Name: a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells
Uploaded: 2017-12-12T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

Vi vil gjerne takke avdeling av Surgery, Duke University School of Medicine og Dekanus kontor for grunnleggende vitenskap, Duke University. Vi takker også medlemmer av Duke Viral vektor kjernen for kommentarer på manuskriptet. Plasmider pLenti CRISPRv2 var gave fra Feng Zhang (bred Institute). LV-pakkesystemet inkludert plasmider psPAX2, var VSV-G, pMD2.G og pRSV-Rev en slags gave fra Didier Trono (EPFL, Sveits). Økonomisk støtte til dette arbeidet ble levert av universitetet av South Carolina School Of Medicine, gi RDF18080-E202 (B.K).

1. dyrking HEK-293T cellene og Seeding for hva
Merk: Menneskelige embryonale nyre 293T (HEK-293T) celler er dyrket i DMEM, høy glukose media med 10% bovin kalv serum supplert med jern og vekst arrangører og 1 x antibiotika-antimycotic løsning (100 x løsningen inneholder 10.000 enheter penicillin 10 mg streptomycin og 25 µg amfotericin B per mL). Media er også supplert med 1 x natrium pyruvate, 1 x ikke-essensielle aminosyren mix og 2 mM L-glutamin (lager 200 mM L-alanyl-L-glutamin dipeptid...

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IDLVs har begynt å dukke opp som bilen av valget for i vivo gen-redigering, spesielt i sammenheng med genetiske sykdommer, på grunn hovedsak til lav risiko for mutagenese forbundet med disse vektorer sammenlignet integrere levering plattformer22 , 28. i gjeldende manuskriptet, vi søkte på detaljer protokollen tilknyttet produksjonen av forbedret alt-i-ett IDLV-CRISPR/Cas9 systemet som ble nylig utviklet i vårt laboratorium28....

