Name: a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno-associated virus assembly-activating proteins
Uploaded: 2018-06-12T13:00:00
Description: Watch this Scientific Journal Video about A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins at JoVE.com

Vi takker Xiao Xiao på University of North Carolina i Chapel Hill for at forsyne os med pEMBL-CMV-NGL plasmid. Vi takke Christopher Cheng og Samuel J. Huang for kritisk læsning af manuskriptet. Dette arbejde blev støttet af Public Health Service tilskud (NIH R01 NS088399 og T32 EY232113).

Bemærk: Opskrifter til løsninger og buffere behov for denne protokol er fastsat i tabel 1. Protokollen beskrevet nedenfor er for VP-AAP cross-komplementering dot skamplet analysen at studere roller af AAP proteiner i kapsid forsamling. Metoden til at mere generiske kvantitative dot skamplet assay for renset AAV vektor titrering er forklaret i afsnittet Repræsentant resultater .
1. opførelse af VP3, AAP og AAV2 Rep udtrykker plasmider
<ol><li>
Opf...

<ol>
	<li>Sonntag, F., Schmidt, K., Kleinschmidt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+assembly+factor+promotes+AAV2+capsid+formation+in+the+nucleolus.">A viral assembly factor promotes AAV2 capsid formation in the nucleolus.</a> Proc Natl Acad Sci U S A. 107 (22), 10220-10225 (2010).</li><li>Sonntag, F., 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+assembly-activating+protein+promotes+capsid+assembly+of+different+adeno-associated+virus+serotypes.">The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes.</a> J Virol. 85 (23), 12686-12697 (2011).</li><li>Naumer, M., 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=Properties+of+the+adeno-associated+virus+assembly-activating+protein.">Properties of the adeno-associated virus assembly-activating protein.</a> J Virol. 86 (23), 13038-13048 (2012).</li><li>Earley, L. F., 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=Identification+and+characterization+of+nuclear+and+nucleolar+localization+signals+in+the+adeno-associated+virus+serotype+2+assembly-activating+protein.">Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein.</a> J Virol. 89 (6), 3038-3048 (2015).</li><li>Kawano, Y., Neeley, S., Adachi, K., Nakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+and+computational+evolution-based+method+to+study+a+mode+of+co-evolution+of+overlapping+open+reading+frames+in+the+AAV2+viral+genome.">An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome.</a> PLoS One. 8 (6), e66211 (2013).</li><li>Earley, L. F., 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=Adeno-associated+Virus+(AAV)+Assembly-Activating+Protein+Is+Not+an+Essential+Requirement+for+Capsid+Assembly+of+AAV+Serotypes+4,+5,+and+11.">Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.</a> J Virol. 91 (3), (2017).</li><li>Aurnhammer, C., 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=Universal+real-time+PCR+for+the+detection+and+quantification+of+adeno-associated+virus+serotype+2-derived+inverted+terminal+repeat+sequences.">Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.</a> Hum Gene Ther Methods. 23 (1), 18-28 (2012).</li><li>Clark, K. R., Liu, X., McGrath, J. P., Johnson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+recombinant+adeno-associated+virus+vectors+are+biologically+active+and+free+of+detectable+helper+and+wild-type+viruses.">Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.</a> Hum Gene Ther. 10 (6), 1031-1039 (1999).</li><li>Samulski, R. J., Chang, L. S., Shenk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-free+stocks+of+recombinant+adeno-associated+viruses:+normal+integration+does+not+require+viral+gene+expression.">Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.</a> J Virol. 63 (9), 3822-3828 (1989).</li><li>Wobus, C. 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=Monoclonal+antibodies+against+the+adeno-associated+virus+type+2+(AAV-2)+capsid:+epitope+mapping+and+identification+of+capsid+domains+involved+in+AAV-2-cell+interaction+and+neutralization+of+AAV-2+infection.">Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.</a> J Virol. 74 (19), 9281-9293 (2000).</li><li>Grimm, D., 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=Titration+of+AAV-2+particles+via+a+novel+capsid+ELISA:+packaging+of+genomes+can+limit+production+of+recombinant+AAV-2.">Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2.