Name: process development for the production and purification of adeno-associated virus (aav)2 vector using baculovirus-insect cell culture system
Uploaded: 2022-01-13T13:00:00
Description: Watch this Scientific Journal Video about Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System at JoVE.com

我们要感谢Robert M. Kotin博士（国家心脏，血液和肺部研究所，NIH）慷慨地为我们提供了AAV质粒，并感谢Danielle Steele和Rebecca Ernst（辛辛那提儿童医院）的技术援助。这项工作得到了辛辛那提儿童研究基金会（Cincinnati Children's Research Foundation）到M.N.的启动基金的支持。

有关概述该协议的插图，请参见图 1。
1. 杆状病毒感染的TIPS细胞的产生
<ol><li>解冻一小瓶Sf9细胞，并立即将其接种在50 mL昆虫细胞培养基中。在轨道振荡器培养箱中以125rpm和28°C在圆底烧瓶中培养Sf9细胞，带有松散的盖子以交换空气8，9。在含有200mL培养基的1L烧瓶中繁殖细胞，以获得足够的细胞用于TIPS细胞?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography-based+purification+of+adeno-associated+virus.">Chromatography-based purification of adeno-associated virus.</a> Methods in Molecular Biology. 434, 37-54 (2008).</li><li>Aslanidi, G., Lamb, K., Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+system+for+highly+efficient+production+of+recombinant+adeno-associated+virus+(rAAV)+vectors+in+insect+Sf9+cells.">An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (13), 5059-5064 (2009).</li><li>Wu, Y., 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=Popularizing+recombinant+baculovirus-derived+OneBac+system+for+laboratory+production+of+all+recombinant+adeno-associated+virus+vector+serotypes.">Popularizing recombinant baculovirus-derived OneBac system for laboratory production of all recombinant adeno-associated virus vector serotypes.</a> Current Gene Therapy. 21 (2), 167-176 (2021).</li><li>Adams, B., Bak, H., Tustian, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+from+the+bench+towards+a+large+scale,+industrial+platform+process+for+adeno-associated+viral+vector+purification.">Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification.</a> Biotechnology and Bioengineering. 117 (10), 3199-3211 (2020).</li><li>Joshi, P. R. 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该协议中用于AAV载体生产，纯化和浓缩过程开发的参数可应用于基因治疗应用的AAV载体的小型和大规模制造。整个上游和下游过程可以在与当前良好生产规范（cGMP）兼容的封闭系统中执行。Sf9-杆状病毒系统的主要优点是可扩展性，能够以可承受的成本进行大规模的GMP级AAV生产。该系统不需要昂贵的质粒和转染试剂，而使用HEK293细胞生产AAV则需要这些质粒和转染试剂。据报道，HEK293细胞和Sf9细胞?...

