Name: production of adeno-associated virus vectors in cell stacks for preclinical studies in large animal models
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Description: Watch this Scientific Journal Video about Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models at JoVE.com

Amira D. Rghei，Brenna A. Y. Stevens，Sylvia P. Thomas和Jacob G. E. Yates是安大略省兽医学院学生津贴以及安大略省研究生奖学金的获得者。Amira D. Rghei是Mitacs Accelerate Studentship的获得者。这项工作由加拿大卫生研究院（CIHR）项目赠款（#66009）和SKW的合作健康研究项目（NSERC合作）赠款（#433339）资助。

1. 细胞堆中HEK293细胞的双质粒转染
<ol><li>在设置为37°C的珠浴中解冻HEK293细胞的冷冻小瓶。 注意：在电芯解冻时将完整的DMEM预热至37°C，以确保电镀时冷温不会冲击电芯。确保细胞具有较低的传代数，理想情况下小于20，以确保最佳生长和转染效率。确保细胞被认证为无支原体。</li>
	<li>将冷冻小瓶的内容物滴入含有10 mL预热完全DMEM的15 mL锥形管中，并以500×g离心细胞5分钟。 </li>
	<li>吸?...

<ol>
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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=A+novel+triple-mutant+AAV6+capsid+induces+rapid+and+potent+transgene+expression+in+the+muscle+and+respiratory+tract+of+mice.">A novel triple-mutant AAV6 capsid induces rapid and potent transgene expression in the muscle and respiratory tract of mice.</a> Molecular Therapy. Methods &amp; Clinical Development. 9, 323-329 (2018).</li><li>Wu, Z., Asokan, A., Grieger, J. C., Govindasamy, L., Agbandje-McKenna, M., Samulski, R. 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本文中描述的重组AAV（rAAV）载体的生产使用大多数分子生物学研究实验室和设施中发现的常见材料，试剂和设备。该论文允许阅读器产生高质量的 体外 和 体内 级rAAV。最重要的是，与涉及氯化铯纯化的更繁琐的方案相比，这种用于rAAV生产的方案是有效的，并且避免了使用超速离心。一旦HEK293细胞被转染，纯化的AAV就可以在5个工作日内使用。
在rAAV生产和纯化?...

