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查看更多产品信息 pAd/CMV/V5-DEST™ Gateway™ Vector Kit - FAQs (V49320)
139 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）。
这里列举了一些可能的原因与解决方案：
•所用检测法可能不适当或不够灵敏： ◦我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
•筛选到的克隆数不够：至少筛选出20个克隆。
•在稳转筛选中使用了不适当的抗生素浓度：请确保正确获取了抗生素的杀死曲线。由于某一既定抗生素的效力依赖于细胞类型，血清，培养基和培养技术，因此必须在每次进行稳定筛选的时候确定抗生素的用量。如果采用的培养基或血清条件明显不同，则即使是我们所提供的稳转细胞系对于我们推荐的剂量也可能出现更敏感或更不敏感的情况。
•基因产物（即使低水平）的表达可能与该细胞系的生长不相容（如毒性基因）：使用一个可诱导的表达系统。
•阴性克隆可能由基因表达的关键载体位点处优先发生了线性化所致：在一个不影响表达的位点实施载体线性化，如在细菌抗药性标志物序列中。
这里列举了一些可能的原因与解决方案：
•尝试试剂盒自带的表达对照。
•可能的检测问题：
◦检测瞬转的表达蛋白可能有难度，因为转染效率可能过低，以致用于整个转染群体的评估手段无法成功实现检测。我们推荐您通过稳转筛选或采用能够逐个检测单一细胞的技术手段来优化您的转染操作。您也可尝试通过改变启动子或细胞类型来提高表达水平。
◦细胞中的蛋白表达水平对于所选择的检测方法来说可能过低。我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
◾蛋白可能降解或截短了：使用Northern杂交进行检测。
◾可能的时程问题：由于蛋白表达随时间延长而发生的变化依赖该蛋白的天然属性，我们一般推荐您先获取一份表达的时程曲线。尝试进行一次时程分析将帮助您确定最优的表达时间窗。
◾可能的克隆问题：通过限制性酶切和/或测序来验证克隆。
不可以；新霉素对哺乳动物细胞有毒性。我们推荐您使用Geneticin（又称 G418硫酸盐)，这一产品的毒性较低，是在哺乳动物细胞中进行有效筛选的新霉素的替代品。
即使缺乏Kozak序列，翻译也还是会在核糖体遇到的第一个ATG处启始，不过启始效率可能相对较低。只要处于最初ATG的阅读框内，任何下游的插入序列都可能表达为融合蛋白，不过如果这里没有Kozak保守序列，则蛋白的表达水平预期会比较低。如果载体中包含一个非Kozak型的保守ATG，我们则推荐您将基因克隆至该ATG上游，再包含一个Kozak序列来优化表达效果。
我们提供pJTI R4 Exp CMV EmGFP pA载体，货号A14146，您可使用这一产品来监控转染和表达情况。
我们推荐使用One Shot ccdB Survival 2 T1^R 感受态细胞，货号A10460。该菌株能够耐受ccdB基因的毒性效应。
注意： 请勿使用常规的大肠杆菌克隆株 - 包括TOP10或DH5α - 来进行扩增和培养，因为这些菌株均对ccdB的效应很敏感。
在小鼠细胞系中，人们已知CMV启动子的效率会随时间延长而逐渐下降。因此，我们推荐您使用一款非CMV型的载体，如EF1α或UbC启动子，以在小鼠细胞系中长时间表达蛋白。
保守的Kozak序列为A/G NNATGG，其中的ATG表示起始密码子。ATG周围的核苷酸点突变会影响翻译效率。尽管我们通常情况下都推荐加入一段Kozak保守序列，不过这一操作的必要性还是基于具体的目的基因，一般只需ATG就足以高效地启始翻译过程。最佳的建议是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有具有N-融合表达的表达载体都已经包含了一个翻译起始位点。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
理论上，pDONR载体在BP反应时对插入片段没有大小的限制。我们自己测试过的最大片段是12 kb。TOPO载体对插入片段大小更敏感一些，要获得较高的克隆效率其插入片段长度的上限是3-5 kb。
在得到attB-PCR产物之后，我们建议对产物进行纯化以去除PCR缓冲液，残留的dNTP，attB引物，以及attB引物二聚体。引物和引物二聚体在BP反应中会高效的与供体载体重组，因而会增加转化E. coli时的背景，而残留的PCR缓冲液可能会抑制BP反应。使用酚/氯仿抽提，加醋酸铵和乙醇或异丙醇沉淀的标准PCR产物纯化方案不适合对attB-PCR产物进行纯化，因为这些实验方案通常仅能去除小于100 bp的杂质，而在去除较大的引物二聚体时效果不佳。我们推荐一种PEG纯化方案（请参见使用Clonase II的Gateway技术手册第17页）。如果使用上述实验方案您的attB-PCR产物仍然不够纯，您可以进一步对其进行凝胶纯化。我们推荐使用Purelink Quick 凝胶纯化试剂盒。
请检查您所用的菌株的基因型。我们的Gateway目的载体通常含有一个ccdB基因元件，该元件如果不被破坏，则E. Coli生长将受到抑制。因此，未进行克隆的载体应该在ccdB survival菌株如我们的ccdB Survival 2 T1R感受态细胞中扩增。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
目的基因必须两端带有合适的att位点，或者是入门克隆中的attL (100 bp)位点，或者是PCR产物中的 attB (25 bp)位点。对于入门克隆而言，所有位于attL位点之间的部分都将被转移到含有attR位点的Gateway目的载体中，而两端带有attB位点的PCR产物需被转移到一个含有attP位点的供体载体，例如pDONR221。
