Name: dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells
Uploaded: 2018-01-15T12:30:00
Description: Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Vi takker Lucia Sammarco og hendes Lulu limonade stå for inspiration, motivation og støtte. Dette arbejde blev støttet i en del af NIH R01 AI102893 og NCI R01 CA179363 (S.M.); NHLBI-HL087951 (S.R.); NIH-CA151893-K08 (M.J.R.); NCI 1R01CA164225 (L.W); Alex Lemonade Stand Foundation (S.M.); Programmet HRHM MACC fondens (S.M.; S.R.; M.S.T); Nicholas Family Foundation (S.M.); Gardetto familie (S.M.); Hyundai lærde Program (M.S.T.); Hyundai håb på hjul (S.R.); MACC fond (M.S.T. og S.M.); children's Research Institute, MCW (S.R.); og Kathy Duffey Fogerty Award (M.J.R.).

Alle dyr protokoller fulgte den humane og etiske behandling af dyr og blev godkendt af institutionelle Animal Care og brug udvalg (IACUC) inden for biomedicinsk forskning Center (BRC) af den Medical College of Wisconsin (MCW), Milwaukee, WI. Brug af humant perifert blod mononukleære celler (PBMC) blev godkendt af institutionelle Review Board (IRB) af blod Research Institute of Wisconsin blod-Center, Milwaukee, WI.
1. mus, cellelinjer og vektorer
<ol><li>Hente C57BL/6 mus fra kommercielle le...

<ol>
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M., Garnier, A., Durocher, Y., Ansorge, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+protocol+for+lentiviral+vector+mass+production.">New protocol for lentiviral vector mass production.</a> Methods Mol. Biol. 614, 39-52 (2010).</li><li>Rajasekaran, 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=Signaling+by+Fyn-ADAP+via+the+Carma1-Bcl-10-MAP3K7+signalosome+exclusively+regulates+inflammatory+cytokine+production+in+NK+cells.">Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells.</a> Nat. Immunol. 14, 1127-1136 (2013).</li><li>Regunathan, J., Chen, Y., Wang, D., Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+receptor-mediated+NK+cell+function+is+regulated+by+inhibitory+Ly49+receptors.">NKG2D receptor-mediated NK cell function is regulated by inhibitory Ly49 receptors.</a> Blood. 105, 233-240 (2005).</li><li>Awasthi, 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=Rap1b+facilitates+NK+cell+functions+via+IQGAP1-mediated+signalosomes.">Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes.</a> J. Exp. 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Denne undersøgelse viser at brugen af dextran som en kationiske polymer agent effektiviserer lentiviral transduktion af både murine og menneskelige primære NK celler. Derudover har andre kationiske stoffer, såsom Pb eller PS, nogen mærkbar virkning på levering af virale vektorer i menneskelige primære NK celler. Det er tidligere påvist, at Pb kan forøge gen transduktion i human T celler17. Disse resultater tyder imidlertid på at hverken Pb eller PS har en lignende effektivitet på mennes...

Natural killer (NK) celler er den største lymfatisk befolkning af medfødte immunsystem1. NK-celler fungerer som first-line forsvarere af vært immunrespons mod tumorer og infektioner2,3,4. NK-celler også spille en central rolle i udviklingen af tolerance gennem udskillelsen af potente cytokiner og kemokiner5. På grund af deres potent evne til at målrette og fjerne tumorceller, er flere kliniske forsøg gennemføres for at vurdere donor-stammer menneskelige NK celler som en adoptive immunterapi for kræft6,7. I modsætning til T-celler endnu NK-celler udviklingsmæssige biologi ikke være godt karakteriseret8. Dette manglende kendskab er delvist på grund af manglende effektive teknikker, der levere gener af interesse til musen eller menneskelige primære NK celler. Af disse grunde, er de fleste NK-celle undersøgelser blevet gennemført i cellelinjer og ikke i primærelementer. Behovet for en pålidelig og effektiv protokol til transduce primære NK celler med gener af interesse er derfor afgørende.
Det overordnede mål med denne undersøgelse var at formulere en konsekvent og pålidelig metode som primære menneskelige eller murine NK celler kunne være transduced med lenti- eller retrovira.
Tidligere undersøgelser, som har forsøgt at løse dette problem er blevet udført, i vid udstrækning benytter den forbigående omdannelse af primære NK celler. Dette omfatter plasmid Transfektion9,10, Epstein - Barr Virus (EBV) / retroviral hybrid vektor11, vaccinia vektorer12,13, og Ad5/F35 kimære adenoviral vektorer14. Trods den beskedne effektivitet af disse teknikker gør transduktion forbigående karakter dem uegnet til den langsigtede udnyttelse af genetisk modificeret NK celler. Nogle nylige undersøgelser har brugt retroviral vektorer til transduce NK celler, der kræver flere cyklusser af infektion at opnå et acceptabelt niveau af gen expression11,15. I modsætning til retroviral vektorer, kan lentiviral vektorer bruge vært-celle nukleare import maskiner til translocate viral pre integration komplekset ind i kernen. Dette er en stor begrænsende faktor i replikering af virus i ikke-dividere celler, som omfatter primær NK celler.
Interaktioner mellem forskellige celle-overflade receptorer og viruspartikler tillade viral udbredelse ind i cellen. De første sammenstød mellem de virale kuvert proteiner og deres beslægtet vært receptorer kunne være begrænset på grund af de potentielle negative afgifter mellem disse to. Rationalet bag mange transduktion teknikker er, at tilsætning af kationiske polymerer, såsom polybrene (Pb), protamine-sulfat (PS) eller dextran, kan give en positiv ladning på celle-overflade receptorer og dermed øge bindingen af virale kuvert proteiner. Dette vil øge fusion effektivitet og udbredelsen af de virale partikler af celler16. Selv om det er blevet rapporteret, at Pb eller PS kan forbedre genoverførsel i T celler17, har deres program ikke nogen effekt i transduktion effektiviteten af primære NK celler. Desuden en komparativ analyse mellem disse reagenser ved hjælp af primære NK celler ikke er blevet udført. I denne undersøgelse, blev transduktion effektiviteten af de tre kationiske polymerer sammenlignet. Resultaterne viser, at blandt disse tre kationiske polymerer, kun dextran betydeligt forbedrer effektiv viral signaltransduktion i både mus og menneskelige primære NK celler.

