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

We thank Xiao Xiao at the University of North Carolina at Chapel Hill for providing us with the pEMBL-CMV-GFP plasmid. We thank Christopher Cheng and Samuel J. Huang for critical reading of the manuscript. This work was supported by Public Health Service grants (NIH R01 NS088399 and T32 EY232113).

NOTE: Recipes for the solutions and buffers needed for this protocol are provided in Table 1. The protocol described below is for the VP-AAP cross-complementation dot blot assay to study the roles of the AAP proteins in capsid assembly. The method for the more generic quantitative dot blot assay for purified AAV vector titration is explained in the Representative Results section.
1. Construction of VP3, AAP, and AAV2 Rep Expressing Plasmids
<ol>
	<li><strong...

<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>

In this report, the utility of quantitative dot blot assays to study AAV AAPs and their role in capsid assembly is described. Knowledge gained from these studies can provide detailed insights into the innate differences in the process of AAV capsid assembly and the functional role of AAPs between different serotypes. In this respect, the AAV VP3-AAP cross-complementation dot blot assay revealed that Snake AAV VP3 displayed a strict dependency on the co-expression of its cognate AAP for capsid assembly and that Snake AAP ...

Adeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA virus with a genome of approximately 4.7 kilobases (kb). The AAV genome contains open-reading frames (ORFs) for the rep and cap genes. In 2010, a previously unidentified nonstructural protein encoded by a +1 frame-shifted ORF within the AAV2 cap gene was discovered by Sonntag et al. and found to play a critical role in the assembly of AAV2 capsid VP monomer proteins into a viral capsid1. This novel protein has been named assembly-activating protein (AAP) after the role it plays in promoting capsid assembly1.
The ORFs for AAP have been identified bioinformatically in the genomes of all parvoviruses within the genus Dependoparvovirus, but not within the genomes of viruses of different genera of the parvovirus family1,2. Functional studies of this novel protein were initially focused on the AAP from the prototype AAV2 (AAP2), which has established the essential role of AAP2 in targeting unassembled VP proteins to the nucleolus for their accumulation and formation into fully assembled capsids1,3,4. The inability of the AAV2 capsids to assemble in the absence of AAP expression has been independently confirmed by multiple groups, including ours1,2,3,4,5. Subsequent studies on AAV serotypes 1, 8, and 9 corroborated the critical role of AAPs in capsid assembly, as VP3 monomer proteins of AAV1, 8, and 9 were unable to form a fully assembled capsid in the absence of co-expression of AAP2.
Recently, through approaches that include the use of quantitative dot blot assays, we investigated the ability of AAV1 to 12 VP3 monomers to assemble into capsids in the absence of AAP expression and the ability of AAP1 to 12 to promote assembly of VP3 monomers from heterologous serotypes. This study has revealed that AAV4, 5, and 11 VP3 monomers can assemble without AAP. Additionally, it was found that eight out of the twelve AAP serotypes we examined (i.e., all but AAP4, 5, 11, and 12) displayed a broad ability to support capsid assembly of heterologous AAV serotype capsids, while AAP4, 5, 11 and 12 displayed a substantially limited ability in this regard6. These four serotypes are phylogenetically distant from the other AAP serotypes2,3. Moreover, the study has uncovered significant heterogeneity in subcellular localizations of different AAPs6. Furthermore, the study has suggested that the tight association of AAP with assembled capsids and the nucleolus, the hallmark of AAV2 capsid assembly, cannot necessarily be extended to other serotypes including AAV5, 8, and 9, which display nucleolar exclusion of assembled capsids6. Thus, the information gained from the study of any particular serotype AAP is not broadly applicable to all AAP biology. Such puzzling nature of AAP biology underscores the need to investigate the role and function of each AAP from both canonical and non-canonical AAV serotypes.