腺相关病毒（AAV）是非包膜的人类细小病毒，含有4.6 kb的单链DNA。对于基因治疗应用，AAV载体比其他病毒载体具有几个优点1，2，3，4。AAV自然是复制无能的，因此，需要帮助病毒和主机进行复制。AAV不会引起任何疾病，并且在受感染的宿主3，5中具有低免疫原性。AAV可以感染静止和主动分裂的细胞，并且可以持续作为表观体而不整合到宿主细胞的基因组中（AAV很少整合到宿主基因组中）1，3。这些特性使AAV成为基因治疗应用的理想工具。
为了生成AAV基因转移载体，在两个内部末端重复序列（ITR）之间克隆转基因盒，包括治疗基因，这两个重复序列通常来自AAV血清型2。从5'ITR到3'ITR的最大尺寸，包括转基因序列，为4.6 kb6。不同的衣壳可能具有不同的细胞或组织向性。因此，应根据拟用AAV载体靶向的组织或细胞类型选择衣壳7。
重组AAV载体通常在哺乳动物细胞系中产生，例如人胚胎肾细胞，HEK293通过AAV基因转移载体，AAV rep-cap和辅助病毒质粒2，3的瞬时转染。然而，通过贴壁HEK293细胞的瞬时转染进行大规模AAV生产存在一些局限性。首先，需要大量的电池堆或滚瓶。其次，需要高质量的质粒DNA和转染试剂，这增加了制造成本。最后，当使用贴壁HEK293细胞时，血清经常需要最佳生产，使下游处理1，2，3复杂化。AAV制造的替代方法涉及使用昆虫细胞系，Spodoptera frugiperda（Sf9）细胞，以及称为重组Autographa californica多衣壳核多肝病病毒（AcMNPV或杆状病毒）的昆虫病毒8，9，10。Sf9细胞在无血清悬浮培养物中生长，易于放大，并且与当前大规模的良好生产规范（cGMP）生产兼容，不需要质粒或转染试剂。此外，使用Sf9-杆状病毒系统的AAV生产成本低于使用质粒瞬时转染到HEK293细胞的成本11。
原来的rAAV生产系统使用杆状病毒-Sf9细胞，使用三种杆状病毒：一种是含有基因转移盒的杆状病毒，第二种是含有重复基因的杆状病毒，第三种杆状病毒含有血清型特异性衣壳基因12、13。然而，含有重复构建体的杆状病毒在多轮传代时遗传不稳定，这阻止了杆状病毒在大规模AAV生产中的扩增。为了解决这个问题，开发了一种新型的rAAV载体系统，其中包含两种杆状病毒（TwoBac）：一种含有AAV基因转移盒的杆状病毒和另一种含有AAV重集基因的杆状病毒，它们在遗传上比原始系统更稳定，并且由于使用TwoBac而不是三个14，因此更方便产生rAAV。 15.OneBac系统使用单个杆状病毒中的AAV基因转移盒和rep-cap基因，由于使用一种杆状病毒而不是使用TwoBac或ThreeBac2，16，17，因此更方便地产生rAAV。在我们的研究中，TwoBac系统用于优化。
用于AAV生产的杆状病毒系统也有局限性：杆状病毒颗粒在无血清培养基11中长期储存不稳定，如果杆状病毒滴度低，则需要大量的杆状病毒上清液，这可能在AAV生产过程中对Sf9细胞的生长产生毒性（个人观察）。使用无滴度感染细胞保存和放大（TIPS）细胞或杆状病毒感染的昆虫细胞（BIIC）为AAV生产提供了一个很好的选择，其中制备杆状病毒感染的Sf9细胞，冷冻保存，随后用于感染新鲜的Sf9细胞。另一个优点是冷冻保存后Sf9细胞中杆状病毒（BV）的稳定性增加10，11。
产生两种类型的TIPS细胞以实现AAV的产生：第一种是通过感染具有BV-AAV2-GFP或治疗基因的Sf9细胞，第二种是通过具有BV-AAV2-rep-cap的Sf9细胞感染的。TIPS细胞被冷冻保存在小型和即用型等分试样中。AAV载体通过共培养产生杆状病毒和新鲜Sf9细胞的TIPS细胞，在置于轨道振荡器或Wave生物反应器的烧瓶中以无血清悬浮培养物产生。Sf9细胞被携带AAV2-GFP载体和重复帽序列以产生AAV的杆状病毒感染。四到五天后，当AAV产量最高时，用洗涤剂裂解生产细胞以释放AAV颗粒。随后通过低速离心和过滤澄清细胞裂解物。AAV颗粒通过AVB琼脂糖柱色谱法从裂解物中纯化。最后，使用TFF浓缩AAV载体。该协议描述了AAV的小规模生产，可用于研究和临床前研究。然而，这些方法是可扩展的，并且与制造用于基因治疗应用的临床级AAV载体兼容。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1 N Sodium Hydroxide</td>
			<td >Sigma-Aldrich</td>
			<td >1.09137</td>
			<td >For Akta Avant cleaning</td>
		</tr>
		<tr >
			<td >2 L flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>431281</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr >
			<td >50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of supernatants</td>
		</tr>
		<tr >
			<td >24-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Adherent cell culture plate</td>
		</tr>
		<tr >
			<td >250 mL flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr >
			<td >Akta Avant 150 with Unicorn Software</td>
			<td>Cytiva</td>
			<td>28976337</td>
			<td>Chromatography system</td>
		</tr>
		<tr >
			<td >AVB Sepharose High Performance</td>
			<td>Cytiva</td>
			<td>28411210</td>
			<td>Chromatography medium</td>
		</tr>
		<tr >
			<td >Baculovirus-AAV-2 GFP</td>
			<td>In-house</td>
			<td>non-catalog</td>
			<td>AAV transfer vector</td>
		</tr>
		<tr >
			<td >Baculovirus-AAV-2 rep-cap</td>
			<td>In-house</td>
			<td>non-catalog</td>
