+of+lentiviral+vector+integration.">Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.</a> Nat. Biotechnol. 24, 687-696 (2006).</li><li>Montini, 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=The+genotoxic+potential+of+retroviral+vectors+is+strongly+modulated+by+vector+design+and+integration+site+selection+in+a+mouse+model+of+HSC+gene+therapy.">The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.</a> J. Clin. Invest. 119, 964-975 (1172).</li><li>Hu, B., Tai, A., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+delivered+by+lentiviral+vectors+for+cancer+and+infectious+diseases.">Immunization delivered by lentiviral vectors for cancer and infectious diseases.</a> Immunological Reviews. 239, 45-61 (2011).</li><li>Broussau, S., 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=Inducible+packaging+cells+for+large-scale+production+of+lentiviral+vectors+in+serum-free+suspension+culture.">Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 16, 500-507 (2008).</li><li>Cockrell, A. S., Ma, H., Fu, K., McCown, T. J., Kafri, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trans-lentiviral+packaging+cell+line+for+high-titer+conditional+self-inactivating+HIV-1+vectors.">A trans-lentiviral packaging cell line for high-titer conditional self-inactivating HIV-1 vectors.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 14, 276-284 (2006).</li><li>Ikeda, Y., 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=Continuous+high-titer+HIV-1+vector+production.">Continuous high-titer HIV-1 vector production.</a> Nature Biotechnology. 21, 569-572 (2003).</li><li>Kafri, T., van Praag, H., Ouyang, L., Gage, F. H., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+packaging+cell+line+for+lentivirus+vectors.">A packaging cell line for lentivirus vectors.</a> Journal of Virology. 73, 576-584 (1999).</li><li>Strang, B. L., 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=Human+immunodeficiency+virus+type+1+vectors+with+alphavirus+envelope+glycoproteins+produced+from+stable+packaging+cells.">Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells.</a> Journal of Virology. 79, 1765-1771 (2005).</li><li>Throm, R. 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=Efficient+construction+of+producer+cell+lines+for+a+SIN+lentiviral+vector+for+SCID-X1+gene+therapy+by+concatemeric+array+transfection.">Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.</a> Blood. 113, 5104-5110 (2009).</li><li>Lee, C. L., Chou, M., Dai, B., Xiao, L., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+stable+producer+cells+to+make+high-titer+lentiviral+vectors+for+dendritic+cell-based+vaccination.">Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination.</a> Biotechnol. Bioeng. 109, 1551-1560 (2012).</li><li>Steinman, R. M., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+dendritic+cells+into+medicine.">Taking dendritic cells into medicine.</a> Nature. 449, 419-426 (2007).</li><li>Yang, L., 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=Engineered+lentivector+targeting+of+dendritic+cells+for+in+vivo+immunization.">Engineered lentivector targeting of dendritic cells for in vivo immunization.</a> Nature Biotechnology. 26, 326-334 (2008).</li><li>Hanawa, H., 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=Efficient+gene+transfer+into+rhesus+repopulating+hematopoietic+stem+cells+using+a+simian+immunodeficiency+virus-based+lentiviral+vector+system.">Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.</a> Blood. 103, 4062-4069 (2004).</li><li>Yang, L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+provision+of+antigen-specific+T+cell+immunity+by+programming+hematopoietic+stem+cells.">Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 4518-4523 (2005).</li><li>Dai, B., 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=HIV-1+Gag-specific+immunity+induced+by+a+lentivector-based+vaccine+directed+to+dendritic+cells.">HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 20382-20387 (2009).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J. Vis. Exp. (63), e4031 (2012).</li><li>Kochan, G., Escors, D., Stephenson, H., Breckpot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Grade+Lentiviral+Vectors.">Clinical Grade Lentiviral Vectors.</a> Lentiviral Vectors and Gene Therapy. , 69-85 (2012).</li><li>Loew, R., 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+new+PG13-based+packaging+cell+line+for+stable+production+of+clinical-grade+self-inactivating+gamma-retroviral+vectors+using+targeted+integration.">A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration.</a> Gene Ther. 17, 272-280 (2010).</li><li>Gaspar, H. B., 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=Gene+therapy+of+X-linked+severe+combined+immunodeficiency+by+use+of+a+pseudotyped+gammaretroviral+vector.">Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.</a> Lancet. 364, 2181-2187 (2004).</li><li>Cornetta, K., 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=Replication-competent+lentivirus+analysis+of+clinical+grade+vector+products.">Replication-competent lentivirus analysis of clinical grade vector products.</a> Molecular Therapy: the journal of the American Society of Gene Therapy. 19, 557-566 (2011).</li><li>Stewart, H. J., 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+stable+producer+cell+line+for+the+manufacture+of+a+lentiviral+vector+for+gene+therapy+of+Parkinson's+disease.">A stable producer cell line for the manufacture of a lentiviral vector for gene therapy of Parkinson's disease.</a> Human Gene Therapy. 22, 357-369 (2011).</li><li>Coroadinha, A. S., 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=Retrovirus+producer+cell+line+metabolism:+implications+on+viral+productivity.">Retrovirus producer cell line metabolism: implications on viral productivity.</a> Appl. Microbiol. Biotechnol. 72, 1125-1135 (2006).</li></ol>

Her har vi skitseret en fremgangsmåde til fremstilling af store mængder af lentivirusvektorer hjælp 293T celler stabilt transduceret med alle lentivirale komponenter under tet off reguleringssystem. Til dato er afhængige fleste protokoller til at producere lentivirusvektorer på standard calciumphosphat transient transfektion (se 18, for eksempel). Denne tilgang har været en succes på en klinisk skala, men den kan lide af nogle begrænsninger ikke kan være til stede i opskalering produktion fra stabile...

