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查看更多产品信息 ViraPower™ Adenoviral Promoterless Gateway™ Expression Kit - FAQs (K494000)
72 个常见问题解答
在BP/LR Clonase反应的一步法实验方案中，不建议用BP Clonase酶和LR Clonase酶替代BP Clonase II 酶/LR Clonase II酶，因为这样的重组效率非常低。
有的，我们能提供针对BP/LR Clonase反应的一步式实验方案DNA可以在一步反应后被克隆到目的载体中，从而节省了您的时间和金钱。
建议使用一个供体载体进行一次BP反应以获得一个入门克隆。然后将这一入门克隆和目的载体进行一次LR反应以获得新的表达克隆。
5X LR Clonase缓冲液或5X BP Clonase缓冲液不作为单独产品出售。它们作为酶试剂盒的一部分进行销售。
我们不提供任何用于在植物内表达的Gateway载体。
以下为一些可能的原因与解决方案：
-使用了过量的原始病毒母液 ：
-减少用于转导的原始病毒母液，或对原病毒母液进行稀释。
-扩增腺病毒储液。
-对粗的病毒储液进行浓缩。
-野生型RCA（具有复制能力的腺病毒）污染：筛查RCA污染。噬菌斑纯化以分离重组的腺病毒，或制备全新的腺病毒母液。
-目的基因对细胞有毒性：不推荐构建包含激活型致癌基因或潜在有害基因的载体。
以下为一些可能的原因与解决方案：
-低转导效率：
-哺乳动物细胞不够健康：请确保您用的细胞在转导前保持健康状态。
-使用了非分裂型细胞：以更高的MOI比例值向您的细胞中转导腺病毒。
-MOI值过低：以更高的MOI比例向您用的细胞中转导腺病毒。
-病毒滴度过低：使用手册（https://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf）第20页的步骤扩增腺病毒母液。
-腺病毒母液受RCA（具有复制能力的腺病毒）污染：
-筛查RCA污染。
-制备全新的腺病毒母液或噬菌斑纯化以分离重组的腺病毒。
-转导操作后过早收获细胞：请至少在转导24小时之后收获细胞。
-转导操作后过晚收获细胞：对于分裂活跃的细胞，请在转导5天内检测重组蛋白表达的最高水平。
-目的基因对细胞有毒性：不推荐构建包含激活型致癌基因或潜在有害基因的载体。
以下为一些可能的原因与解决方案：
-病毒母液未正确储存： 分装并将母液保存于–80°C。请勿将其冻融超过10次。
-目的基因中含有一个Pac I位点：进行突变操作，来改变或去除PacI位点。
这可能由病毒上清液的稀释度不足所致。我们推荐您使用从10-4至10-9续列稀释度来检测腺病毒滴度。
以下为一些可能的原因与解决方案：
-病毒母液未正确储存： 分装并将母液保存于–80°C。请勿将其冻融超过10次。
-使用错误的细胞系进行病毒滴度测定：请按照 手册（https://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf）第23页中所讨论的属性选择细胞系，或直接使用293A细胞系。
-未正确制备琼脂糖覆层：请确保向细胞加入的琼脂糖不会过热；过热的琼脂糖将杀死细胞。
-病毒储存液的滴度过低或过高：以更宽广的续列稀释度（以10倍为单位，如10-2至10-8）来测定腺病毒的滴度。
以下为一些可能的原因与解决方案：
-低转染效率：
-腺病毒Destination载体发生了断裂：小心操作腺病毒Destination载体。不要进行过多可能造成DNA断裂的操作（如涡旋或剧烈吸打溶液）
-不彻底的Pac I消化效果或消化后的DNA含有苯酚、乙醇或盐类：重新进行Pac I消化处理。请确保使用未含苯酚，乙醇或盐类的纯净DNA。
-使用了不健康的293A细胞；细胞活力较低：使用健康的293A细胞；不要让细胞过度生长
-293A在转染前一天培养密度过低：转染时刻细胞（密度）应处于90-95%的融汇度
-质粒DNA与转染试剂之间的比例错误： 将质粒DNA（μg为单位）:Lipofectamine 2000（μL为单位）的比例设置为1:2到1:3。如果您正在使用另一款转染试剂，请按照生产商的推荐用法进行条件优化
-病毒上清液稀释过度： 通过CsCl离心或任意可选方法对病毒进行浓缩处理。
-病毒上清液经多次冻融：请勿将病毒上清液冻融超过10次。
-目的基因过长：病毒滴度通常随着插入片段长度的增加而降低；不推荐使用超过6 kb（在pAd/CMV/V5-DEST中）和7.5 kb（在pAd/PL-DEST中）的插入片段。
-目的基因对细胞有毒性：不推荐构建包含激活型致癌基因或潜在有害基因的载体。
以下为一些可能的原因与解决方案：
-将LR反应体系转化至包含F'游离体和ccdA基因的大肠杆菌菌株中：使用不含F'游离体的大肠杆菌菌株进行转化（如TOP10、DH5α-T1R）。