<table>
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATALANTA</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Corning</td>
			<td>MT25000CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodding staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cell negative selection kit</td>
			<td>Stem Cell</td>
			<td>19855</td>
			<td></td>
		</tr>
		<tr>
			<td>Yac-1</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>K562</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jakson</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Cornnig</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>antibody-based negative selection kits</td>
			<td>Stem Cell</td>
			<td>19055</td>
			<td></td>
		</tr>
		<tr>
			<td>51Chromium (Cr)-release assays</td>
			<td>perkin elmer&#39;s</td>
			<td>NEZ030</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma</td>
			<td>5887-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polyethylenimine</td>
			<td>polysciences</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Life Science</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

Den effektive transduktion af specifikke gener i natural killer (NK) celler har været en stor udfordring. Vellykket transductions er afgørende for definitionen af gen interesse for udvikling, differentiering og funktion af NK celler. De seneste fremskridt i forbindelse med kimære antigen receptorer (biler) i cancer immunterapi fremhæve behovet for en effektiv metode til at levere udefrakommende gener til effektor lymfocytter. Effektiviteten af lentiviral-medieret gen transductions til primære menneskelige eller mus NK celler forbliver betydeligt lav, hvilket er en stor begrænsende faktor. De seneste fremskridt ved hjælp af kationiske polymerer, såsom polybrene, vise en forbedret gen transduktion effektivitet i T-celler. Disse produkter blev dog ikke bedre transduktion effektivitetsgevinster af NK celler. Dette arbejde viser, at dextran, en forgrenet glucan polysaccharid, væsentligt forbedrer transduktion effektivitet af menneskelige og musen primære NK celler. Denne meget reproducerbare transduktion metode giver en kompetente værktøj for transducing menneskelige primære NK celler, som kan langt bedre klinisk gen sikkerhedsudvidede programmer og dermed NK cellebaserede cancer immunterapi.

Dextran inducerer den effektive genoverførsel af lentiviral vektor i primære menneskelige og murine NK celler
NK celler blev isoleret og renset fra PBMC (med en renhed på mere end 85%) og inkuberes natten over med rIL-2 300 U/mL. Disse primære NK celler blev derefter transduced med normal god landbrugspraksis lentivirus på varieret multiplicities af infektion (MOI; 3, 10 og 20 IU pr. celle) i 24-godt plader 8 µg/mL Pb, PS ell...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Målet med denne undersøgelse var at formulere teknologier, der muliggør vellykket gen transduktion i primære natural killer (NK) celler. Dextran-medieret lentiviral transduktion af menneskelige eller musen primære NK celler resultater i højere gen expression effektivitet. Denne metode af genet transduktion vil langt forbedre NK celler genetisk manipulation.

Dextran effektiviserer Lentiviral transduktion af Murine og menneskelige primære NK-celler

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

Generering af inducerede regulatoriske T-celler fra primære humane naive og memory T-celler

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Ændring af primære humane T-celler

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolation og intravenøs injektion af murine knoglemarv Afledte Monocytter

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Pålidelig og High Efficiency Udvinding af Nyre immunceller

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Retroviral transduktion af Helper T-celler som en genetisk tilgang til at studere Mekanismer Controlling deres Differentiering og funktion

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

En Flow flowcytometri-baserede cytotoksicitet analyse til vurdering af menneskers NK celleaktivitet

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Vurdering af Synaptic grænsefladen af primære menneskelige T celler fra perifert blod og lymfoide væv

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

Generation af Knock-out primære og udvidet menneskelige NK celler ved hjælp af Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Høj overførselshastighed måling af Plasma membran genforsegling effektivitet i pattedyrceller

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...