利用腺相关病毒（AAV）载体的基因治疗在过去三十年中取得了巨大的进步1，2。在各种遗传疾病（包括先天性失明，血友病以及肌肉骨骼和中枢神经系统疾病）中表现出的改善，使AAV基因治疗成为临床研究的前沿3，4。2012年，欧洲药品管理局（EMA）批准了Glybera，一种表达脂蛋白脂肪酶（LPL）的AAV1载体，用于治疗LPL缺乏症，使其成为欧洲或美国基因治疗的首个上市授权5。从那时起，另外两种AAV基因疗法Luxturna6和Zolgensma7已获得FDA批准，预计到2025年，市场将迅速扩大，预计到2025年将有多达10-20种基因疗法8。现有的临床数据表明，AAV基因治疗是一种安全、耐受性良好且有效的方法，使其成为最有前途的病毒载体之一，超过244项涉及AAV的临床试验已 ClinicalTrials.gov 注册。对涉及AAV载体的临床应用的兴趣日益增加，需要强大且可扩展的生产方法来促进大型动物模型中AAV疗法的评估，因为这是转化管道中的关键步骤9。
对于AAV载体生产，两个主要要求是AAV基因组和衣壳。野生型（wt）-AAV的基因组是单链DNA，长度约为4.7 kb10。wt-AAV基因组包括在基因组两端发现的倒置末端重复序列（ITR），这对包装很重要，并且rep和cap基因11。基因组复制，病毒衣壳组装以及将基因组封装到病毒衣壳中所必需的rep和cap基因从病毒基因组中移除并以反式形式提供用于AAV载体生产12。从病毒基因组中去除这些基因为治疗性转基因和所有必要的调节元件（包括启动子和polyA信号）提供了空间。ITR保留在载体基因组中，以确保适当的基因组复制和病毒封装13，14。为了改善转基因表达的动力学，可以将AAV载体基因组设计为自互补，这减轻了AAV基因组复制期间从单链DNA转换到双链DNA转换的需求，但将编码能力降低到〜2.4 kb15。
除了AAV基因组设计之外，衣壳血清型选择还决定了体内AAV载体2的组织和细胞向性。除了组织向性外，不同的AAV血清型已被证明可以显示不同的基因表达动力学16。例如，Zincarelli等人17将不同的AAV血清型分为低表达血清型（AAV2，3，4，5），中等表达血清型（AAV1，6，8）和高表达血清型（AAV7和9）。他们还将AAV血清型分为慢发表达（AAV2，3，4，5）或快速发作表达（AAV1，6，7，8和9）。这些不同的趋向性和基因表达动力学是由于衣壳蛋白中的氨基酸变异，衣壳蛋白的形成以及与宿主细胞受体/共受体的相互作用18。一些AAV衣壳具有额外的有益特征，例如在血管内给药（AAV9）后穿过血脑屏障的能力或驻留在长寿的肌肉细胞中以持久转基因表达（AAV6，6.2FF，8和9）19，20。
本文旨在详细介绍一种生产高纯度、高滴度、研究级AAV载体的具有成本效益的方法，用于临床前大型动物模型。使用该协议生产AAV是使用双质粒转染到在细胞堆叠中生长的贴壁人胚胎肾（HEK）293细胞来实现的。此外，该研究描述了硫酸肝素亲和色谱纯化的方案，可用于含有肝素结合结构域的AAV血清型，包括AAV2，3，6，6.2FF，13和DJ21，22。
许多包装系统可用于生产AAV载体。其中，使用双质粒共转染系统，其中Rep和Cap基因以及Ad辅助基因（E1A，E1B55K，E2A，E4orf6和VA RNA）包含在一个质粒（pHelper）中，与常见的三质粒（三重）转染方法相比具有一些实际优势，包括降低质粒生产成本23，24.含有转基因表达盒（pTransgene）的AAV基因组质粒必须由ITR两侧放置，并且长度不得超过约4.7 kb。由于转染过程中潜在的细胞毒性作用，载体滴度和纯度可能受到转基因的影响。本文描述了载体纯度的评估。在小鼠，仓鼠和绵羊动物模型中评估使用该方法生产的载体，每个载体产生1 x10 13 vg / mL。
表1：所需溶液的组成。整个协议中各种解决方案所需组件的必要信息，包括百分比和体积。 <a href="https://www.jove.com/files/ftp_upload/62727/Table%201-%2062727_R2.xlsx" target="_blank">请点击此处下载此表格。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millipore Sigma</td>
			<td>S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Fisher Scientific</td>
			<td>SM2001C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Butanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A399-4</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>10 chamber cellstack</td>
			<td>Corning</td>
			<td>3271</td>
			<td></td>
		</tr>
		<tr>
			<td>1L PETG bottle</td>
			<td>Thermo Fisher Scientific</td>
			<td>2019-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis Solution</td>
			<td>Bio-Rad</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well skirted plate</td>
			<td>FroggaBio</td>
			<td>FS-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive plate seals</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Blood and Tissue Clean up Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>Use for DNA clean up in section 4.6 of protocol</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Fisher Scientific</td>
			<td>B392-5</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Cell Culture Dishes</td>
			<td>Greiner bio-one</td>
			<td>7000232</td>
			<td>15 cm plates</td>
		</tr>
		<tr>
			<td>Culture Conical Tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td>15 mL conical tube</td>
		</tr>
		<tr>
			<td>Culture Conical Tube</td>
			<td>Fisher Scientific</td>
			<td>14955240</td>
			<td>50 mL conical tube</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Western Blotting Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32209</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Greenfield</td>
			<td>P016EA95</td>
			<td>Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38-500</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Mg2+ and Ca2+</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH302268.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS without Mg2+ and Ca2+</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30588.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>American Tissue Culture Collection</td>
			<td>CRL-1573</td>
			<td>Upon receipt, thaw the cells and culture as described in manufacturer&#8217;s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots</td>
		</tr>
		<tr>
			<td>HEK293 host cell protein ELISA kit</td>
			<td>Cygnus Technologies</td>
			<td>F650S</td>
			<td>Follow manufacturer&#8217;s instructions</td>
		</tr>
		<tr>
			<td>Heparin sulfate column</td>
			<td>Cytiva Life Sciences</td>
			<td>17040703</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Thermo Fisher Scientific</td>
			<td>KC34120</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30034.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Volume Centrifuge Tube Support Cushion</td>
			<td>Corning</td>
			<td>CLS431124</td>
			<td>Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions</td>
		</tr>
		<tr>
			<td>Large Volume Centrifuge Tubes</td>
			<td>Corning</td>
			<td>CLS431123-6EA</td>
			<td>500 mL centrifuge tubes</td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>1.5 mL microcentrifuge tube, sterilize prior to use</td>
		</tr>
		<tr>
			<td>Molecular Grade Water</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30538.03</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>L5125</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP35810</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem, reduced serum medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td>Sterilize prior to use</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td>Dilute to 1x for use on cells</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Cytiva Life Sciences</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>pHelper plasmid</td>
			<td>De novo design or obtained from plasmid repository</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet basin</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-681-502</td>
			<td>Purchase sterile pipet basins</td>
		</tr>
		<tr>
			<td>Polyethylene glycol tert-octylphenyl ether (Triton X-100)</td>
			<td>Thermo Fisher Scientific</td>
			<td>9002-93-1</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td>Follow manufacturer&#8217;s instructions to produce a 1L solution. 0.22&#956;m filter and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Polypropylene semi-skirted PCR Plate</td>
			<td>FroggaBio</td>
			<td>WS-96</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP337-100</td>
			<td>CAUTION. Wear gloves</td>
		</tr>
		<tr>
			<td>polyvinylidene difluoride (PVDF) membrane</td>
			<td>Cytiva Life Sciences</td>
			<td>10600023</td>
			<td>Use forceps to manipulate. Wear gloves.</td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td>Progen</td>
			<td>65158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Ladder</td>
			<td>FroggaBio</td>
			<td>PM008-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2546</td>
			<td></td>
		</tr>
		<tr>
			<td>pTrangene plasmid</td>
			<td>De novo design or obtained from plasmid repository</td>
			<td>NA</td>
			<td>Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0</td>
		</tr>
		<tr>
			<td>Pump tubing</td>
			<td>Cole-Parmer</td>
			<td>RK-96440-14</td>
			<td>Optimize length of tubing and containment of virus in fractions E1-E5</td>
		</tr>
		<tr>
			<td>RQ1 Dnase 10 Reaction Buffer</td>
			<td>Promega</td>
			<td>M6101</td>
			<td>Use at 10x concentration in protocol from section 4.0</td>
		</tr>
		<tr>
			<td>RQ1 Rnase-free Dnase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample dilutent</td>
			<td>Cygnus Technologies</td>
			<td>I700</td>
			<td>Must be purchased separately for use with HEK293 host cell protein ELISA kit</td>
		</tr>
		<tr>
			<td>Secondary antibody, HRP</td>
			<td>Thermo Fisher Scientific</td>
			<td>G-21040</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Oxoid</td>
			<td>LP0033B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>28312</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SS266-4</td>
			<td></td>
		</tr>
		<tr>
			<td>SV40 polyA primer probe</td>
			<td>IDT</td>
			<td></td>
			<td>Use sequence in Table X for quote from IDT for synthesis</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15524010</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Bio-Rad</td>
			<td>1450021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Filter</td>
			<td>Millipore Sigma</td>
			<td>UFC810024</td>
			<td>Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff</td>
		</tr>
		<tr>
			<td>Universal Nuclease for cell lysis</td>
			<td>Thermo Fisher Scientific</td>
			<td>88702</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal qPCR master mix</td>
			<td>NEB</td>
			<td>M3003L</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Paper</td>
			<td>Millipore Sigma</td>
			<td>WHA1001325</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>21985023</td>
			<td>CAUTION. Use under a laminar flow hood. Wear gloves</td>
		</tr>
		<tr>
			<td>CAUTION:&#160;Refer to the Materials Table for guidelines on the use of dangerous chemicals.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

production of adeno-associated virus vectors in cell stacks for preclinical studies in large animal models

腺相关病毒（AAV）载体是临床上最先进的基因治疗载体之一，有三种AAV基因疗法被批准用于人类。AAV新应用的临床进展涉及从小动物模型（如小鼠）过渡到较大的动物模型，包括狗，绵羊和非人类灵长类动物。对大型动物施用AAV的局限性之一是需要大量高滴度病毒。虽然悬浮细胞培养是AAV载体生产的可扩展方法，但很少有研究实验室拥有设备（例如，生物反应器）或知道如何以这种方式生产AAV。此外，与贴壁HEK293细胞相比，在悬浮HEK 293细胞中产生AAV滴度通常显着降低。这里描述的是一种使用细胞堆栈生产大量高滴度AAV的方法。还描述了用于滴定AAV的详细方案以及验证载体纯度的方法。最后，介绍了AAV介导的转基因表达在绵羊模型中的代表性结果。这种用于在贴壁细胞中大规模生产AAV载体的优化方案将使分子生物学实验室能够在更大的动物模型中推进其新型AAV疗法的测试。

从小型啮齿动物模型到大型动物模型的转换以及最终的临床应用提出了重大挑战，因为需要大量的AAV来转导大型动物并达到治疗效果。为了比较合理设计的AAV6.2FF衣壳的转导效率，先前证明与AAV63相比，小鼠肌细胞的转导效率提高了101倍，小鼠，仓鼠和羔羊都给予AAV6.2FF表达人单克隆抗体（hIgG）。AAV6.2FF-hIgG表达载体含有CASI启动子4、伍德查克肝炎病毒转录后调节...

Watch this Scientific Journal Video about Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models at JoVE.com

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

在这里，我们提供了使用在细胞堆叠中生长的贴壁HEK 293细胞和亲和色谱纯化大规模生产研究级AAV载体的详细程序。该方案始终如一地产生>1 x 1013 个载体基因组/ mL，提供适合大型动物研究的载体量。

在细胞堆中生产腺相关病毒载体，用于大型动物模型的临床前研究

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

该生产AAV病毒载体的方案具有成本效益，可产生高纯度，高滴度，研究级AAV，用于临床前大型动物模型。该技术在细胞培养室中使用贴壁HEK293细胞，这具有时间效率并且较少的动手操作，从而减少了对细胞的破坏。该协议可以极大地帮助基因治疗领域的病毒载体的产生，也可用于生产其他也结合硫酸乙酰肝素的AAV血清型。首先，当培养物达到80%汇合度时，从培养物中收集HEK293细胞。吸出培养基，然后用三毫升PBS轻轻洗涤细胞培养板。然后吸出PBS并加入三毫升胰蛋白酶。将板在37摄氏度下孵育两分钟。孵育后，通过将7毫升完整的DMEM加入到板中来中和胰蛋白酶，并将上清液收集在50毫升锥形管中。轻轻倒置管子以确保细胞均匀。为了确定细胞密度，将10微升细胞样品与10微升台盼蓝混合。并将混合物添加到细胞计数载玻片中，以便在细胞计数器中进行分析。将细胞悬浮液与一升预热的完整DMEM混合以查看培养室。轻轻旋转腔室以均匀分布细胞。将腔室在37摄氏度下孵育5%的二氧化碳。除细胞培养室外，培养细胞以每百厘米10至第四个细胞的平方放入15厘米的板中作为汇合的参考。孵育65小时后，当细胞汇合度为80%至90%时，缓慢地将聚乙烯亚胺-DNA和DMEM混合物加入细胞培养室端口。将液体均匀地分布到所有行中，然后在37摄氏度和5%二氧化碳下孵育72小时。孵育后，用力摇动细胞培养室以移开细胞，直到培养基出现浑浊，并将其倒入4或500毫升离心管中。通过在4摄氏度下以18， 000倍G离心30分钟来沉淀细胞。将澄清的上清液倒入一升PETG瓶中，并将细胞沉淀重悬于500毫升离心管中的50毫升许可缓冲液中。将管子在37摄氏度下孵育60分钟。孵育后，将试管以18， 000倍G离心30分钟，并将上清液转移到相同的一升PETG瓶中。储存在零下80摄氏度。在四摄氏度下解冻粗裂解物过夜。解冻后，使用0.22微米过滤器过滤。在纯化步骤之前，通过为所使用的每个肝素 - 琼脂糖柱添加4毫升过滤预处理缓冲液来钝化离心浓缩器。将管道置于蠕动泵中。按顺序运行解决方案和介质。通过将5毫升肝素 - 琼脂糖柱连接到管子上，并通过柱运行25毫升Bazell DMEM来准备色谱。然后将0.2微摩尔的过滤粗裂解物以每秒一到两滴的流速加载到柱上。依次洗涤色谱柱，用300毫摩尔氯化钠HBSS洗脱五次，其中镁和钙，并将五个洗脱物标记为E1至E5。准备就绪后，以900倍G旋转含有预处理缓冲液的离心浓缩器两分钟。丢弃流出。用四毫升HBSS用镁和钙洗涤离心浓缩器过滤器，以1000倍G旋转两分钟。然后将洗脱液E2加入离心浓缩器中，并以1000倍G旋转五分钟。丢弃流出。同样地将洗脱液E3旋转到离心浓缩器中，直到浓缩的病毒约为一毫升。使用P200过滤的尖端从离心浓缩器中取出浓缩的病毒，并将其收集到无菌的1.5毫升离心管中。收集病毒后，用200微升HBSS镁和钙冲洗离心浓缩器。用力上下移液30秒，以移走粘附在膜上的任何病毒，并将浓缩的病毒收集在同一个1.5毫升离心管中。将管子混合均匀。洗涤色谱柱并用20%乙醇饱和，然后储存在4摄氏度。将泵管储存在一摩尔氢氧化钠中。肌内给药后28天比较小鼠，仓鼠和羔羊中AAV6.2FF衣壳的转导效率。小鼠给予每公斤AAV6.2FF人IgG的第12个载体基因组10倍，血清中每毫升人IgG表达171至237微克。仓鼠以每公斤AAV6.2FF人类IgG向第13个载体基因组施用两次10，表达的人类IgG水平远高于小鼠。而大型动物模型能够在血浆中平均每毫升表达35微克的人类IgG水平。大型动物模型可以确定AAV转基因在体内的表达，并确定转基因是否对感兴趣的疾病或疾病具有治疗作用。该研究展示了AAV6.2FF（骨骼肌中一种新型AAV 6血清型载体）的巨大转导能力，以及AAV病毒载体在体内的低免疫原性。

Research

JoVE Journal

Immunology and Infection

Estimating Virus Production Rates in Aquatic Systems

估计在水生系统的病毒产率

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

科研

免疫与感染

Visualizing Dengue Virus through Alexa Fluor Labeling

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Influenza A Virus Studies in a Mouse Model of Infection

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...