</a> Gene Ther. 6 (7), 1322-1330 (1999).</li><li>Sommer, J. M., 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=Quantification+of+adeno-associated+virus+particles+and+empty+capsids+by+optical+density+measurement.">Quantification of adeno-associated virus particles and empty capsids by optical density measurement.</a> Mol Ther. 7 (1), 122-128 (2003).</li><li>Fagone, P., 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=Systemic+errors+in+quantitative+polymerase+chain+reaction+titration+of+self-complementary+adeno-associated+viral+vectors+and+improved+alternative+methods.">Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods.</a> Hum Gene Ther Methods. 23 (1), 1-7 (2012).</li><li>Farkas, S. 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=A+parvovirus+isolated+from+royal+python+(Python+regius)+is+a+member+of+the+genus+Dependovirus.">A parvovirus isolated from royal python (Python regius) is a member of the genus Dependovirus.</a> J Gen Virol. 85 (Pt 3), 555-561 (2004).</li><li>NEB, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2017-2018 Catalog &amp; Technical Reference. , 302-368 (2017).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2012).</li><li>. Bacterial Transformation Available from: <a target="_blank" href="https://www.addgene.org/protocols/bacterial-transformation/">https://www.addgene.org/protocols/bacterial-transformation/</a> (2017)</li><li>. Sequence Analysis of your Addgene Plasmid Available from: <a target="_blank" href="https://www.addgene.org/recipient-instructions/sequence-analysis/">https://www.addgene.org/recipient-instructions/sequence-analysis/</a> (2017)</li><li>Burton, M., 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=Coexpression+of+factor+VIII+heavy+and+light+chain+adeno-associated+viral+vectors+produces+biologically+active+protein.">Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.</a> Proc Natl Acad Sci U S A. 96 (22), 12725-12730 (1999).</li><li>Grimm, D., Pandey, K., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vectors+for+short+hairpin+RNA+expression.">Adeno-associated virus vectors for short hairpin RNA expression.</a> Methods Enzymol. 392, 381-405 (2005).</li><li>Leonard, J. N., Ferstl, P., Delgado, A., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+preparation+of+adeno-associated+viral+vectors+by+using+high+hydrostatic+pressure+to+selectively+inactivate+helper+adenovirus.">Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus.</a> Biotechnol Bioeng. 97 (5), 1170-1179 (2007).</li><li>Lock, M., Alvira, M. R., Chen, S. J., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+determination+of+single-stranded+and+self-complementary+adeno-associated+viral+vector+genome+titers+by+droplet+digital+PCR.">Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.</a> Hum Gene Ther Methods. 25 (2), 115-125 (2014).</li><li>Werling, N. J., Satkunanathan, S., Thorpe, R., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Comparison+and+Validation+of+Quantitative+Real-Time+PCR+Methods+for+the+Quantitation+of+Adeno-Associated+Viral+Products.">Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products.</a> Hum Gene Ther Methods. 26 (3), 82-92 (2015).</li><li>Kafatos, F. C., Jones, C. W., Efstratiadis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+nucleic+acid+sequence+homologies+and+relative+concentrations+by+a+dot+hybridization+procedure.">Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure.</a> Nucleic Acids Res. 7 (6), 1541-1552 (1979).</li><li>Reue, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+quantitation+techniques:+considerations+for+experimental+design+and+application.">mRNA quantitation techniques: considerations for experimental design and application.</a> J Nutr. 128 (11), 2038-2044 (1998).</li><li>Clementi, M., 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=Quantitative+PCR+and+RT-PCR+in+virology.">Quantitative PCR and RT-PCR in virology.</a> PCR Methods Appl. 2 (3), 191-196 (1993).</li><li>Mezzina, M., Merten, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viruses.">Adeno-associated viruses.</a> Methods Mol Biol. 737, 211-234 (2011).</li><li>Chen, W. J., Liao, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+bovine+pancreatic+deoxyribonuclease+I.">Structure and function of bovine pancreatic deoxyribonuclease I.</a> Protein Pept Lett. 13 (5), 447-453 (2006).</li></ol>