The biological role of AAP in capsid assembly can be assessed by determining the fully-packaged AAV viral particle titers produced in human embryonic kidney (HEK) 293 cells, the most commonly used cell line for AAV vector production, with or without AAP protein expression. The standard methods for AAV quantitation are quantitative PCR (qPCR)-based assays7,8 and quantitative dot blot-based assays9. Other methods for AAV viral particle quantitation such as enzyme-linked immunosorbent assay10,11 or optical density measurement12 are not ideal for samples derived from many different AAV serotypes or samples contaminated with impurities (crude lysates or culture media), which are often the samples used for AAV research. Currently, qPCR is most widely used for AAV quantitation; however, it is necessary to acknowledge potential caveats of the qPCR-based assay, as the assay can result in systemic errors and significant titer variations13,14. PCR-based assays are affected by a number of potentially confounding factors, such as the presence of covalently closed terminal hairpins in PCR templates that inhibit amplification13. Even an experienced individual can introduce potential confounding factors into a qPCR-based assay unknowingly13. In contrast, quantitative dot blot assays are a classical molecular biology technique that does not involve genome amplification and uses a much simpler principle with a minimal risk of errors as compared to qPCR-based assays. The method is less technically challenging; therefore, the assay results are reasonably reproducible even by inexperienced individuals.
In this report, we describe the methodological details of a quantitative dot blot assay we routinely use for AAV vector quantitation and provide an example of how to apply the assay to study the assembly-promoting role of AAPs in common serotypes (AAV5 and AAV9) and a previously uncharacterized AAP from Snake AAV14. In nature, AAV VP proteins and AAP proteins are expressed in cis from a single gene (i.e., VP-AAP cis-complementation), while in the assay described here, VP and AAP proteins are supplied in trans from two separate plasmids (i.e., VP-AAP trans-complementation). Since each VP or AAP protein from different serotypes can be expressed from each independent plasmid, it becomes possible to test heterologous VP-AAP combinations for capsid assembly (i.e., VP-AAP cross-complementation). Briefly, AAV VP3 from various serotypes is expressed in HEK 293 cells by plasmid DNA transfection to package an AAV vector genome in the presence or absence of the cognate serotype AAP, or in the presence of a heterologous serotype AAP. Following production, culture media and cell lysates are subjected to a dot blot assay to quantify the viral genome within the capsid shell. The first step of the dot blot assay is to treat samples with a nuclease to remove contaminating plasmid DNAs and unpackaged AAV genomes in samples. Failure to do so would increase the background signals in particular when unpurified samples are assayed. This is then followed by a protease treatment to break viral capsids and release nuclease-resistant viral genomes into sample solutions. Next, viral genomes are denatured, blotted on a membrane, and hybridized with a viral genome-specific DNA probe for quantitation. In the example assay reported here, we demonstrate that Snake AAV VP3 requires Snake AAP for capsid assembly and that Snake AAP does not promote the assembly of AAV9 capsids unlike many of the AAPs derived from AAP-dependent serotypes that can also promote assembly of heterologous serotype capsids. Lastly, we report an important caveat to qPCR or dot blot-based AAV quantitation assays that the choice of nuclease significantly affects the assay results.

