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

我们感谢小肖在北卡罗来纳大学教堂山为我们提供了 pEMBL-CMV-GFP 质粒。我们感谢克里斯托弗和塞缪尔. 黄对手稿的批判性阅读。这项工作得到公共卫生服务补助金 (NIH R01 NS088399 和 T32 EY232113) 的支持。

注意:表 1提供了此协议所需的解决方案和缓冲区的配方。下面描述的协议是为副校长交叉互补斑点杂交试验研究的作用, 在衣壳组装的蛋白。对纯化 AAV 矢量滴定法进行了较为通用的定量斑点杂交试验, 并在代表性结果部分作了说明。
1. 构建 VP3、AAV2 和表达质粒的代表
<ol><li>
pCMV-AAVx-VP3 的构建 (x = 血清型)
	<ol>
<li>PCR-放大整个 VP3 ORF (1.6 kb)...

<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>

本文介绍了定量斑点杂交检测 AAV AAPs 的应用及其在衣壳装配中的作用。从这些研究中获得的知识可以对 AAV 衣壳组装过程中的先天差异和不同血清型间 AAPs 的功能作用提供详细的见解。在这方面, AAV VP3-AAP 交叉互补斑点杂交法揭示, 蛇 AAV VP3 显示了严格依赖于其同源的联合表达的衣壳组装和蛇, 不促进外源血清型衣壳组装.这一观察很有趣, 因为以前调查过的 AAV 血清型 (AAV1、2、3、6、7、8、9和 12) 都...

腺相关病毒 (AAV) 是一种小的、非封闭的单链 DNA 病毒, 其基因组约为 4.7 kilobases (kb)。AAV 基因组包含开放阅读框架 (码) 的代表和cap基因。在 2010年, 一个先前不明的结构性蛋白编码由 +1 帧转移的 ORF 在 AAV2帽基因中发现了报等, 并发现发挥关键作用, 在组装 AAV2 衣壳副总裁单体蛋白成病毒衣壳1。这种新的蛋白质被命名为组装活化蛋白 () 后, 它发挥作用, 促进衣壳组装1。
码 bioinformatically 在Dependoparvovirus的所有细小的基因组中被识别出来, 但不在细小病毒家族1、2的不同属的基因组中。本文首先从原型 AAV2 (AAP2) 对该蛋白的功能研究进行了初步研究, 确立了 AAP2 在靶向未组装 VP 蛋白对核仁的聚集和形成中的重要作用, 从而充分组装衣壳1,3,4。AAV2 衣壳无法在没有该表达式的情况下组装, 已由多个组独立确认, 包括我们的1、2、3、4、5.随后对 AAV 血清型1、8和9的研究证实了 AAPs 在衣壳组装中的关键作用, 因为 AAV1、8和9的 VP3 单体蛋白在没有联合表达的情况下无法形成完全组装的衣壳.
最近, 通过包括使用定量斑点杂交检测的方法, 我们研究了 AAV1 12 VP3 单体组装成衣壳的能力, 在缺乏表达和 AAP1 12 的能力, 以促进 VP3 单体组装异源血清型。这项研究表明, AAV4, 5 和 11 VP3 单体可以组装, 不用的。另外, 据发现, 我们研究的十二种血清蛋白中有八 (即除了 AAP4、5、11和 12) 显示了广泛的能力, 支持外源 AAV 血清型衣壳的衣壳组装, 而 AAP4, 5, 11 和12显示了极大的有限的能力在这方面6。这四种血清型是 phylogenetically 远离其他的儿科血清2,3。此外, 该研究发现不同 AAPs6的亚细胞定位有显著的异质性。此外, 研究还表明, 与组装的衣壳和核仁, AAV2 衣壳组装的标志紧密关联, 不一定可以扩展到其他血清型包括 AAV5, 8 和 9, 这显示核仁排除组装衣壳6。因此, 从研究任何特定的血清学杂志获得的信息并不广泛适用于所有的儿科生物学。此类令人费解的生物学特性突显出, 需要从规范和非规范的 AAV 血清型中研究每个标准的角色和功能。
通过测定人胚肾 (HEK) 293 细胞中所产生的完全包装的 AAV 病毒微粒滴度, 可以评估在衣壳组装中的生物作用, 其中最常用的细胞系为 AAV 向量生产, 有或没有蛋白表达。标准的 AAV 定量方法是定量 PCR (qPCR) 为基础的化验7,8和定量斑点杂交为基础的化验9。其他 AAV 病毒微粒定量的方法, 如酶联免疫吸附试验10,11或光学密度测量12不理想的样本来自许多不同的 AAV 血清学或样品被杂质污染 (粗裂解物或培养基), 通常是用于 AAV 研究的样品。目前, qPCR 被广泛应用于 AAV 定量;然而, 有必要承认 qPCR 的检测可能的警告, 因为该检测可能导致系统性错误和显著的效价变化13,14。基于 pcr 的化验受到一些潜在的混杂因素的影响, 例如在 pcr 模板中存在共价键闭合终端发夹, 抑制放大13。即使有经验的个体也可以在不知不觉的13中引入潜在的混杂因素到 qPCR 的检测中。相比之下, 定量斑点杂交检测是一种经典的分子生物学技术, 不涉及基因组扩增, 并使用一个简单的原理, 与 qPCR 的检测相比, 错误的风险最小。该方法在技术上缺乏挑战性;因此, 即使没有经验的人也能合理地重现化验结果。
在本报告中, 我们描述了一个定量斑点杂交检测的方法学细节, 我们经常使用 AAV 向量定量, 并提供了一个例子, 如何应用该方法来研究 AAPs 在共同的血清学 (AAV5 和 AAV9) 的组装促进作用和以前 uncharacterized 从蛇 AAV14。在自然界中, AAV vp 蛋白和蛋白的表达在 cis 从一个单一的基因 (即, 副总裁的 cis 互补), 而在这里描述的化验, vp 和儿科蛋白是从两个独立的质粒提供反相 (即, vp跨互补)。由于来自不同血清型的每种 vp 或蛋白都可以从每个独立的质粒中表达出来, 因此就有可能测试外源副总裁--在衣壳装配上的组合 (即, 副校长交叉互补)。简单地, AAV VP3 从不同的血清学表达在 HEK 293 细胞通过质粒 DNA 转染包裹一个 AAV 向量基因组在存在或没有同源血清学, 或在存在的异种血清学。继生产后, 培养基和细胞裂解物受到斑点印迹检测, 以量化在衣壳内的病毒基因组。斑点杂交法的第一步是用核酸酶处理样品, 去除污染的质粒 dna 和无包装的 AAV 基因组。如果不这样做, 将增加背景信号, 特别是当粗样品被检测。接下来是蛋白酶治疗, 以打破病毒衣壳和释放核酸酶病毒基因组的样本解决方案。其次, 病毒基因组变性, 在膜上涂抹, 并与病毒基因组特异的 DNA 探针杂交进行定量。在这里报告的例子化验中, 我们证明蛇 AAV VP3 需要蛇的电泳为衣壳装配, 并且蛇的附属程序不促进 AAV9 衣壳的汇编, 不同于由依赖于异源血清衣壳。最后, 我们报告了一个重要的告诫, qPCR 或斑点印迹为基础的 AAV 定量分析的选择核酸酶显着影响的化验结果。