Presis genet redigering utgjør hjørnesteinen i store biomedisinsk fremskritt som involverer utviklingen av romanen strategier for å takle genetiske sykdommer. I forkant av gen-redigering teknologiene er metoden stole på bruk av den clustered regularly -jegnterspaced short palindromic repeats (CRISPR) / Cas9 system som ble identifisert som en del av bakteriell immunitet mot invasjonen av viral genetiske materiale (omtalt i referanser1,2). En stor fordel av CRISPR/Cas9 over andre gen-redigeringsverktøy, som sink-finger nucleases (ZFNs) og transkripsjon aktivator som effektor nucleases (TALENs) (omtalt i referanse3), er den relative enkelheten av plasmider design og bygging av CRISPR komponenter, en funksjon som har drevet utvidelse av gen-redigering av noen få spesialiserte laboratories til en mye bredere forskningsmiljø. I tillegg har enkelheten til CRISPR/Cas9 programmering og dens kapasitet for multiplex målet anerkjennelse ytterligere fueled sin popularitet som en kostnadseffektiv og lett-å-bruke teknologi. Blant de ulike metodene tilgjengelig for forskere å levere slike gen-redigering komponenter til celler, fortsatt viral vektorer langt den mest populære og effektive systemet.
Lentiviral vektorer (LVs) har dukket opp som den ønsker å levere komponentene i CRISPR/Cas9 systemet i vivo for diverse programmer4,5,6,7. Flere viktige funksjoner gjør LVs et populært valg for denne prosessen inkluderer deres evne til å infisere både dele og ikke dele celler, lav immunogenisitet og minimal mobilnettet toksisitet (omtalt i referanse8). Som et resultat, har LV-mediert genterapi vært ansatt i behandling av infeksjonssykdommer, som HIV-1 og HBV HSV-1, og i korrigering av feil underliggende menneskelige arvelige sykdommer, som cystisk fibrose og neo-vaskulær macular degenerasjon 4 , 5 , 7 , 9 , 10 , 11. videre, LVs har endret effektivt for å utføre multiplex genet redigering på tydelige genomisk loci bruker en enkelt vektor systemet12.
Men den iboende egenskapen av LVs å integrere i vert genomet kan mutagene og ofte handicap deres nytte som transgene levering biler, spesielt i klinisk innstillinger. Videre siden stabilt integrert LVs uttrykke deres effekter av transgener på bærekraftig høye nivåer, er dette systemet dårlig tilpasset for levering av gen-redigering komponenter som CRISPR/Cas9; overuttrykte Cas9-guide RNA (gRNA) og lignende proteiner som ZFNs, er forbundet med forhøyede nivåer av off-målet effekter, blant annet uønsket mutasjoner13,14,15,16 , 17 og kan potensielt øke cytotoksisitet18. Derfor, for å oppnå presis gen-redigering med minimal off-målet effekter, er det viktig å design systemer som tillater for forbigående uttrykket av genet redigeringskomponenter.
De siste årene, en rekke levering plattformer er utviklet for å uttrykke transiently CRISPR/Cas9 i cellene16,19,20,21 (omtalt i referanse22). Disse omfatter metoder som bruker direkte innføre renset Cas9 sammen med riktig veiledning RNAs i celler, som viste seg å være mer effektiv på målrettet gen-redigering i forhold til plasmider-mediert transfection16. Studier har vist at ribonucleoprotein (RNP) komplekser bestående av guide RNA/Cas9 partikler er raskt snudd etter formidling DNA cleavage på sine mål, antyder at kortsiktige uttrykket av disse komponentene er tilstrekkelig for å oppnå robust genet redigering16. Tenkes, ikke integrert viral vektor plattformer som adeno-assosiert virus vektorer (AAVs) kan gi et levedyktig alternativ å levere gen-redigering maskiner til cellene. Dessverre AAV capsids har betydelig lavere emballasje kapasiteter enn LVs (< 5kb), som sterkt hindrer deres evne til å pakke multi-komponent CRISPR verktøysettet innenfor en enkelt vektor (omtalt i referanse8). Det er verdt å merke seg at tillegg av forbindelser som hemme histone deacetylases (f.eks, natrium butyrate23) eller hindre cellen syklus (f.eks, koffein24) har vist seg å øke lentiviral titers. Til tross for framskritt, er forbigående uttrykk systemer utviklet så langt fortsatt hindret av flere mangler, for eksempel lavere effektivitet, som fører til redusert viral titers, og lav signaltransduksjon effektiviteten av virus generert gjennom slike tilnærminger25.