-（完全或部分）删除腺病毒Destination载体中的ccdB基因：
-腺病毒Destination载体以溶液形式提供，可直接应用于LR反应。不过，如果您希望扩增这些载体，我们推荐您使用One Shot ccdB Survival 2 T1R化学感受态细胞（货号A10460）。
-在含有50–100 μg/mL氨苄青霉素和15–30 μg/mL氯霉素的培养基中筛选转化子，以维持载体的完整性。
-从一个或多个克隆制备质粒DNA，并在使用前验证载体的完整性。
以下为一些可能的原因与解决方案：
-使用不正确的抗生素来筛选转化子：在含有100 μg/mL氨苄青霉素的LB琼脂糖平板上筛选转化子。
-未使用蛋白酶K处理LR重组反应体系：在转化前使用蛋白酶K处理LR重组反应体系。
-在LR反应中使用了过多的入门克隆DNA：在LR反应中使用50-150 ng入门克隆DNA。
-在LR反应中使用不当的入门克隆:DEST载体比例：入门克隆:DEST载体的摩尔比应为1:1。
- 对插入片段长于5 kb的LR重组反应体系只孵育1小时：对插入片段长度超过5 kb的片段，我们建议将LR反应体系孵育过夜。注意：过夜孵育也能提升小插入片段的克隆数目。
-腺病毒Destination载体DNA发生了断裂：务必小心操作腺病毒Destination载体。不要进行过多可能造成DNA断裂的操作（如涡旋或剧烈吸打溶液）。
-没有用推荐数量的LR Clonase II酶混合物，或LR Clonase II酶混合物已失活：
-请确保将LR Clonase II酶混合物保存于–20°C。
-请勿将LR Clonase II酶混合物冻融超过10次。
-请使用推荐用量的LR Clonase II酶混合物（参见使用手册第14页）。
-请尝试另一管分装的LR Clonase II酶混合物产品。
-转化时使用的LR反应体系不足：使用适当的大肠杆菌感受态菌株来转化2-3 μL LR反应体系。使用转化效率 >1 x 108 cfu/μg的大肠杆菌细胞。
-使用不足量的转化混合物进行铺板：增加大肠杆菌的铺板用量。
ViraPower腺病毒表达系统有以下特性，提升了其生物安全性：
•腺病毒表达载体（pAd/CMV/V5-DEST和pAd/PLDEST）中删除了整条E1基因，由293A生产细胞系来提供（这一基因）。由于E1的表达（E1a与E1b）对于其他腺病毒基因（如晚期基因）的表达是必需的，因此在任何不表达E1a和E1b的哺乳动物细胞中使用这一系统生产的腺病毒都不具备复制能力。
•E3基因在体外应用中可完全省略，因此也被从腺病毒表达载体骨架中删去。
•在转导过程中腺病毒不会整合进宿主基因组中。由于该病毒是复制缺陷型的，因此病毒基因组的存在是瞬时的，而且最终会随着细胞分裂而逐渐被稀释。
尽管有以上安全特性方面的设计，所生成的腺病毒仍具有某些生物安全方面的风险，因为它们仍具有转导人体原代细胞的能力。基于此种原因，我们强烈建议您按照二级生物安全（BL-2）标准来操作本系统生成的腺病毒母液，并严格遵守全部已发表的BL-2准则。此外，当创建的腺病毒中携带有潜在毒性或有害基因（如激活型致癌基因）时，或大规模制备病毒时，需格外小心（参见使用手册第10页。
如需了解更多关于BL-2准则以及腺病毒操作的详细信息，请参见由疾病控制中心（CDC）出版的文件《微生物学与生物医学实验室的生物安全（Biosafety in Microbiological and Biomedical Laboratories）》第四版（www.cdc.gov/biosafety/publications/index.h）。
获得细胞转染和观察到丰富的病毒转导效果是两件不同的事情。如果您的培养物中只有一两个细胞在产生病毒，就可能需要等很长时间才能通过肉眼观察到（比人们一般可接受的等待时间更长）。转染效率与病毒得率有关，因为你转染的细胞越多，在第一周或第二周观察到病毒生成的机会就越高。如果您的转染效率较低，尽管您最终还是会看到病毒生成效果，但等待的时间就会更长。
pAd/CMV/V5-DEST或pAd/PL-DEST腺病毒载体不会整合到宿主基因组。一旦转导进入目的哺乳动物细胞中，您的重组蛋白就会伴随病毒基因组的存在一直持续表达。对于分裂活跃的细胞而言，转基因表达会随着时间流逝而逐渐降低，转导2周后降至背景水平。在不分裂的CD34+细胞或动物组织等（骨骼肌、神经元、肝脏）中，转基因表达效果非常稳定，可在转导后持续6个月之久。
在分裂活跃的细胞（每24小时倍增一次）中，我们发现通常可在转导24小时后观察到转基因表达，转导48-96小时（2-4天）时观察到最大量蛋白表达。表达水平通常在转导5天后开始下降。在倍增时间更长或非分裂型的细胞中，高水平的转基因表达会持续更长时间。