			<td>AAV packaging vector</td>
		</tr>
		<tr >
			<td >Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr >
			<td >Blocking buffer</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>516214</td>
			<td>Blocking to prevent non-specific antibody binding to cells</td>
		</tr>
		<tr >
			<td >Cell lysis buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 % Titron X-100</td>
		</tr>
		<tr >
			<td >Cellbag, 2 L and 10 L</td>
			<td>Cytiva</td>
			<td>28937662</td>
			<td>Bioreactor bag</td>
		</tr>
		<tr >
			<td >Cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>100 mM citric acid (pH 2.1)&#160;</td>
		</tr>
		<tr >
			<td >Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For cryopreservation</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr >
			<td >Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>50 mM sodium citrate buffer (pH 3.0)</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For disinfection and storage of the chromatography</td>
		</tr>
		<tr >
			<td >Filtration unit&#160;</td>
			<td>Pall Corporation</td>
			<td>12941</td>
			<td>Membrane filter</td>
		</tr>
		<tr >
			<td >HT1080 cell line</td>
			<td>ATCC</td>
			<td >CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr >
			<td >HyClone&#8482; SFX-Insect culture media</td>
			<td>Cytiva</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr >
			<td >Peristaltic Pump</td>
			<td>Pall Corporation</td>
			<td>Non-catalog item</td>
			<td>TFF pump</td>
		</tr>
		<tr >
			<td >MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Nikon</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr >
			<td >Mouse anti-baculovirus gp64 PE antibody</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>65498 PE</td>
			<td>Monitoring baculovirus infection in Sf9 cells</td>
		</tr>
		<tr >
			<td >Oxygen tank</td>
			<td>Praxair</td>
			<td>Non-catalog item</td>
			<td>40 % Oxygen supply is needed for Sf9 cell growth</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>ThermoFisher Scientific</td>
			<td >20012027</td>
			<td>Wash buffer</td>
		</tr>
		<tr >
			<td >Silver Staining kit</td>
			<td >ThermoFisher Scientific</td>
			<td >LC6100</td>
			<td >Staining AAV capsid proteins</td>
		</tr>
		<tr >
			<td >Sf9 cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr >
			<td >Steile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr >
			<td >Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr >
			<td >Tangential Flow Filtration (TFF) cartridge</td>
			<td>Pall Corporation</td>
			<td>OA100C12</td>
			<td>TFF cartridge to concentrate the AAV</td>
		</tr>
		<tr >
			<td >Tris-HCl, pH 8.0</td>
			<td>ThermoFisher</td>
			<td>15568025</td>
			<td>Alkaline buffer to neutralize the eluted AAV</td>
		</tr>
		<tr >
			<td >WAVE Bioreactor System 20/50</td>
			<td>Cytiva</td>
			<td>28-9378-00</td>
			<td>Bioreactor</td>
		</tr>
	</tbody>
</table>