Mange vektorsystemer er blevet udviklet til genlevering. Vektorer baseret på lentivira har været blandt de mest almindeligt undersøgte viral system. Disse vektorer er fordelagtige, fordi de effektivt kan transducere både dividere og ikke-delende celler 1, opnå langsigtet udtryk grundet integration i værtsgenomet, udvise lav naturlige anti-vektor immunitet i de fleste menneskelige befolkninger 2, og har et lavt potentiale for genotoksicitet fra insertionsmutagenese 3,4. Produktion af lentivira har altid været farvet af sikkerhedsmæssige bekymringer. Lentivirusvektorer er generelt afledt fra HIV-1, det ætiologiske agens for AIDS. Transient transfektion af enkelte komponenter i lentivector (overførsel, konvolutten, og emballage plasmider) er et almindeligt og fleksibelt middel til levering af genetisk materiale i laboratoriet indstillinger. Men opskalering forbigående transfektioner til kliniske anvendelser er tung og may føre til udvikling af replikationskompetent lentivirus 5,6. For at overvinde disse forhindringer har flere stabile emballage og producerende cellelinier blevet udviklet 6-11. En af disse linjer, GPR pakkelinie 11, har den attraktive fordel reguleres af tetracyclin. I dette papir, viser vi, hvordan du tilpasse dette system til at producere selv inaktiveringsbetingelser lentivirusvektorer, der er specifikt målrettet mod dendritiske celler (DC-LVS) 12. Dendritiske celler (DC&#39;er) er de mest robuste antigenpræsenterende celler i immunsystemet. De har været genstand for stor interesse i cancer-vaccine udvikling, fordi de er direkte iværksætte, program og regulere tumor-specifikke immunreaktioner 13.. Omfatter en vaccinationsprotokol at omfatte DC&#39;er har potentiale til at fremkalde en stærkere antitumor immunrespons end peptid-eller DNA vacciner. For nylig har vi udviklet et lentivirusvektor at specifically mål dendritiske celler gennem en modificeret Sindbis virus glycoprotein SVGmu 14.. Disse vektorer er unikke i, at de viser en høj specificitet til dendritiske celler og generere kraftigere immunrespons end uspecifik, VSVG-pseudotype vektorer. Her rapporterer vi en metode til at producere store mængder af disse DC-målrettede lentivirusvektorer. Vi viser, at disse DC-LVS kan inficere DC&#39;er og frembringer stærke CD8 + T-celle immunrespons. Alle procedurer, der involverer dyr blev udført humant, under godkendelsen af ​​USC Intitutional Animal Care og brug Udvalg. For at udføre in vivo og kliniske forsøg, er det afgørende at skabe en cellelinie, som kan producere virus i en høj titer. Udførelse af transduktion og transfektion trin nøjagtigt som beskrevet, vil maksimere chancerne for at generere en sådan klon.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentivirusvektorer (LVS), er et effektivt middel til at levere genetisk materiale til mange typer af celler. På grund af sikkerhedsmæssige bekymringer forbundet med disse HIV-1 afledte vektorer, der producerer store mængder af LVS er udfordrende. I dette papir, rapporterer vi en metode til fremstilling høje titre af selv-inaktiverende LVS. Vi retroviralt transducere tet-off stabil producent-cellelinje GPR at generere en cellelinie, GPRS, som kan udtrykke alle de virale komponenter, herunder en dendritisk celle-specifik glycoprotein SVGmu. Så vi bruger concatemeric DNA-transfektion til transfektion LV transferplasmidet koder et reportergen GFP i kombination med en selekterbar markør. Flere af de resulterende kloner kan producere LV på en titer 10 gange større end hvad vi opnår med transient transfektion. Plus, disse vira effektivt transducere dendritiske celler in vitro og generere en stærk T-celle immunrespons til vores reporter antigen. Denne metode kan være en god mulighed for producerer stærke LV-baserede vacciner til kliniske studier af kræft eller infektionssygdomme.

Den stabile cellelinie beskrevet i denne metode kan producere store mængder af lentivirale vektorer, der er specifikt rettet mod dendritiske celler. Som vist i figur 1B, isolering af individuelle kloner gav stabile cellelinjer af varierende kvalitet 12.. Mellem 26 testede kloner, produceret 8 lentivirale partikler ved en titer på mere end 10 6 transduktion enheder pr ml (TU / ml), som er et typisk benchmark for SVG-pseudotype lentivirusvektorer fremstillet ved transient transfekt...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Her bruger vi retroviral transduktion og concatemeric transfektion til at skabe en cellelinie, der kan udtrykke komponenterne i en lentivirusvektor (LV) i fravær af tetracyclin. Dette LV koder GFP og pseudotype med et glycoprotein, SVGmu, som er specifik for en receptor på dendritiske celler.

En Tetracyklin-regulerede cellelinje producerer high-titer lentivirusvektorer som specifikt rettet dendritiske celler

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

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