I denne betænkning, er nytten af kvantitative dot skamplet assays til at studere AAV AAPs og deres rolle i kapsid forsamling beskrevet. Viden fra disse undersøgelser kan give detaljeret indsigt i de medfødte forskelle i AAV kapsid forsamling og AAPs funktionelle rolle mellem forskellige serotyper. I denne henseende AAV VP3-AAP cross-komplementering dot duppes assay afsløret, at slangen AAV VP3 vises en streng afhængighed på fælles udtryk for dens beslægtet AAP for kapsid forsamling og slange AAP ikke fremmer kaps...

Adeno-associeret virus (AAV) er en lille, ikke-kappebærende, enkeltstrenget DNA virus med en genom af ca 4,7 kilobases (kb). AAV genom indeholder åbne læserammer (ORFs) for rep og cap generne. I 2010, blev en tidligere uidentificerede nonstructural protein kodet af et + 1 frame-skiftet ORF i AAV2 cap genet opdaget af Sonntag et al. og fundet for at spille en afgørende rolle i forsamlingen af AAV2 kapsid VP monomer proteiner i en viral kapsid1. Denne roman protein er blevet udnævnt forsamling-aktivering protein (AAP) efter den rolle, det spiller i fremme af kapsid forsamling1.
ORFs for AAP har været identificeret bioinformatically i genomer af alle parvoviruses inden for slægten Dependoparvovirus, men ikke i genomer af vira af forskellige slægter af parvovirus familie1,2. Funktionelle studier af denne roman protein var oprindeligt fokuseret på AAP fra prototype AAV2 (AAP2), som har etableret AAP2 afgørende rolle i at målrette usamlet VP proteiner til nucleolus for deres ophobning og dannelse i fuldt samlet capsids1,3,4. AAV2 capsids manglende evne til at samle i mangel af AAP udtryk har været uafhængigt bekræftet af flere grupper, herunder vores1,2,3,4,5 . Efterfølgende studier på AAV serotyper 1, 8 og 9 praktikanttid AAPs kritiske rolle i kapsid forsamling, som VP3 monomer proteiner af AAV1, 8 og 9 ikke var i stand til at danne en færdigsamlet kapsid i mangel af fælles udtryk af AAP2.
For nylig, gennem strategier, der omfatter anvendelse af kvantitative dot duppes assays, vi undersøgt AAV1 evne til 12 VP3 monomerer til at samle ind i capsids i mangel af AAP udtryk og mulighed for AAP1 til 12 for at fremme montering af VP3 monomerer fra heterolog serotyper. Denne undersøgelse har vist, at AAV4, 5 og 11 VP3 monomerer kan samle uden AAP. Desuden blev det konstateret, at otte ud af de tolv AAP serotyper vi undersøgt (dvs.alle undtagen AAP4, 5, 11 og 12) vises en bred evnen til at understøtte kapsid forsamling af heterolog AAV serotype capsids, mens AAP4, 5, 11 og 12 vises et væsentligt begrænsede evne i denne henseende6. Disse fire serotyper er Fylogenetisk fjernt fra de andre AAP serotyper2,3. Derudover har undersøgelsen afdækket signifikant heterogenitet i subcellulært lokaliseringer af forskellige AAPs6. Desuden undersøgelsen har foreslået, at den stramme sammenslutning af AAP med samlet capsids og nucleolus, kendetegnende for AAV2 kapsid forsamling, nødvendigvis ikke kan udvides til andre serotyper, herunder AAV5, 8 og 9, som viser nucleolar udelukkelse af samlet capsids6. De oplysninger fra studiet af nogen bestemt serotype AAP er således, ikke generelt gælder for alle AAP biologi. Så gådefuldt karakter af AAP biologi understreger behovet for at undersøge den rolle og funktion af hver AAP fra canonical og ikke-kanoniske AAV serotyper.
AAP biologiske rolle i kapsid forsamling kan vurderes ved at bestemme fuldt pakket AAV viral partikel titers produceret i menneskelige embryonale nyre (HEK) 293 celler, de mest almindeligt anvendte cellelinie for AAV vektor produktion, med eller uden AAP protein udtryk. Standardmetoder for AAV kvantitering er kvantitativ PCR (qPCR)-baseret assays7,8 og kvantitative dot skamplet-baserede undersøgelser vedrørende9. Andre metoder til AAV viral partikel kvantitering som enzymmaerket assay10,11 eller optisk tæthed måling12 er ikke ideel til prøverne stammer fra mange forskellige AAV serotyper eller prøver forurenet med urenheder (rå lysates eller kultur medier), som ofte prøver anvendes til AAV forskning. I øjeblikket, er qPCR mest udbredt for AAV kvantitering; Det er imidlertid nødvendigt at anerkende potentielle forbehold af qPCR-baseret analysen, som analysen kan resultere i systemisk fejl og væsentlig titer variationer13,14. PCR-baserede analyser er påvirket af en række potentielt forstyrrende faktorer, såsom tilstedeværelsen af kovalent lukkede terminal Hårnåle i PCR-skabeloner, der hæmmer forstærkning13. Selv en erfaren person kan indføre potentiale konfunderende faktorer i en qPCR-baserede assay ubevidst13. Derimod er kvantitative dot skamplet assays en klassisk Molekylærbiologi teknik, der involverer ikke genom forstærkning og bruger en meget enklere princippet med en minimal risiko for fejl i forhold til qPCR-baserede assays. Metoden er mindre teknisk udfordrende; Derfor er assay resultater rimeligt reproducerbare selv af uerfarne personer.
I denne betænkning beskriver vi de metodologiske oplysninger om en kvantitativ dot skamplet assay vi rutinemæssigt brug for AAV vektor kvantitering og Giv et eksempel på, hvordan at anvende assay for at studere rollen forsamling-fremme af AAPs fælles serotyperne (AAV5 og AAV9) og et tidligere uncharacterized AAP fra Snake AAV14. I naturen, AAV VP proteiner og AAP proteiner er udtrykt i cis fra et enkelt gen (dvs., VP-AAP cis-komplementering), mens i analysen beskrives her, VP og AAP proteiner leveres i trans fra to separate plasmider (dvs., VP-AAP Trans-komplementering). Da hver VP eller AAP protein fra forskellige serotyper kan udtrykkes fra hver uafhængig plasmid, bliver det muligt at teste heterolog VP-AAP kombinationer for kapsid Forsamling (dvs., VP-AAP cross-komplementering). Kort, AAV VP3 fra forskellige serotyper udtrykkes i HEK 293 celler af plasmid DNA Transfektion til at pakke en AAV vektor genom i tilstedeværelse eller fravær af den beslægtet serotype AAP, eller i nærværelse af en heterolog serotype AAP. Efter produktion, dyrkningsmedier og cellelysater bliver underkastet en dot skamplet analyse til kvantificering af det virale genom i kapsid shell. Det første trin i dot skamplet assay er at behandle prøver med en nukleasen fjerne forurenende plasmid DNAs og uemballerede AAV genomer i prøver. Undladelse af at gøre det ville øge baggrunden signaler navnlig når urenset prøver er analyseret. Dette efterfølges af en protease behandling at bryde viral capsids og frigive nukleasen-resistente viral genomer i stikprøven løsninger. Næste, viral genomer er denatureret, renset på en membran og hybridiseret med en virale genom-specifikke DNA-sonder til kvantitering. I eksempel assay rapporteret her, vise vi at slange AAV VP3 kræver slange AAP for kapsid forsamling og slange AAP ikke fremmer forsamlingen af AAV9 capsids i modsætning til mange af AAPs stammer fra AAP-afhængige serotyper, der også kan fremme samling af heterolog serotype capsids. Endelig, vi rapporterer en vigtig advarsel til qPCR eller dot skamplet-baserede AAV kvantitering-undersøgelser, at valget af nukleasen væsentligt påvirker assay resultater.

<table>
	<tbody>
		<tr>
			<td>Benzonase</td>
			<td>MilliporeSigma</td>
			<td>1016970001</td>
			<td>Referred to as Serratia marcescen endonuclease in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>Referred to as DNase I Enzyme A in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Referred to as DNase I Enzyme B in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Biolabs</td>
			<td>M0303L</td>
			<td>Referred to as DNase I Enzyme C in the main text.</td>
		</tr>
		<tr>
			<td>1.5 mL Attached O-Ring Screw Cap Microcentrifuge Tubes</td>
			<td>Corning</td>
			<td>430909</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M Sodium Acetate</td>
			<td>Teknova</td>
			<td>S0298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV-293 Cells</td>
			<td>Agilent</td>
			<td>240073</td>
			<td>HEK-293 cell line optimized for the packaging of AAV virions.</td>
		</tr>
		<tr>
			<td>AccuGENE 0.5 M EDTA Solution</td>
			<td>Lonza</td>
			<td>51234</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1 M Tris-HCl pH 8.0</td>
			<td>Lonza</td>
			<td>51238</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 10% SDS</td>
			<td>Lonza</td>
			<td>51213</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1X TE Buffer</td>
			<td>Lonza</td>
			<td>51235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot Apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MilliporeSigma</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MilliporeSigma</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Extractor Kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>295-50201</td>
			<td>This is used for quantitative dot blots on purified virus. We use only a half volume of all the reagents in each step recommended for the Protocol Scheme 2 in the manual provided by the manufacturer.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)-high glucose (4.5 g/L)</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>ElectroMAX DH10B Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>18290015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Alfa Aesar</td>
			<td>B22095</td>
			<td>Referred to as Polysucrose 400.</td>
		</tr>
		<tr>
			<td>Fluorescence&#160;Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 40 CFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td></td>
		</tr>
		<tr>
			<td>Herring Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15634-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-247-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant TL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>ImageQuant TL</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Choloride Hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroPulser Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Quick Spin DNA Columns</td>
			<td>MilliporeSigma</td>
			<td>11814419001</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-RC2 Vector</td>
			<td>Cell Biolabs Inc</td>
			<td>VPK-422</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10K/10K</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Exposure Cassette</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Pfx DNA Polymerase</td>
			<td>Invitrogen</td>
			<td>11708013</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences Inc</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>MilliporeSigma</td>
			<td>P5288</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime-It II Random Primer Labeling Kit</td>
			<td>Agilent Technologies</td>
			<td>300385</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Forward (N25 nucleotides from the 4th nucleotide in the AAP ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACCATGGACTACAAGGACGACGATGACAAA-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Forward (N25 the first 25 nucleotides of the VP3 ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACC-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K Solution</td>
			<td>Invitrogen</td>
			<td>25530-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction Enzymes</td>
			<td>New England Biolabs</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-678-11D / 13-678-11E / 13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Tribasic Dihydrate</td>
			<td>MilliporeSigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>6321000515</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture Treated 6-well Plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28955809</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Buffer-Saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-047</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Spectroline</td>
			<td>XLE-1000</td>
			<td>Optimal Crosslink mode is used, providing a UV energy dosage of 120 mJ/cm2.</td>
		</tr>
		<tr>
			<td>Zeta-Probe Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0165</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno-associated virus assembly-activating proteins