翻译起始位点的位置，终止子，或者用于表达的融合标签必须在最开始的克隆设计中考虑到。例如，如果您的目的载体包含一个N末端标记而非C末端标记，则该载体应当已经带有合适的翻译起始位点，但是终止子应当被包含在插入片段当中。
小抽（碱裂解）纯化的DNA即适用在Gateway克隆反应中。重要的一点是要将RNA污染去除干净以便得到精确的定量。推荐使用通过我们的S.N.A.P. 核酸纯化试剂盒，ChargeSwitch试剂盒，或PureLink试剂盒纯化的质粒DNA。
理论上没有片段大小限制。长度在100 bp到11 kb之间的PCR产物可以被直接克隆到pDONR Gateway载体中。其它DNA片段如带有att位点的150 kb DNA片段可以成功和一个Gateway兼容载体发生重组。对于大的插入片段，推荐进行过夜孵育反应。
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).
Here are possible causes and solutions:
Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.
Here are possible causes and solutions:
- Try the control expression that is included in the kit
Possible detection problem:
- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
Protein might be degraded or truncated: Check on a Northern.
Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
Possible cloning issues: Verify clones by restriction digestion and/or sequencing.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.
Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.
We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.
We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.
The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.
Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.
LR Clonase II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway LR reactions. LR Clonase and LR Clonase II enzyme mixes are not recommended for MultiSite Gateway LR recombination reactions, but LR Clonase II Plus is compatible with both multi-site and single-site LR recombination reactions.
When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.
No. The ViraPower system uses adenovirus type 5. Adenoviruses (Adenoviridae) and adeno-associated viruses (Parvoviridae) are completely different. Adeno-associated viruses are often associated with adenovirus infections, hence the name. Since they are thought to be virtually non-pathogenic, they are attractive vectors for gene therapy. The disadvantage is that they can package only about half the foreign DNA that adenoviruses can.
Clone your gene of interest into the pAd/CMV/V5-DEST (or pAd-PL-DEST if you want to use your own promoter). Prior to cloning, if desired, propagate this vector in One Shot ccdB Survival 2 T1R Competent Cells (Cat. No. A10460) as described below. After cloning your gene of interest, propagate in E. coli strain TOP10. pAd/CMV/V5-GW/lacZ is provided as a positive control vector for expression.