<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

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 biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

A representative result of quantitative dot blots for quantitation of purified AAV vector stocks produced on a large scale is shown in Figure 1. With this dot blot assay, the titer of a double-stranded AAV2G9-CMV-GFP vector stock was determined. The vector was purified by two rounds of cesium chloride (CsCl) density-gradient ultracentrifugation followed by dialysis as previously described20. In general, for purified AAV vector stocks, ...

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

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

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 ...

This method can determine both purified and the non-purified AV vac titers and assess the ability for the AAV AAP to promote assembly of cognate and heterologous AV serotype-capsis. The main advantage of this technique is that it is a straightforward inexpensive method that avoids the drawbacks of other AAV titration methods. Though this method has commonly been used for AAV vector quantitation, it can also be applied to address various fundamental questions about AAV biology as shown in this protocol.On day zero, after culturing HEK 293 cells to 90%confluency, prepare the transfection reagent by mixing the plasmids in 96 microliters of PBS without calcium or magnesium in sterile 1.5 milliliter micro-centrifuge tubes. As indicated in this table, the total amount of DNA is two micrograms per well. Add four microliters of PEI solution to the PBS plasmid DNA mix.The final volume is approximately 100 microliters or 5%volume of the culture medium. Vortex the tubes briefly and incubate the DNA PEI mixture at room temperature for 15 minutes. Following the incubation, briefly spin the tubes in a micro centrifuge to collect the liquid to the bottom.And add the DNA PEI mixture drop-wise to the culture medium in each well of the HEK 293 culture plate. Then gently agitate the plates and return them to a 37 degree Celsius incubator with 5%CO2. Transfection of HEK 293 cell is a critical component of this protocol for high-titer virus production.Healthy, low-passage number cells should be used and care should be taken not to disrupt the cells'monolayer. On days one and two, under an inverted fluorescence microscope, observe cells transfected with the PCMVGFP plasmid to assess the transfection efficiency. On day five, pipette the cells and virus-containing medium up and down or use a cell scraper to scrape all the cells.Then, transfer the suspension into 15 milliliter polypropylene conical tubes. Store the samples at minus 80 degree Celsius until use. To recover the viral particles, quickly thaw the frozen tubes in a 37 degree Celsius water bath.Then, vortex the tubes vigorously for one minute to maximize the recovery of AAV from the cells. Pellet the cell debris by centrifugation at 3, 700 times G and four degrees Celsius for 10 minutes. Then transfer 200 microliters of the supernatant from each tube into labeled micro-centrifuge tubes with screw caps for the dot blot assay.To treat the samples with Serratia marcescens endonuclease, prepare mix A and mix B reagents. Add 10 microliters of each reagent to each sample tube. Vortex the samples for five seconds and briefly spin them to collect the liquid to the bottom of the tubes.Then incubate the tubes at 37 degrees Celsius for at least one hour. After briefly spinning the samples, carry out proteinase K treatment by preparing mix C reagent and adding 180 microliters to each tube. Then after vortexing and briefly spinning the tubes again, incubate them at 55 degrees Celsius for one hour.Once the samples have returned to room temperature, add 200 microliters of phenol-chloroform and vortex the tubes for one minute. Then spin the samples in a micro-centrifuge at 16, 100 times G and room temperature for at least five minutes. Transfer 320 microliters of the aqueous layer or 80%of the aqueous fluid volume to a new standard micro-centrifuge tube.This transfer step is critically important as the same volume must be taken from the sample to maintain accuracy for AAV quantitation. Care should be taken to avoid the organic phase while removing the aqueous layer. Prepare the mix D reagent and add 833 microliters to each sample tube.After vortexing the tubes, incubate them at minus 80 degrees Celsius for at least 20 minutes. Following centrifugation, pour off supernatant and blot the tubes once on a clean paper towel. Pipette approximately 500 microliters of 70%ethanol into each tube and vortex the tubes for five seconds.After spinning the samples again, pour off the supernatant and blot the tubes once on a clean paper towel. Then dry the pellets at 65 degrees Celsius before resuspending them in TE buffer. To carry out the dot blot assay, prepare plasmid DNA standards by diluting AAV vectored genome plasmid DNA to 10 nanograms per microliter in water or Tris-HCL buffer.Transfer a 25 microliter aliquot of the diluted plasmid DNA to a fresh tube and linearize it with an appropriate restriction enzyme in a 50 microliter reaction volume for one hour. Add 450 microliters of water or TE to the tube containing the digested plasmid DNA standard and mix thoroughly. Then transfer 70 microliters of this mixture to a new 1.5 milliliter micro-centrifuge tube with 1, 330 microliters of water or TE to make a diluted plasmid standard.To set up the dot blot apparatus, using scissors, cut the blotting membrane to an appropriate size for the number of samples and standards. Soak the membrane with water for 10 minutes before placing it on the dot blot apparatus. Then, cover the unused wells.Add water to the wells to which the samples will be loaded. Apply a vacuum, pull the water through the wells, check for errors, and then retighten the screws. Retest if necessary.Apply 400 microliters of each diluted plasmid DNA standard to each well and run four lanes of standard dilutions. Use two separate aliquots of the standard digest and load each