Mens adeno-associeret virus (AAV) er bredt accepteret som en attraktiv vektor for genterapi, fungerer det også som en model virus for at forstå virus biologi. I sidstnævnte henseende, har den nylige opdagelse af en ikke-strukturelle AAV protein, kaldes forsamling-aktivering protein (AAP), kaste nyt lys over de processer, der er involveret i montage af viral kapsid VP proteiner i en kapsid. Selvom mange AAV serotyper kræver AAP for forsamlingen, har vi for nylig rapporteret, at AAV4, 5, og 11 er undtagelser fra denne regel. Vi viste desuden, at AAPs og samlet capsids af forskellige serotyper lokalisere til forskellige subcellulært rum. Denne uventede heterogenitet i de biologiske egenskaber og funktionelle roller for AAPs blandt forskellige AAV serotyperne understreger betydningen af undersøgelser på AAPs stammer fra forskellige serotyper. Dette manuskript detaljer en ligetil dot skamplet assay for AAV kvantitering og dens anvendelse til at vurdere AAP afhængighed og serotype specificitet i kapsid forsamling. For at påvise nytten af denne dot skamplet assay, satte vi sig for at karakterisere kapsid forsamling og AAP afhængighed af slange AAV, en tidligere uncharacterized krybdyr AAV, samt AAV5 og AAV9, som har tidligere vist sig at være AAP-uafhængig og AAP-afhængige serotyperne, henholdsvis. Analysen viste, at slangen AAV kapsid forsamling kræver slange AAP og ikke kan fremmes ved AAPs fra AAV5 og AAV9. Analysen viste også, at i modsætning til mange af de fælles serotype AAPs, der fremmer heterolog kapsid forsamling af cross-komplementering, slange AAP ikke fremmer forsamling af AAV9 capsids. Derudover viser vi, at valget af nukleasen væsentligt påvirker udlæsning af dot skamplet assay, og således, at vælge en optimal enzym er afgørende for vellykket vurdering af AAV titers.

Et repræsentativt resultat af kvantitative dot blotting til kvantitering af renset AAV vektor bestande produceres i stor målestok er vist i figur 1. Med denne dot skamplet assay, blev titer af en dobbelt-strenget AAV2G9-CMV-NGL vektor bestand fastsat. Vektoren blev renset af to runder af caesiumchloridoploesning (CsCl) densitet-gradient ultracentrifugering efterfulgt af dialyse som tidligere beskrevet20. Generelt for renset AAV vekto...

Watch this Scientific Journal Video about A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins at JoVE.com

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

Dette manuskript detaljer en ligetil dot skamplet assay til kvantitering af adeno-associeret virus (AAV) titers og dens anvendelse til at studere rolle aktivering af forsamling proteiner (AAPs), en roman klasse af ikke-strukturelle virale proteiner, der findes i alle AAV serotyper, fremme samling af capsids stammer fra beslægtet og heterolog AAV serotyper.

En kvantitativ Dot skamplet Assay for AAV titrering og dets anvendelse til funktionel vurdering af Adeno-associeret Virus forsamling-aktivering proteiner

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Research

JoVE Journal

Biology

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

En kvantitativ analyse til Insulin-udtrykkende kolonidannende stamceller

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Anvendelse af en robot til Højkapacitetsforskning Krystallisation af membranproteiner i Lipidiske mesofaser

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

En hybrid DNA Extraction Metode til kvalitativ og kvantitativ vurdering af bakteriel Fællesskabers fra fjerkræproduktion Prøver

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

En protokol til brug af gen sæt berigelse analyse til at identificere den passende dyremodel for Translationel forskning

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Immunofluorescerende mærkning af plantevirus og insektvektorproteiner i hemipteran tarme

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...