Digest recombinant plasmid with Pac I to expose the ITRs (inverted terminal repeats).
Transfect (we recommend Lipofectamine 2000 reagent) E1-containing cells (293A cells) with linear DNA (only 10% of transfected cells will make virus).
Infected cells will ball up, and release virus to surrounding cells, which in turn will be killed and ball up. Look for plaques in the monolayer created by areas cleared by detaching, balled up cells (it takes 8-10 days to see visible plaques from this initial transfection).
Collect a crude viral lysate.
Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate. Harvest virus after 2-3 days when cells ball up. Determine the titer of the adenoviral stock by performing a plaque assay. The virus generated is adenovirus type 5 (subclass C).
Add the viral supernatant to your mammalian cell line of interest to transduce cells.
Assay for recombinant protein of interest.
Once you have your gene of interest in the adenoviral vector, you can simply re-amplify when you need more of the virus. You do not need to repeat cloning steps and transfections each time.
When cloning or propagating DNA with unstable inserts (such as lentiviral DNA containing direct repeats), we recommend using the following modifications to reduce the chance of recombination between direct repeats:
- Select and culture transformants at 25-30 degrees C.
- Do not use "rich" bacterial media as they tend to give rise to a greater number of unwanted recombinants.
-If your plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 µg/mL chloramphenicol in addition to the antibiotic appropriate for selection of your plasmid.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Ultracentrifugation is the most commonly used approach and is typically very successful (see Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037; Reiser (2000) Gene Ther 7:910-913). Others have used PEG precipitation. Some purification methods are covered by patents issued to the University of California and Chiron.
Adenovirus is concentrated using CsCl density gradient centrifugation (there is a reference for this procedure in our adenovirus manual) or commercially available columns.
This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.
We use the following plasticware for 293A and 293FT cells:
T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 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).
Viral vectors:
Store lentiviral and adenoviral expression vectors (plasmid DNA) at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely. Glycerol stocks of vectors transformed into bacteria should always be stored at -80 degrees C.
Virus:
Both adenovirus and lentivirus particles should be aliquoted immediately after production and stored at -80 degrees C.
Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.
Adenovirus particles can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.
When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Thermo Fisher Scientific has also engineered specific safety features into the lentiviral system.
Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If you're 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're looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. 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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.
For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST vector: 6 kb
pLenti6/V5-DEST vector: 6 kb
pLenti6/V5/D-TOPO vector: 6 kb
pLenti6/UbC/V5-DEST vector: 5.6 kb
For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST vector: 6 kb
pAd/PL-DEST vector: 7.5 kb
In most cases, there will not be enough pENTR vector DNA present to go directly from TOPO cloning into an LR reaction. You need between 100-300 ng of pENTR vector for an efficient LR reaction, and miniprep of a colony from the TOPO transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO reaction using pENTR/D, or SD TOPO, or pCR8/GW/TOPO vectors:
1. Heat inactivate the topoisomerase after the TOPO cloning reaction by incubating the reaction at 85 degrees C for 15 minutes.
2. Use the entire reaction (6 µL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 µL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO reaction is very inefficient and will result in a very low colony number, if any at all.
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO adapted Gateway entry vectors. All TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No, not directly. The attB-PCR product must first be cloned, via a BP Clonase reaction, into a pDONR vector which creates an "Entry Clone" with attL sites. This clone can then be recombined, via an LR Clonase reaction, with a Destination vector containing attR sites. However, It is possible to perform both of these reactions in one step using the "One-Tube Protocol" described in the manual entitled "Gateway Technology with Clonase II".
Yes, this can be done using the Multisite Gateway Technology. MultiSite Gateway Pro Technology enables you to efficiently and conveniently assemble multiple DNA fragments - including genes of interest, promoters, and IRES sequences - in the desired order and orientation into a Gateway Expression vector. Using specifically designed att sites for recombinational cloning, you can clone two, three, or four DNA fragments into any Gateway Destination vector containing attR1 and attR2 sites. The resulting expression clone is ready for downstream expression and analysis applications.