<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

虽然腺相关病毒 (AAV) 被广泛接受为一种有吸引力的基因治疗载体, 但它也作为一种了解病毒生物学的模型病毒。在后者方面, 最近发现一种非结构化的 AAV 蛋白 (称为组装活化蛋白), 已经为病毒衣壳副总裁蛋白质组装过程中所涉及的进程注入了新的光。虽然许多 AAV 血清型需要用于程序集, 我们最近报告说, AAV4, 5 和11是这个规则的例外。此外, 我们还证明了不同血清型的 AAPs 和组装衣壳不同的亚细胞间隔。这种意想不到的异质性的生物学性质和功能作用的 AAPs 之间的不同 AAV 血清学强调了研究的重要性, AAPs 来自不同的血清型。本手稿详细介绍了 AAV 定量的直接斑点印迹法及其在衣壳体组装中对其依赖性和血清型特异性的评估。为了证明这种斑点杂交化验的效用, 我们着手描述了蛇 AAV, 以前的 uncharacterized 爬行动物 AAV, 以及 AAV5 和 AAV9 的衣壳组装和儿科的依赖性, 这些以前被证明是独立于血清型, 分别。该化验结果显示, 蛇 AAV 衣壳组件需要蛇的 AAPs, 不能由 AAV5 和 AAV9 促进。该检测还表明, 不同于许多常见的血清 AAPs, 促进异种衣壳组装交叉互补, 蛇的儿科学会不促进组装 AAV9 衣壳。此外, 我们还表明, 核酸酶的选择对斑点印迹检测的读数有显著影响, 因此, 选择最佳酶对 AAV 滴度的成功评估是至关重要的。

如图 1所示, 在大规模生产的纯化 AAV 矢量种群定量斑点印迹的一个代表性结果。用这种斑点杂交法测定了双绞股 AAV2G9-CMV-GFP 载体的滴度。用两轮氯化铯 (中海) 密度梯度离心和前述20的透析纯化了该载体。一般而言, 对于纯化的 AAV 向量股, 每 AAV 矢量股的三种不同体积 (例如, 0.3 µL、0.1 µL 和0.03 µL) 都受到上面描述的斑点杂交?...

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

本手稿详细介绍了一种直接斑点印迹法定量的腺相关病毒 (AAV) 的效价及其应用研究组装活化蛋白 (AAPs) 的作用, 一种新型的非结构化病毒蛋白在所有 AAV 发现血清型, 促进衣壳的组装从同源和异源 AAV 血清型。

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

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

走在大鼠作为定性分析方法使用Rotorod

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

科研

生物学

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

梯级步行任务：一个评分系统，其实际应用。

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

微探针：操作和使用的局部表面处理

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

使用油脂介孔结构的测定结晶膜蛋白

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

多重PCR和反向线杂交检测（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

一个定量测定胰岛素表达细胞集落形成先觉

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

使用机器人在油脂中间相高通量膜蛋白的结晶

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

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

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

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

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

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

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 ...