process development for the production and purification of adeno-associated virus (aav)2 vector using baculovirus-insect cell culture system

腺相关病毒（AAV）是基因治疗应用的有前途的载体。在这里，AAV2载体是通过在无血清悬浮培养物中共培养 Spodoptera frugiperda（Sf9） 细胞与感染杆状病毒（BV）-AAV2-GFP（或治疗基因）和BV-AAV2-rep-cap的Sf9细胞共培养产生的。细胞在轨道振荡器或Wave生物反应器的烧瓶中培养。为了释放AAV颗粒，将生产者细胞裂解在含有洗涤剂的缓冲液中，随后通过低速离心和过滤进行澄清。使用AVB Sepharose柱色谱法从细胞裂解物中纯化AAV颗粒，其结合AAV颗粒。用PBS洗涤结合的颗粒以除去污染物，并使用柠檬酸钠缓冲液在pH 3.0下从树脂中洗脱。酸性洗脱液用碱性Tris-HCl缓冲液（pH 8.0）中和，用磷酸盐缓冲盐水（PBS）稀释，并用切向流动过滤（TFF）进一步浓缩。该协议描述了小规模的临床前载体生产与用于人类基因治疗应用的大规模临床级AAV制造相容。

这里展示了使用Sf9昆虫细胞系统生产和纯化AAV载体的工艺开发的代表性结果。该方法包括将Sf9细胞与杆状病毒感染的TIPS细胞共培养，用生长培养基喂养细胞，收获和裂解生产细胞以释放AAV颗粒，用核酸酶处理澄清细胞裂解物，离心和过滤，使用AVB Sepharose亲和色谱法纯化AAV，并用TFF浓缩（图1）。
TIPS细胞通过感染BV-AAV2-GFP或治疗基因和BV-AAV2-rep-cap分别进?...

Watch this Scientific Journal Video about Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System at JoVE.com

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

在该协议中，通过将 Spodoptera frugiperda（Sf9） 昆虫细胞与杆状病毒（BV）-AAV2-绿色荧光蛋白（GFP）或治疗基因和BV-AAV2-rep-cap感染的Sf9细胞在悬浮培养物中共同培养来产生AAV2载体。使用洗涤剂从细胞中释放AAV颗粒，澄清，通过亲和柱色谱纯化，并通过切向流过滤浓缩。