For the BP reaction, approximately 5-10% of the starting material is converted into product. For the LR reaction, approximately 30% of the starting material is converted into product.
The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.
We do have an alternative method called the "attB Adapter PCR" Protocol in which you make your gene specific primer with only 12 additional attB bases and use attB universal adapter primers. This protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. You can find the sequence of these primers in the protocol on page 45 of the "Gateway Technology with Clonase II" manual.
There is a protocol in which all 4 primers mentioned above are in a single PCR reaction. You can find this protocol at in the following article: Quest vol. 1, Issue 2, 2004. https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/quest-archive.reg.in.html. The best ratio of the first gene-specific and the second attB primers was 1:10.
We do not offer pre-made primers, but we can recommend the following sequences that can be ordered as custom primers for sequencing of pDONR201:
Forward primer, proximal to attL1: 5'- TCGCGTTAACGCTAGCATGGATCTC
Reverse primer, proximal to attL2: 5'-GTAACATCAGAGATTTTGAGACAC
1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.
2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.
3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).
4. High-throughput Yeast Two-Hybrid Assays for Large-Scale Protein Interaction mapping. Methods: A Companion to Methods in Enzymology 24(3), pp.297-306
5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.
6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.
7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.
8. Protein-over expression and crystallography Evdokimov, A.G., Anderson, D.E., Routzahn, K.M. & Waugh, D.S.
9. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis. Acta Crystallography (2000) D56, 1676-1679.
10. Evdokimov, et al. Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallographica D57, 793-799 (2001).
11. Lao, G. et al. Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells. Cryobiology 43(2):106-113 (2001).
12. High-throughput cloning and expression Albertha J. M. Walhout, Gary F. Temple, Michael A. Brasch, James L. Hartley, Monique A. Lorson, Sander Van Den Huevel, and Marc Vidal.
13. Gateway Recombinational Cloning: Application to the Cloning of Large Numbers of Open Reading Frames or ORFeomes. Methods in Enzymology, Vol. 328, 575-592.
14. Wiemann, S. et.al., Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs, Genome Research (March 2001) Vol. 11, Issue 3, pp.422-435
15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination
16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)
17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs
18. Loftus S K et al. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 31;8(5):221 (2001).
19. James L. Hartley, Gary F. Temple and Michael A. Brasch. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research (2000) 10(11), pp. 1788-1795.
20. Reboul et al. Open-reading frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nature Genetics 27(3):332-226 (2001).
21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).
The attP1 sequence (pDONR) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]
The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.
The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN
attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
The sequence in brackets comes from attB, and N is your gene-specific sequence.
Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway Entry vectors. The backbone in the case of PCR BP cloning is pDONR201.
There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO adapted Gateway Entry vector is approximately 5 kb. A Gateway recombination reaction can occur between DNA fragments that are as large as 150 kb.
Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag), the N-terminus of an expressed protein will contain an additional 9 amino acids from the attB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.
Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.
Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case, the attB sites do not interfere with transcription or translation.
Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.
Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.
Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence
No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
The Gateway nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.
The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway compatible TOPO Cloning vectors such as pCR8/GW/TOPO and pENTR/D-TOPO. The final option is to use restriction enzymes to clone into a pENTR Dual Selection vector.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.
BP Clonase II and LR Clonase II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.
These enzymes are more stable than the original BP and LR Clonase and can be stored at -20 degrees C for 6 months.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P. nucleic acid purification kits, ChargeSwitch kits, or PureLink kits are recommended.
A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.
This depends on whether you are expressing a fusion or a native protein in the Gateway destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.
The Gateway attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).
Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.
No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.
The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.
There is no theoretical size limitation. PCR products between 100 bp and 11 Kb have been readily cloned into a pDONR Gateway vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway-compatible vector. Overnight incubation is recommended for large inserts.
Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway cloning (about 50 bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.
Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
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