Integrase dårlig lentiviral vektorer (IDLVs) representerer et stort fremskritt i utviklingen av genet levering biler, som de kombinerer emballasje evnen til LVs med fordelen av AAV-lignende episomal vedlikehold i celler. Disse funksjonene hjelper IDLVs i stor grad omgå de store problemene forbundet med å integrere vektorer, vis-à-vis kontinuerlig overuttrykte potensielt gentoksisk elementer og integrasjon-mediert genetisk virkning. Det ble tidligere vist at IDLVs kan endres med hell for å forbedre episomal gene expression26,27. Med hensyn til IDLV-mediert CRISPR/Cas9 levering begrenser lav produksjon titers og lavere uttrykk for episome-borne genomer i forhold til integrase-dyktige lentiviral systemer deres nytte som bona fide verktøy for å levere genomet-redigering transgene konstruksjoner. Vi har nylig vist at både transgene uttrykk og viral titers knyttet IDLV produksjon er betydelig forbedret ved inkludering av bindende områder for transkripsjon factor Sp1 i viral uttrykk kassett28. De endrede IDLVs støttet robust CRISPR-mediert genet redigering både i vitro (i HEK-293T celler) og i vivo (på etter mitotisk hjernen nevroner), mens inducing minimal off-målet mutasjoner sammenlignet med tilsvarende ICLV-mediert systemer28. Samlet, vi utviklet en roman, kompakt, alle-inne-ettall CRISPR toolkit gjennomført på en IDLV plattform og skissert ulike fordelene med å bruke slike en levering kjøretøy for forbedret genet redigering.
Her er produksjon protokollen av IDLV-CRISPR/Cas9 beskrevet, inkludert de ulike trinnene involvert i samlingen, renselse, konsentrasjon og titrering av IDLVs, samt strategier for å validere gen-redigering effekten av disse vektorer. Denne protokollen er lett skalerbare for å møte behovene til ulike etterforskere og er utformet for å kunne generere LV vektorer med titers i området fra 1 x 1010 transducing enheter (TU) / mL. Vektorer generert gjennom denne protokollen kan brukes til å effektivt infisere flere ulike celletyper, inkludert vanskelig å transduce embryonic stilk celler, blodkreft cellene (T-celler og makrofager) og kultivert og i vivo- injisert neurons. Videre er protokollen like godt egnet for produksjon av integrase-kompetent lentiviral vektorer i lignende mengder.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56915/56915fig1.jpg"> Figur 1: IDLV emballering. (a) skjematisk av wild type integrase protein (b) den endrede plasmider ble avledet fra psPAX2 (se metoder, plasmider konstruksjon for detaljer). Representant agarose gel bilde av kloner vist for muterte integrase kloner. DNA-prøver tilberedt med en standard plasmider DNA isolasjon mini kit ble analysert av fordøyelsen med EcoRV og SphI. Riktig fordøyd klone (nummer 5, stiplet rød boks) ble ytterligere bekreftet ved direkte (Sanger) sekvenser for D64E substitusjon i INT. Integrase-mangelfull emballasje kassetten ble kalt pBK43. (c) skjematisk av forbigående transfection protokollen ansatt til å generere IDLV-CRISPR/Cas9 vektorer, viser transfekterte 293T celler med VSV-G, emballasje og transgene kassetter (Sp1-CRISPR/Cas9 alt-i-ett plasmider). Virus partikler som bud ut fra cellemembranen inneholder den fulle RNA av vektoren (uttrykt fra transgene kassetten). Den andre generasjonen av IDLV-emballasje systemet ble brukt, som inkluderer regulatoriske proteiner Tat og rev Rev uttrykk er ytterligere supplert fra en egen kassett (RSV-REV-plasmider). ABBREV: LTR-lang-terminal gjenta, VSV-G, vesicular stomatitt virus G-protein, pCMV-cytomegalovirus promoter; Rous sarkomspesialitet virus (RSV) selskapet; RRE-(Rev svar Element). Andre regulerende elementer på uttrykk kassetten inkluderer Sp1 bindende områder, Rev Response element (RRE), hakkespett hepatitt Virus Posttranscriptional regulatorisk Element (WPRE), en kjerne-forlengelse faktor 1α promoter (EFS-NC), vektor emballasjen elementet ψ (psi), menneskelige Cytomegalovirus (hCMV) arrangøren og menneskelige U6 promoter. <a href="//cloudflare.jove.com/files/ftp_upload/56915/56915fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a>

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN-80 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A99839</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 25R tabletop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369434</td>
			<td></td>
		</tr>
		<tr>
			<td>xMark Microplate Absorbance plate reader</td>
			<td>Bio-Rad</td>
			<td>1681150</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS</td>
			<td>Becton Dickinson</td>
			<td>338960</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DM IRB2</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-&#956;m filter unit, 500mL</td>
			<td>Corning</td>
			<td>430773</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical bottom ultracentrifugation tubes</td>
			<td>Seton Scientific</td>
			<td>5067</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube adapters</td>
			<td>Seton Scientific</td>
			<td>PN 4230</td>
			<td></td>
		</tr>
		<tr>
			<td>SW32Ti swinging-bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>369650</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>High-binding 96-well plates</td>
			<td>Corning</td>
			<td>3366</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm TC-Treated Cell Culture dishes with 20 mm Grid</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;M filter unit, 1L</td>
			<td>Corning</td>
			<td>430513</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (50)</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 5 mL</td>
			<td>Corning</td>
			<td>4487</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture pipettes, 25 mL</td>
			<td>Corning</td>
			<td>4489</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer with cover slips</td>
			<td>Cole-Parmer</td>
			<td>UX-79001-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human embryonic kidney 293T (HEK 293T) cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>293FT cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose media</td>
			<td>Gibco</td>
			<td>11965</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution, 100X</td>
			<td>Sigma Aldrich</td>
			<td>A5955-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acid (NEAA)</td>
			<td>Hyclone</td>
			<td>SH30087.04</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)</td>
			<td>Sigma Aldrich</td>
			<td>B9879 - BES</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1800-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>p24 ELISA reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>3537</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV-1 standards</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP968F</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-p24 antibody</td>
			<td>NIH AIDS Research and Reference Reagent Program</td>
			<td>SP451T</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit horseradish peroxidase IgG</td>
			<td>Sigma Aldrich</td>
			<td>12-348</td>
			<td>Working concentration 1:1500</td>
		</tr>
		<tr>
			<td>Normal mouse serum, Sterile, 500mL</td>
			<td>Equitech-Bio</td>
			<td>SM30-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum, Sterile, 10mL</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr>
			<td>TMB peroxidase substrate</td>
			<td>KPL</td>
			<td>5120-0076</td>
			<td>Working concentration 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiCRISPR v2</td>
			<td>Addgene</td>
			<td>52961</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BsrGI</td>
			<td>New England Biolabs</td>
			<td>R0575S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England Biolabs</td>
			<td>R0195S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R0547S</td>
			<td></td>
		</tr>
		<tr>
			<td>SphI</td>
			<td>New England Biolabs</td>
			<td>R0182S</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells

Lentiviral vektorer er et ideelt valg for å levere gen-redigering komponenter til på grunn av deres evne til stabilt transducing et bredt spekter av celler og formidling av høye nivåer av genuttrykk. Men deres evne til å integrere i verten celle genomet øker risikoen for insertional genetisk virkning og dermed øker sikkerheten bekymringer og begrenser deres bruk i klinisk innstillinger. Videre levert vedvarende uttrykket av gen-redigering komponenter av disse integrasjon-kompetent lentiviral vektorer (ICLVs) øker sannsynligheten for promiskuøse genet målrette. Som et alternativ, har en ny generasjon integrase dårlig lentiviral vektorer (IDLVs) blitt utviklet som løser mange av disse bekymringene. Her produksjon protokollen av en ny og forbedret IDLV plattform for CRISPR-mediert genet redigering og listen trinnene involvert i rensing og konsentrasjonen av slike vektorer er beskrevet og signaltransduksjon og gen-redigering effektivitet ved hjelp HEK-293T cellene ble demonstrert. Denne protokollen er lett skalerbare og kan brukes til å generere høy titer IDLVs som kan transducing celler i vitro og i vivo. Dessuten, denne protokollen kan lett tilpasses for produksjon av ICLVs.

Validering av knockout-effektiviteten av IDLV-CRISPR/Cas9 vektorer Vi brukte GFP-uttrykke 293T celler som en modell for å validere effektiviteten av CRISPR/Cas9-mediert genet knockout. GFP + celler ble generert av signaltransduksjon av HEK-293T-celler med pLenti-GFP (vBK201a) på en MOI på 0,5 (figur 3b, "no-virus" panel). SgRNA-til-GFP/Cas9 alt-i-ett vektor kassetten ble pakket inn i IDLV eller ICLV partikler og produksjonen effektivite...

Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

En protokoll for produksjon av Integrase dårlig Lentiviral vektorer for CRISPR/Cas9-mediert Gene Knockout i dele celler

Vi beskriver produksjon strategi integrase dårlig lentiviral vektorer (IDLVs) som kjøretøy for å levere CRISPR/Cas9 til cellene. Med en evne å megle rask og robust genet redigering i celler, IDLVs presenterer en tryggere og like effektivt vektor plattform for gen levering sammenlignet integrase-kompetent vektorer.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

The overall goal of this protocol is to describe the production and potential applications of optimized integrase-deficient lentiviral vectors capable of delivering CRISPR/Cas9 transgenes to cells in vivo and in vitro for rapid and efficient gene editing. CRISPR/Cas was a highly-efficient lab for gene editing manipulation. Here is a protocol outline in the production of a highly-concentrated optimized integrase-deficient lentiviral vectors for efficient delivery of CRISPR/Cas gene editing tools.The technique has proven to be very efficient in the causal cell and post-mitotic neurons. Seed the HEK-293T cells a day before the transfection so that the confluency reaches between 70 to 80%the next day. Before transfection, aspirate the old media from the culture plates and add freshly-prepared media lacking serum.In a 15-milliliter conical tube, add all the four plasmids to prepare a plasmid mix for transfecting a 15-centimeter dish. Then, add 312.5 microliters of one molar calcium chloride to the plasmid mix and adjust the volume to 1.25 milliliters with sterile double-distilled water. Keep adding drops of 2X BES buffered solution gradually while vortexing.And then, incubate the mixture at room temperature for 30 minutes. Add 2.5 milliliters of plasmid mixture in drops to each of the 15-centimeter plates. Then, gently swirl the plates and incubate at 37 degrees Celsius with 5%carbon dioxide for two to three hours.After three hours, add 2.5 milliliters of 10%serum to each plate and continue for overnight incubation. Use a 10-milliliter tissue culture pipette to carefully aspirate the supernatant from the transfected cells seeded on each culture dish. Then, pool the entire supernatant in 50-milliliter centrifuge tubes.Place the tube inside a tabletop centrifuge and start the centrifugation. Then, filter the supernatant through a 0.45-micrometer vacuum filter unit. Load the conical ultracentrifugation tubes with different sucrose concentrations to form layers with sucrose gradient.Carefully add the virus containing supernatant to the sucrose gradient. To process the entire 100-milliliter volume of viral supernatant, use six ultracentrifugation tubes in each spin. Then, balance the ultracentrifuge tubes with 1X PBS buffer and spin the samples at 70, 000 G for two hours at 17 degrees Celsius.In clean tubes, collect the 30%and 60%sucrose fractions and add cold 1X PBS buffer to adjust the volume to 100 milliliters. Mix the samples by pipetting it up and down several times. Then, add 20 to 25 milliliters of the viral solution on top of four milliliters of 20%sucrose in 1X PBS solution to prepare the sucrose cushion.Adjust the volume with sterile 1X PBS buffer if the tubes are less than 3/4 full and balance it. Then, spin the samples for two hours at 17 degrees Celsius. Then, pour the supernatant off and drain the remaining liquid by inverting the tubes on paper towels.After aspirating the last remaining droplets, a pellet containing the virus is barely visible as a translucent spot. Next, resuspend the pellet in 70 microliters of 1X PBS by thoroughly pipetting the suspension. Again, transfer the suspension to another tube and thoroughly mix to completely dissolve the pellet.Then, rinse the tube with an additional 50 microliters of cold 1X PBS buffer and mix. Keep the samples on a tabletop microcentrifuge and centrifuge at 10, 000 G for 30 seconds. After centrifugation, add 10 microliter aliquots of the supernatant to each fresh microfuge tube and store them at negative 80 degrees Celsius.Coat a high-binding 96-well plate with 100 microliters of monoclonal anti-p24 antibody at a dilution of one to 1, 500 according to NIH AIDS vaccine program for HIV-1 p24 antigen capture assay kit. Incubate the plate overnight at four degrees Celsius. The next day, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times.To avoid non-specific binding, block the plate with 200 microliters of 1%bovine serum albumin for one hour at room temperature. Then, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times. Then, prepare the concentrated and non-concentrated vector samples to a final concentration of 10%by diluting the vector with double-distilled water and Triton X-100.Next, prepare the HIV-1 standards using two-fold serial dilution starting at five nanograms per milliliter. After diluting the concentrated and non-concentrated vector samples, pipette it on the plate in triplicates and incubate at four degrees Celsius overnight. The next day, wash the plate, then add 100 microliters of polyclonal rabbit anti-p24 antibody on each plate and incubate at 37 degrees Celsius for four hours.After six washes, incubate the plate with goat anti-rabbit horseradish peroxidase at 37 degrees Celsius for one hour. After multiple washing, incubate the plate with TMB peroxidase substrate at room temperature for 15 minutes. Add one normal hydrochloric acid to stop the reaction.And read the absorbance at 450 nanometers with an absorbance plate reader. Seed the cells in a six-well plate. Next day, when the cells reach 90%confluency, transduce with purified virus at a predetermined multiplicity of infection ranging from one to 10.Then, incubate the plate at 37 degrees Celsius with 5%carbon dioxide. Monitor the plates to record difference in the GFP signals at regular intervals for one to seven days. Look for GFP positive cells under fluorescent microscope fitted with GFP filter set with excitation and emission wavelengths at 470 and 525 nanometers respectively.To differentiate between the population of GFP positive and negative cells, use untransduced cells. A representative agarose gel showing the positive mutated integrase clone. The double-digested clone with restriction enzymes EcoRV and SPH1 in red-dashed box represents the mutated integrase clone.The clone was further sequenced to check the mutation of aspartic acid at position 64 to glutamic acid. A representative bar graph for p24 ELISA assay has been shown depicting the viral titers for SP1-integrase-deficient lentiviral vector CRISPR/Cas9 plotted in the black bar and SP1-integration-competent lentiviral vectors CRISPR/Cas9 plotted in the white bar. The bar graph shows integrase-deficient and competent viral titers in the range of one times 10 to the 10.The bar plots represent the copy number of viral particles per milliliter. Representative fluorescent images showing the mean fluorescent intensity of GFP positive cells under viral treatment. The cells were transduced with integration-competent lentiviral vectors CRISPR or integrase-deficient lentiviral vector CRISPR viral components.A sharp reduction in GFP signal was observed at multiplicity of infection one and five respectively after transducing the integrase-competent or deficient lentiviral components with respect to the untransduced control cells. The main advantage of this technique stem from the ability of integrase-deficient lentiviral vectors to a deliver genome editing tool transiently which significantly enhance its specificity and the safety. If properly conducted, the protocols should take one week to complete.Conducting this protocol, one should expect to generate highly-concentrated lentiviral vectors in the range of 10 to the nine, 10 to the 10 viral genome parameter.

Research

JoVE Journal

Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Ved hjelp av en Fluorescent PCR-kapillar-gelelektroforese teknikken for å genotype CRISPR / Cas9-mediert knockout-mutanter i en high-throughput-format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Utvalgsavhengig og uavhengig generasjon av CRISPR / Cas9-medierte Gene Knockouts i mammale celler

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

En universell protokoll for store gRNA biblioteket produksjon fra en DNA-kilde

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediert målrettet integrering i Vivo med en homologi-mediert slutten begynte-basert strategi

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Effektiv produksjon og identifikasjon av CRISPR/Cas9-generert Gene Knockouts i modellsystem Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Production and Purification of Baculovirus for Gene Therapy Application

Produksjon og rensing av Baculovirus for genterapi program

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Svært effektiv Gene avbrudd i Murine og menneskelig blodkreft stamfar celler av CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Lentiviral formidlet produksjon av transgene mus: en enkel og svært effektiv metode for direkte av grunnleggerne

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Høy gjennomstrømming identifikasjon av regulatoriske gensekvenser med neste generasjons sekvensering av runde kromosom konformasjon fange (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

In vivo anvendelsen av fjernkontroll systemet for manipulering av endogene genuttrykk

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...