人类5型腺病毒（Ad5）能够与柯萨奇病毒与腺病毒受体相结合，进而通过整联蛋白介导的内吞途径进入靶细胞。由于CAR/整联蛋白广泛分布于哺乳动物细胞中，因此腺病毒能够转导非常广谱的细胞类型。如果您特异性的细胞类型表达CAR的水平极低，腺病毒的转导效率就会下降，这时您可能需要使用非常高的MOI（MOI=100）值才能获得良好的表达效果。
我们的ViraPower腺病毒表达载体的骨架源自5型人类腺病毒（Ad5）。Ad5能够与柯萨奇病毒与腺病毒受体相结合，进而通过整联蛋白介导的内吞途径进入细胞。使用充分表达CAR受体并活跃分裂的靶细胞，以及足量的MOI值，就可能达到80-90%的腺病毒转导效率。
注意：不同类型细胞的转导效率会有所不同。举例：在HT1080细胞中，以MOI=1进行转导，转导效率可达90%左右。在某些种类的细胞中，您可能需要使用10倍以上MOI才能获得同等转导效率。
未扩增的腺病毒的滴度通常在1 x 107–1 x 108噬菌斑形成单位（pfu）/mL。您可使用这一母液感染一批新的293A细胞来生产更高滴度的病毒液（即扩增病毒）。扩增操作能够生成范围在1 x 108至-1 x 109 pfu/mL滴度的病毒液。腺病毒可通过多种方法（如CsCl纯化）浓缩至1 x 1011 pfu/mL的滴度。
腺病毒可通过多种方法（如CsCl纯化；请参考手册第25页的参考文献）浓缩至1 x 1011 pfu/mL的滴度。
一旦您获得了病毒母液，您就可应用这一母液感染一批新的293A细胞来生产更高滴度的病毒（扩增病毒）。用于转染293A细胞的原始病毒母液滴度通常在1 x 107至1 x 108噬菌斑形成单位（pfu）/mL。扩增操作能够生成范围在1 x 108至1 x 109 pfu/mL滴度的病毒，我们通常推荐用户进行这一操作。请参见使用手册第19页对于病毒扩增操作的说明。
注意：其他293细胞系以及表达E1蛋白的细胞系均适用于病毒扩增操作。
我们推荐您立即将所生成的腺病毒母液分装为小的工作体积，之后在–80°C条件下长期保存。由于腺病毒不具有外包膜，因此病毒母液的相对稳定性较好，可耐受一定量的冻融操作。我们不推荐对病毒母液冻融超过10次，这可能会造成病毒滴度的损失。如经良好保存，拥有适当滴度的病毒母液可持续使用一年以上。经长期保存后，我们推荐您在使用前重新测定病毒滴度。
大多数腺病毒包含在漂浮的细胞中，细胞破裂之前它们并不会释放到培养基中。我们推荐每三天左右更换一次培养基，直至大量细胞变大变圆，并从塑料培养皿上脱离下来。一旦细胞发生破裂，游离的病毒就会迅速感染邻近的细胞。如果您很担心在更换培养基的过程中损失这些受到感染的细胞（及内在的病毒），您可保留含有漂浮细胞的培养基，冻/融三次后将其中的一小部分（可能1/10左右）与新鲜培养基一道重新加回新鲜培养基中。或者，您也可使用新鲜培养基，以显著高于每三天一次的频率进行半换液。
任何293来源的细胞系及其他表达E1蛋白的细胞系均适用于腺病毒的生产。293A中A代表“贴壁”，是因为293A细胞（只是常规293细胞的一个单细胞克隆）具有贴壁的倾向，在组织培养皿中会形成平铺生长良好的单层细胞。这就是为什么这些细胞很适合应用于噬斑测定实验。普通的293细胞不会形成这样的单层细胞；它们生长所形成的细胞层会出现孔洞和空白之处。
我们使用经测试不含支原体的Gibco FBS（货号16000-044），并用下列塑料器皿进行293A细胞的培养：
T175—Fisher 货号 10-126-13；这是一款Falcon培养瓶，带有0.2 μm透气孔的密封盖。
T75—Fisher 货号 07-200-68；这是一款Costar培养瓶，带有0.2 μm透气孔的密封盖。
100 mm 平板—Fisher 货号 08-772E；这是一款针对组织培养应用进行预先处理的的Falcon聚苯乙烯平板。
我们在日常的细胞培养/维持过程（除在293A细胞中生成腺病毒时的细胞裂解过程）中使用这些平板获得了绝佳的贴壁效果。
野生型5型腺病毒基因组的大小在35.9 kb左右。研究表明，重组型腺病毒由于使用了E1-和E3-删除的载体，其中能够有效包装的序列长度能够达到野生型病毒的108%。用户需针对每一腺病毒目的载体的表达应用，将相关的元件尺寸都计算在内，从而确保目的基因插入序列的长度不会超过有效包装的大小限制（请参见每一独立载体的包装限制）：
pAd/CMV/V5-DEST: 6 kb
pAd/PL-DEST: 7.5 kb
注意：病毒滴度通常随着插入片段长度的增加而减小。
您可在表达细胞系（或293A细胞）中转染您的腺病毒构建序列，来观察蛋白是否能够成功表达，而无需等待两周（病毒包装时间）。由于质粒较大，转染效率会较低，所以用户可能需要遵循ViraPower腺病毒表达系统手册中的说明，来向培养基中加入更多的脂质体-DNA复合物。这一操作过程中请勿使用Pac I消化腺病毒质粒，因为超螺旋质粒的转染效率更高。如需在293A细胞中检测蛋白表达情况，请在转染后2-3天收获细胞。
在将表达克隆转染进293A细胞之前，您须暴露载体上左侧和右侧的病毒反向末端重复序列（LTRs），以辅助病毒复制和包装过程的顺利进行。同时，这一操作也将去除细菌序列（即pUC来源和氨苄青霉素抗性基因）。pAd/CMV/V5-DEST和pAd/PL-DEST载体均包含Pac I限制性酶切位点（请分别参见使用手册第20页和第22页图谱中的Pac I位点）。
注意：请确保你的目的DNA序列中不含任何Pac I限制性酶切位点。如果您无法应用Pac I位点进行处理，您也可换用Swa I位点。
一旦您构建了pAd/CMV/V5-DEST或pAd/PL-DEST表达克隆，您就可以使用任意方法来制备纯化的质粒DNA了。我们推荐用户使用PureLink HiPure质粒中抽试剂盒（Plasmid Midiprep Kit，货号K210004）或CsCl密度梯度离心法来分离质粒DNA。 注意：我们推荐用户在制备质粒后通过一个限制性酶切分析来验证表达载体的完整性。
我们推荐用户在–20°C条件下储存腺病毒表达载体。由于它们相对较大，我们并不推荐在–80°C保存这些载体，因为在–80°C条件下，载体溶液将彻底冻结，–80°C条件下的过多反复冻融将会影响克隆效率。
pAd-DEST质粒很大（>34 kb），过多的操作有可能造成DNA的断裂，进而导致LR的重组效率降低。操作pAd-DEST质粒时，请勿剧烈涡旋或吹打质粒溶液。这些载体是以超螺旋的形式提供的，冻干处理和室温保存都可能造成质粒受损。只要能够有效避免DNA的断裂，冻融操作是可行的。
不，我们的系统使用的是5型人类腺病毒（Ad5）。
我们的ViraPower腺病毒表达载体的骨架源自5型人类腺病毒（Ad5）。
如果您希望实现稳定整合和进行筛选，请选择慢病毒系统。我们提供定向的TOPO（D-TOPO）和Gateway两种类型试剂盒，便于用户基因克隆操作的灵活性。如果您在寻找瞬时基因表达系统，请选择腺病毒系统。我们为此提供Gateway克隆方法。 不过还需要注意，这两种系统中的基因表达通常可在转导后24-48小时内进行检测，所以其实两种系统均可用于瞬时性质的实验。两者之间的主要区别在于慢病毒会整合于宿主细胞基因组中，而腺病毒不会。腺病毒能够获得更高的病毒滴度。
MOI（multiplicity of infection）是感染复数。理论上，MOI=1意味着平板中生长的每一个细胞被一个病毒颗粒所感染，MOI=10意味着每个细胞被10个病毒颗粒所感染。不过，许多因素都会影响最佳的MOI，如哺乳动物细胞系的天然属性，（分裂与不分裂细胞），转导效率，目的用途以及您的目的蛋白等。
当您第一次将构建的腺病毒或慢病毒转导进所选的哺乳动物细胞系中时，我们建议您尝试使用一系列MOI（0,0.5,1,2,5,10,50），以确定获得最佳基因表达效果所需的MOI值（使用慢病毒转导神经元时，我们一般使用超过50的MOI值——如MOI 100）。当您确定了获得最佳基因表达效果所需的MOI值后，后续的转导实验就都可应用这一优化的MOI值。
腺病毒表达通常适用于瞬时表达，而慢病毒表达一般用于长时间表达。腺病毒可在293A细胞中扩增数次，而浓缩慢病毒的方法一般只有离心。腺病毒需要宿主细胞表达CAR受体才能实现有效的转导，由于慢病毒颗粒上包被了VSVG膜蛋白，这些病毒能够适用于更广泛的哺乳动物细胞类型。
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
Here are possible causes and solutions:
- Too much crude viral stock used:
-Reduce the amount of crude viral stock used for transduction or dilute the crude viral stock.
-Amplify the adenoviral stock.
-Concentrate the crude viral stock.
- Wild-type RCA (replication-competent adenovirus) contamination: Screen for RCA contamination. Plaque purify to isolate recombinant adenovirus or prepare a new adenoviral stock.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Here are possible causes and solutions:
- Poor transduction efficiency due to:
-Mammalian cells not healthy: Make sure that your cells are healthy before transduction.
-Non-dividing cell type used: Transduce your adenoviral construct into cells using a higher MOI.
- MOI too low: Transduce your adenoviral construct into cells using a higher MOI.
- Low viral titer: Amplify the adenoviral stock using the procedure on page 20 of the manual (http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf).
- Adenoviral stock contaminated with RCA (replication-competent adenovirus):
-Screen for RCA contamination.
-Prepare a new adenoviral stock or plaque purify to isolate recombinant adenovirus.
- Cells harvested too soon after transduction: Do not harvest cells until at least 24 hours after transduction.
- Cells harvested too long after transduction: For actively dividing cells, assay for maximal levels of recombinant protein expression within 5 days of transduction.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Here are possible causes and solutions:
- Viral stocks stored incorrectly: Aliquot and store stocks at –80 degrees C. Do not freeze/thaw more than 10 times.
- Gene of interest contains a PacI site: Perform mutagenesis to change or remove the PacI site.
This could be due to insufficient dilution of the viral supernatant. We recommend titering the adenovirus stock using 10-fold serial dilutions ranging from 10e-4 to 10e-9.
Here are possible causes and solutions:
- Viral stocks stored incorrectly:
Aliquot and store stocks at –80 degrees C. Do not freeze/thaw more than 10 times.
- Incorrect titering of cell line used: Use the 293A cell line or any cell line with the characteristics discussed on page 23 of the manual http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf).
- Agarose overlay incorrectly prepared: Make sure that the agarose is not too hot before addition to the cells; hot agarose will kill the cells.
- Viral stock with very low titer or very high titer: Titer adenovirus using a wider range of 10-fold serial dilutions (e.g.,10e2 to 10e8).
Here are possible causes and solutions:
- Low transfection efficiency due to:
-Shearing of adenoviral Destination vector DNA: Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.
-Incomplete PacI digestion or digested DNA contaminated with phenol, ethanol, or salts: Repeat the Pac I digestion. Make sure purified DNA is not contaminated with phenol, ethanol, or salts.
-Unhealthy 293A cells; cells exhibit low viability: Use healthy 293A cells; do not overgrow cells.
-293A cells plated too sparsely on the day before transfection: Cells should be 90-95% confluent at the time of transfection.
-Plasmid DNA: transfection reagent ratio incorrect: Optimize such that plasmid DNA (in µg):Lipofectamine 2000 (in µL) ratio ranges from 1:2 to 1:3. If you are using another transfection reagent, optimize according to the manufacturer's recommendations.
- Viral supernatant too dilute: Concentrate virus using CsCl purification or any method of choice.
- Viral supernatant frozen and thawed multiple times: Do not freeze/thaw viral supernatant more than 10 times.
- Gene of interest is large: Viral titers generally decrease as the size of the insert increases; inserts larger than 6 kb (for pAd/CMV/V5-DEST) and 7.5 kb (for pAd/PL-DEST) are not recommended.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Here are possible causes and solutions:
- LR reaction transformed into an E. coli strain containing the F' episome and the ccdA gene: Use an E. coli strain that does not contain the
F' episome for transformation (e.g.,TOP10, DH5α-T1R).
- Deletions (full or partial) of the ccdB gene from adenoviral Destination vector:
- The adenoviral Destination vectors are provided in solution and are ready to use in an LR reaction. However, if you wish to propagate them, we recommend using One Shot ccdB Survival2 T1R Chemically Competent Cells (Cat. No. A10460).
- Select for transformants in media containing 50-100 µg/mL ampicillin and 15-30 µg/mL chloramphenicol, to maintain the integrity of the vector.
- Prepare plasmid DNA from one or more colonies and verify the integrity of the vector before use.
Here are possible causes and solutions:
- Incorrect antibiotic used to select for transformants: Select for transformants on LB agar plates containing 100 µg/mL ampicillin.
- LR recombination reaction not treated with proteinase K: Treat reaction with proteinase K before transformation.
- Too much entry clone DNA used in the LR reaction: Use 50-150 ng of the entry clone in the LR reaction.
- Inappropriate ratio of entry clone:DEST vector used in the LR reaction: Aim for a 1:1 molar ratio of entry clone:DEST vector.
- LR recombination of >5 kb insert only incubated for 1 hr: For inserts larger than 5 kb, we recommend to incubate the LR reaction overnight. Note: This overnight incubation will also boost colony count for smaller inserts.
- Adenoviral Destination vector DNA was sheared: Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.
- Didn't use the suggested amount of LR Clonase II enzyme mix or LR Clonase II enzyme mix was inactive:
-Make sure to store the LR Clonase II enzyme mix at –20 degrees C.
-Do not freeze/thaw the LR Clonase II enzyme mix more than 10 times.
-Use the recommended amount of LR Clonase II enzyme mix (see page 14 of the manual [http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf]).
-Test another aliquot of the LR Clonase II enzyme mix.
- Not enough LR reaction transformed: Transform 2-3 µL of the LR reaction into the appropriate competent E. coli strain. Use E. coli cells with a transformation efficiency >1 x 10e8 cfu/µg.
- Not enough transformation mixture plated: Increase the amount of E. coli plated.
The ViraPower Adenoviral Expression System includes the following features designed to enhance its biosafety:
- The entire E1 gene is deleted in the adenoviral expression vectors (pAd/CMV/V5-DEST and pAd/PLDEST) and supplied in trans in the 293A producer cell line. Since expression of E1 (E1a and E1b) proteins is required for the expression of the other adenoviral viral genes (e.g., late genes), adenovirus produced using this system is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins.
- The E3 gene is completely dispensable for in vitro applications and hence is deleted as well from the adenoviral expression vector backbone.
- The adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
Despite the presence of the above safety features, the adenovirus produced can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat adenoviral stocks generated using this system as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating adenovirus carrying potential harmful or toxic genes (e.g., activated oncogenes) or when producing large-scale preparations of virus (see page 10 of the manual [http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf]).
For more information about the BL-2 guidelines and adenovirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories,” 4th Edition, published by the Centers for Disease Control (CDC) (www.cdc.gov/biosafety/publications/index.htm).
Getting a cell transfected and observing productive viral transduction are two different things. If only one or two cells in your lawn are producing virus, it will take quite a while for that to be visible to the naked eye (longer than most are willing to wait). Transfection efficiency is correlated with virus production because the more cells you get DNA into, the higher chance you have of seeing virus production within the first week or two. If your transfection efficiency is low, you will eventually see virus being produced, but you have to wait a long time to see it.
The pAd/CMV/V5-DEST or pAd/PL-DEST adenoviral constructs do not integrate into the host genome. Once transduced into the mammalian cell of interest, your recombinant protein is expressed as long as the viral genome is present. For actively dividing cells, transgene expression decreases over time and can be down to background levels within 2 weeks after transduction. In non-dividing cells such as quiescent CD34+ cells or animal tissues (skeletal muscle, neurons, liver), transgene expression is more stable and can persist for as long as 6 months post-transduction.
In actively dividing cells (doubling time of every 24 hours), we have found that transgene expression is generally detectable within 24 hours of transduction, with maximal expression observed at 48-96 hours (2-4 days) post-transduction. Expression levels generally start to decline after 5 days post-transduction. In cell lines that exhibit longer doubling times or in non-dividing cell lines, high levels of transgene expression persist for a longer period of time.
Human adenovirus type 5 (Ad5) enters target cells via the coxsackie virus and adenovirus receptor (CAR), followed by an integrin-mediated internalization mechanism. CAR/integrin proteins are ubiquitously present on mammalian cells, thus affording adenovirus the ability to transduce a very broad range of cell types. If your specific cell type has very low expression of CAR, adenoviral transduction will be inefficient, in which case you may need to use a very high MOI (in the 100s) to get good expression.
The backbone for our ViraPower adenoviral expression vectors is human adenovirus type 5 (Ad5). Ad5 entry into cells is achieved by binding to the coxsackie virus and adenovirus receptor (CAR), followed by an integrin-mediated internalization mechanism. For target cells that have sufficient expression of the CAR receptor and are actively dividing, it should be possible to get adenovirus transduction efficiencies in the range of 80-90%, as long as an adequate MOI is used.
Note: There is variability in the transduction efficiencies of different cell types. Example: In HT1080 cells, which are readily transduced with adenovirus, transduction efficiencies are around 90% with an MOI of 1. In some cell types, you may need to use a 10-fold higher MOI to get the same transduction efficiency.
Crude adenovirus titers are generally 1 x 10e7 to 1 x 10e8 plaque forming unts (pfu)/mL. You can use this stock to infect a new batch of 293A cells to generate a higher-titer viral stock (i.e., amplify the virus). Amplification allows production of a viral stock with a titer ranging from 1 x 10e8 to 1 x 10e9 pfu/mL. Adenovirus can be concentrated to titers as high as 1 x 10e11 pfu/mL using a variety of methods (e.g., CsCl purification).
Adenovirus can be concentrated to titers as high as 1 x 10e11 pfu/mL using a variety of methods (e.g., CsCl purification; please find a reference on page 25 of the manual [http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf]).
Once you have created a crude viral stock, you can use this stock to infect a new batch of 293A cells to generate a higher-titer viral stock (i.e., amplify the virus). The titer of the initial viral stock obtained from transfecting 293A cells generally ranges from 1 x 10e7 to 1 x 10e8 plaque forming units (pfu)/mL. Amplification allows production of a viral stock with a titer ranging from 1 x 10e8 to 1 x 10e9 pfu/mL and is generally recommended. Please refer to page 19 in the manual (http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf) for specific instructions for amplification.
Note: Other 293 cell lines or cell lines expressing the E1 proteins are suitable for amplification.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend aliquoting adenoviral stocks immediately after production into small working volumes, and storing at –80 degrees C for long-term storage. Since adenovirus is non-enveloped, viral stocks remain relatively stable and some freezing and thawing of the viral stocks is acceptable. We do not recommend freezing and thawing viral stocks more than 10 times, as loss of viral titer can occur. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Most of the adenovirus is contained within the floating cells and is not released into the medium until those cells burst. We recommend changing the medium every 3 days or so until it is obvious that a lot of cells become big and rounded and are detaching from the plastic. Once a cell bursts, the free viruses rapidly infect the neighboring cells. If you're ever worried that you're losing infected cells (and therefore potential virus) in your medium changes, you can always save the medium with the floating cells, freeze/thaw it 3 times and then use a little (maybe 1/10th) and add it back to your culture with fresh media. Or, replace only half of the medium with fresh medium and do this more often than every three days.
Any 293-derived cell line or other cell line that expresses the E1 proteins may be used to produce adenovirus. In 293A cells (recommended for adenovirus production), "A" stands for "adherent" because the 293A cells (which are just a single-cell clone of regular 293) tend to adhere and form nice flat monolayers in tissue culture dishes. This is why they work so well for plaque assays. Regular 293 cells will not form the same type of monolayers; they exhibit holes and gaps during growth.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) and use the following plasticware for 293A cells:
T175: Fisher Cat. No. 10-126-13; this is a Falcon flask with a 0.2 µm vented plug seal cap.
T75: Fisher Cat. No. 07-200-68; this is a Costar flask with a 0.2 µm vented seal cap.
100 mm plate: Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate.
We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).
The size of the wild-type adenovirus type 5 genome is approximately 35.9 kb. Studies have demonstrated that recombinant adenovirus can efficiently package up to 108% of the wild-type virus size from E1- and E3-deleted vectors. Taking into account the size of the elements required for expression from each adenoviral destination vector, make sure that your DNA sequence or gene of interest does not exceed the size indicated for efficient packaging (see below for packaging limits for individual vectors):
pAd/CMV/V5-DEST: 6 kb
pAd/PL-DEST: 7.5 kb
You can transfect your adenoviral construct into your expression cell line (or the 293A cells) to see if the protein will be expressed without waiting the two weeks it takes to make virus. Transfection efficiency will be low due to the large size of the plasmid, so it may require adding more lipid-DNA complexes to the medium than indicated in the ViraPower Adenoviral Expression System manual. The adenoviral construct should not be digested with Pac I when doing this, as supercoiled plasmids transfect more efficiently. If checking expression in 293A cells, harvest 2-3 days post-transfection.
Before you can transfect your expression clone into 293A cells, you must expose the left and right viral inverted terminal repeats (ITRs) on the vector to allow proper viral replication and packaging. This also removes bacterial sequences (i.e., pUC origin and ampicillin resistance gene). Both pAd/CMV/V5-DEST and pAd/PL-DEST ;vectors contain Pac I restriction sites (see maps on pages 20 and 22 of the manual (http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf), respectively, for the location of the Pac I sites).
Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. If you are unable to use the Pac I site, you can use the Swa I site.
Once you have generated your pAd/CMV/V5-DEST or pAd/PL-DEST expression clone, you may use any method of choice to prepare purified plasmid DNA. We recommend isolating plasmid DNA using the PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004) or CsCl gradient centrifugation.
Note: We recommend performing restriction analysis to verify the integrity of your expression construct after plasmid preparation.
We recommend storing adenoviral expression vectors at –20 degrees C. Due to their relatively large size, we do not recommend storing these vectors at –80 degrees C, as the vector solution will completely freeze and too many freeze thaws from –80 degrees C will affect the cloning efficiency.
The pAd-DEST plasmids are large (>34 kb in size) and excessive manipulations can shear the DNA, resulting in reduced LR recombination efficiency. When working with pAd-DEST plasmids, do not vortex or pipet the solution vigorously. These vectors are supplied supercoiled, as lyophilization methods and room temperature storage may result in plasmid damage. Freeze thaws are acceptable as long as shearing is prevented.
No, our system uses human adenovirus type 5 (Ad5).
The backbone for our ViraPoweradenoviral expression vectors is human adenovirus type 5 (Ad5).
If you are interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO (D-TOPO) and Gateway version of the kit to provide flexibility in the cloning of the gene of interest. If you are looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have th CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.
It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
MOI stands for multiplicity of infection. Theoretically, an MOI of 1 will provide 1 virus particle for each cell on a plate, while an MOI of 10 represents ten virus particles per cell. However, several factors can influence the optimal MOI including the nature of your mammalian cell line, (non-dividing vs. dividing), transduction efficiency, your application of interest, and your protein of interest.
When transducing your adenoviral or lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOIs (0, 0.5, 1, 2, 5, 10, 50) to determine the MOI required to obtain optimal gene expression. MOIs greater than 50, such as MOI 100, are common for the transduction of neurons with lentivirus. After you determine the MOI that gives optimal gene expression, subsequent transductions can be performed at the optimal MOI.
Adenoviral expression is used for transient expression, whereas lentiviral expression is used for longer-term expression. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have the CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.