in duplicate. Then apply 200 microliters per well of each viral DNA sample.Apply a gentle vacuum to pull the DNA solutions through the wells. Then once all the wells have emptied, release the vacuum by adjusting the three-way valve. Add 400 microliters of one X alkaline solution to each well.Then wait for five minutes before reapplying the vacuum to empty the wells. Reapply the vacuum and disassemble the dot blot apparatus. Then remove the membrane and rinse it with two X SSC.Place the membrane on a clean paper towel with the DNA-bound side facing up to remove the excess buffer. Use an appropriate UV crosslinker to UV crosslink the blotted DNA to the membrane. The membrane is now ready for hybridization.Place the membrane in a hybridization bottle with the DNA-bound side up. Rinse the membrane with five to 10 milliliters of pre-warmed hybridization buffer and discard the buffer. Then add 10 milliliters of pre-warmed hybridization buffer and place the bottle in a rotating hybridization oven set to 65 degrees Celsius.Rotate the bottle for at least five minutes. Quickly add the denatured sperm DNA and radioactive probe to the hybridization buffer in the bottle and shake it for 10 seconds to mix. Return the bottle to the 65 degree Celsius oven and incubate it with rotation at 65 degrees Celsius for at least four hours.Once hybridization is complete, stop the rotation. Remove the hybridization bottle and then pour the radioactive probe solution into a 50 milliliter conical tube with a leakproof plug seal cap. To wash the membrane, add 20 to 30 microliters of pre-warmed wash buffer to the hybridization bottle and rotate it for five minutes.Repeat this wash two more times. After the third wash, remove the membrane from the hybridization bottle, and with paper towels, remove the excess buffer from the membrane. Then place the membrane in a clear plastic paper holder.With a Geiger counter, check the radioactive signals on the membrane. After exposing the erased phosphor-imaging screen to the membrane, scan the screen using a phosphor-image scanning system and obtain the data on signal intensity of each dot. Carry out data analysis according to the text protocol.This quantitative dot blot for AAV 2G9 vector titration has two sets of standards and three different amounts of an AAV vector preparation, including 0.3, 0.1, and 0.03 microliters in duplicate. The amounts to be assayed on the blot are determined empirically or based on a preliminary assay. It was determined by interpolation that 0.3, 0.1, and 0.03 microliter aliquots of the AAV 2G9 vector had on average a titer of 5.16, 0.27 nanograms equivalent microliters.All possible cognate and heterologous serotype combinations of VP3 cas-pid and AAP proteins from AAV5, AAV9, and snake AAV were assessed for capsid assembly by a quantitative dot blot assay. The results of this graph show that AAV5 VP3 assembles regardless of whether AAP was provided in trans. In addition, AAP5 and AAP9 can promote AAV9 VP3 capsid assembly, although AAP5 functions less effectively than AAP9.Neither AAP5 nor AAP9 exhibits an assembly promoting activity on snake AAV VP3, and snake AAP only promotes assembly of snake AAV VP3. A dot blot analysis was applied to asses nuclease activity of three commercially available nucleases under non-optimized conditions containing cell and culture medium-derived impurities. Enzyme A was found not as active as the other two enzymes, showing that the choice of nuclease could significantly affect the readout of the dot blot assay.This technique is straightforward for AAV vector quantitation and instrumental in assessing the role of AAP in promoting assembly of AAV capsids derived from cognate and heterologous serotypes. Once mastered, this technique can be done in a day if it performed properly once assay samples are obtained in cell culture experiments. While attempting this procedure, it is important to remember to carefully pipette the correct liquid volume to ensure quantitative accuracy.After its development, the application of the quantitative dot blot assay to study AAP have paved the way for researchers in the field of AAV biology to further understand the mechanism of AAV capsid assembly. Please do not forget that working with human cells, viral agents, phenol-chloroform, and radioactive materials can be extremely hazardous. And the use of lab coats, gloves, and goggles are needed to limit exposure.These materials should only be handled in approved areas. After watching this video, you should have a good understanding on how to prepare dot blot samples for AAV quantitation, set up dot blot apparatus, determine AAV titer, and apply the procedure to the study of AAV biology. Following this procedure, other assays like AAV vector transduction assays in culture cells can be performed to answer additional questions, such as the functional titer of vectorized compared to the physical particle titer determined by dot blot assay.

a-quantitative-dot-blot-assay-for-aav-titration-its-use-for

Research

JoVE Journal

Biology

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 elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

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. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

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 lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in 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/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

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

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 analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

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

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

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

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

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 highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

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 ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...