使用杆状病毒-昆虫细胞培养系统生产和纯化腺相关病毒（AAV）2载体的工艺开发

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

该协议对于想要使用杆状病毒 - 昆虫细胞培养系统开发AAV生产和纯化方法的研究人员非常有用。使用杆状病毒-昆虫细胞培养系统生产AAV是一种具有成本效益的方法，易于放大，并且与当前的良好生产规范兼容。该协议的某些部分将由我实验室的研究助理Alan Heizer演示。首先解冻一小瓶Sf9细胞，并立即将它们接种在15毫升昆虫细胞培养基中。在轨道振荡器培养箱中以125 RPM和28摄氏度在圆底烧瓶中以松散的盖子培养Sf9细胞，用于交换空气。在含有200毫升培养基的一升烧瓶中繁殖细胞，以获得足够的细胞用于TIPS细胞生产。在两个烧瓶中，以2倍10向每毫升第6个功率电池添加50毫升Sf9细胞。用0.5毫升杆状病毒-AAV2-GFP感染一个Sf9细胞瓶，用0.5毫升杆状病毒-AAV2-rep-cap感染另一个烧瓶细胞。三天后，收获一小块等分试样的杆状病毒感染的Sf9细胞，也称为TIPS细胞。用0.5微克小鼠抗杆状病毒gp64抗体在100微升封闭缓冲液中在100微升封闭缓冲液中在黑暗的环境温度下染色2次10至5次方的未感染和杆状病毒感染的Sf9细胞。用PBS洗涤细胞以除去抗体后，将染色的细胞重新悬浮在含有0.5牛血清白蛋白的300微升PBS中。使用流式细胞术分析细胞上的杆状病毒gp64表达。当细胞存活80%至90%时，随着直径的增加，收获细胞并在一毫升昆虫培养基中冷冻保存1倍10至7个功率细胞，在负80摄氏度的慢速冷冻容器中用10%胎牛血清和10%二甲基亚砜代替。第二天，将含有TIPS细胞的管转移到液氮冰箱中长期储存。在含有400毫升昆虫细胞培养基的两升烧瓶中，在腺相关病毒或AAV生产所需的振荡器培养箱中，以2倍10至每毫升28摄氏度培养至第6个功率电池，培养并培养幼稚的Sf9细胞。三到四天后，细胞密度增加到每毫升10到第6次方电池的5至6倍。对于在轨道振荡器培养箱中的烧瓶中生产AAV，请使用移液器或蠕动泵以每毫升10至第6个功率细胞的2倍将400毫升Sf9培养物无菌地接种到两升烧瓶中。将轨道振动器设置为125 RPM和28摄氏度。解冻每个杆状病毒-AAV-GFP和杆状病毒-AAV-rep-cap TIPS细胞的一小瓶。用20毫升昆虫培养基稀释细胞，并使用台盼蓝对细胞进行活力计数。相对于在摇瓶或生物反应器中培养的幼稚Sf9细胞，以1比10， 000的比例接种两个TIPS细胞。第二天，无菌收获一毫升Sf9培养物，并用台盼蓝染料染色，以分析细胞计数，活力和形态。在第三天，重复第二天执行的步骤，并在染色Sf9细胞后检查杆状病毒感染的状态。在第五天，重复第二天执行的步骤，然后在Sf9活力降至50%左右时收获培养基和细胞旋转后收集上清液和细胞沉淀，并将其储存在负80摄氏度。一旦AAV生产者细胞沉淀解冻，加入细胞裂解缓冲液并剧烈混合。在环境温度下孵育重新悬浮的沉淀30分钟以释放AAV颗粒。离心后，将细胞裂解物转移到新容器中。解冻后混合细胞裂解物和细胞培养物上清液。向细胞裂解物中每毫升20单位的核酸酶和10毫摩尔氯化镁以消化DNA和RNA，并在37摄氏度下孵育2至4小时。使用泵通过0.8微米和0.2微米聚醚砜双过滤系统过滤裂解物。将澄清的细胞裂解物储存在四摄氏度下过夜，或立即纯化AAV。将10毫升AVB Sepharose柱安装到色谱机上，并将管道插入AAV样品，洗涤缓冲液和洗脱缓冲液中。将管插入色谱机的馏分收集槽中，以收集含有AAV颗粒的柱直通溶液，洗涤缓冲液和洗脱缓冲液的馏分。以每分钟5毫升的流速将AVB Sepharose柱与五列体积的PBS平衡。使用配备样品泵的色谱机将含有AAV颗粒的过滤细胞裂解物加载到AVB Sepharose亲和柱上。以每分钟3毫升的流速运行样品。以每分钟3毫升的流速运行PBS，直到280纳米处的紫外线吸光度曲线返回基线并变得稳定。用50毫摩尔柠檬酸钠缓冲液pH 3以每分钟3毫升的流速洗脱AVB Sepharose柱中的AAV颗粒。立即用五分之一体积的500毫摩尔Tris HCl pH 8.0稀释AAV上清液，以中和含有AAV的酸性洗脱缓冲液。这可以防止酸性pH 3.0洗脱缓冲液可能发生的pH介导的降解。然后用PBS将AAV上清液稀释十倍，使AAV颗粒在生理缓冲液中。储存每列跑过样品的一毫升，洗涤缓冲液和100微升洗脱的AAV上清液，以评估靶细胞感染引起的AAV的存在。设置切向流过滤系统，配备具有100千道尔顿分子量截止值的聚醚砜膜盒以浓缩AAV。运行200毫升PBS10分钟，以平衡切向流过滤模块。通过控制泵的流量来加载AAV样品，直到样品减少到所需的体积。用100毫升PBS透析滞留物。将AAV样品浓缩至所需体积后，收集AAV样品，通过0.2微米聚醚砜过滤器过滤，并等分。然后将AAV样品储存在负80摄氏度。由于多轮感染，大多数Sf9细胞在三到四天内感染杆状病毒，杆状病毒糖蛋白gp64表达证明。大多数细胞也显示出直径的增加。细胞的直径增加，细胞病变效应，大约一半的细胞在感染后五天内死亡，这是AAV生产完成的迹象。在用对应于AAV级分的酸性缓冲液洗脱时观察到蛋白质的峰值。纯化的AAV样品在SDS-PAGE和银染色后显示出三种不同的衣壳蛋白：VP1，VP2和VP3。最关键的步骤是在冷冻保存之前收获TIPS细胞，当大多数细胞感染杆状病毒时，细胞死亡次数最少。我们在这里描述了血清型2的AAV载体的生产和纯化作为模型。然而，该协议也可以应用于其他AAV血清型。

Research

JoVE Journal

Biology

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

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

科研

生物学

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

封闭的系统使用透氧材料科技HYPERStack船只的细胞培养协议

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

The MultiBac Protein Complex Production Platform at the EMBL

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

肥胖的身体器官培养体系中蚊，一个向量 Zika 病毒

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

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

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 ...

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

检测植物宿主中叶蝉载体的病毒和唾液蛋白

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

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

使用组合的 Cre-LoxP、CRISPR-Cas9 和腺相关病毒单向导 RNA 系统生成棕色脂肪特异性敲除小鼠

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive ...