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查看更多产品信息 ViraPower™ II Lentiviral C-Lumio™ Gateway™ Expression System - FAQs (K37020)
177 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。
可能的原因包括：
- 用于转导操作的病毒上清液体积过大
- 细胞对Polybrene试剂敏感
- 使用过多的抗生素进行筛选
- 转导操作后过早应用抗生素
- 目的基因对细胞有毒性
表达量过低可能是由于以下因素导致的： 低转导效率，MOI值过低，使用过多的抗生素进行筛选， 转导操作后过早应用抗生素， 转导操作后过早收获细胞， 目的基因对细胞有毒性，或DNA表达质粒中的LTR区域发生重排。
这里列举了一些可能的原因与解决方案：
- 启动子被沉默：CMV启动子有被沉默的倾向，特别是在小鼠或大鼠细胞中。筛选多个耐受抗生素的细胞克隆，并挑选其中表达水平最高的一个。
- 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3次。
这里列举了一些可能的原因与解决方案：
- 使用过少的抗生素进行筛选：增加抗生素用量。
- 在长满的细胞上进行筛选操作：加入筛选培养基之前，对转导细胞进行胰酶消化，并将其重新种植在更大的组织培养板中
- 病毒上清液未经充分稀释：以更宽的稀释度（以10倍为单位，如10-2至10-8）来测定慢病毒的滴度。
这里列举了一些可能的原因与解决方案：
低转染效率： 请使用高质量的质粒制备试剂盒；使用16次传代以内的健康293FT细胞；转染过程中请去除Geneticin；使用正确的DNA:脂质体比例；以适当的密度种植细胞。
转染的细胞没有培养于含有丙酮酸钠的培养基中：丙酮酸钠能够为细胞提供额外的能量来源。
过早收获病毒上清液：通常在转染48-72小时后收集病毒上清液。
病毒上清液稀释过度： 通过CsCl离心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3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；不推荐插入片段长于5.6 kb。
DNA表达质粒中的LTR区域发生重排：请确保使用Stbl3细胞进行慢病毒质粒的转化。
选择不适当的细胞系开展病毒滴度测定：应使用HT1080细胞或其他类似细胞系。
转导过程中未加入Polybrene试剂：在Polybrene试剂存在的条件下向细胞中转导慢病毒。
脂质体操作不当：确保适当保存和混匀试剂。
应用荧光显微镜来检查HiPerform FastTiter慢病毒的滴度。
如果转染后不久（4小时至过夜）293FT细胞就从平板上脱离下来：
•这可能是Lipofectamine 2000的毒性所致。可能在转染前细胞种植密度过低。
•用户对这些细胞的操作可能不够轻柔（这些细胞会有易于漂浮的倾向）。
•这些细胞可能在室温条件下放置时间过长。
如果在转染48-72小时内细胞脱离：
•如果细胞大片脱离，这可能是慢病毒产生的一个标志。
通过小量制备获得慢病毒DNA的得率一般非常低，这是由于载体骨架中存在着LTR序列的缘故。因此，我们不推荐通过小量制备试剂盒来提取慢病毒质粒。我们推荐用户使用S.N.A.P.中量制备试剂盒（MidiPrep Kit，货号K191001）或PureLink HiPure质粒中量制备试剂盒（货号K210004）来制备慢病毒载体DNA，这两款试剂盒的重悬缓冲液（Resuspension Buffer）中均包括10 mM EDTA。由于慢病毒DNA的中量制备通常得率也较低，我们推荐用户依照专用实验方案进行操作，以提高得率——简言之，慢速培养细胞、每个抽提提柱加入少量细胞、每次DNA中量制备使用100 mL慢病毒培养物。
注意：如果您希望在克隆操作/克隆筛选过程中小量制备慢病毒质粒，我们推荐您使用PureLink HQ小型试剂盒（Mini Kit，货号K210001），并按照说明书来开展实验，只有一处需要优化：使用50 mL TE，pH 8.0缓冲液洗脱一次。通过该方法获得的DNA得率一般极低——100–150 ng/mL（即一共5–7 mg）。其OD 260/280的通常介于1.8和2.1。
我们强烈建议使用Stbl3大肠杆菌来克隆慢病毒载体。Stbl3大肠杆菌的基因组中携带了recA13突变，能够帮助减少LTR之间发生不必要重组事件的可能性。在转化Stbl3大肠杆菌之后，我们推荐用户挑取克隆并通过Afl II和Xho I消化和和小量制备操作来对慢病毒DNA进行验证。我们所推出的全部慢病毒载体的5'和3’LTR中均含有Afl II位点，而MCS的3'端还有一个Xho I位点。假设Afl II仅在LTR位点中进行切割，而插入片段中不含Afl II或Xho I位点，则AfI II + Xho I消化预期会产生3段 DNA片段。任何非预期的DNA片段都将推定为LTR重组的结果。只有符合预期的DNA片段切割模式的克隆会被挑出，而进入后续的中量制备步骤。
ViraPower 慢病毒表达系统在提升生物安全性方面，设计了以下特性：
•pLenti表达载体的3'LTR中被删去了一段序列（ΔU3），这不会影响生产细胞系中病毒基因组的形成，但会在靶细胞转导后形成慢病毒的“自失活”效应（参考文献：Yee JK, Moores JC, Jolly DJ, Wolff JA, Respess JG, Friedmann T (1987) Gene Expression from Transcriptionally Disabled Retroviral Vectors.Proc.Natl.Acad.Sci.USA 84:5197-5201; Yu SF, Ruden Tv, Kantoff PW, Garber C, Seiberg M, Ruther U, Anderson WF, Wagner EF, Gilboa E (1986) Self-Inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells.Proc.Natl.Acad.Sci.USA 83:3194-3198; Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Selfinactivating Lentivirus Vector for Safe and Efficient in vivo Gene Delivery.J. Virol.72:9873-9880).一旦整合进转导的靶细胞，慢病毒基因组就不再能产生可包装的病毒基因组了。
•本系统所采用HIV-1的基因数目被减至三个（即gag，pol和rev）。
•同时使用源自水泡性口膜炎病毒的VSV-G基因代替了HIV-1的包膜（参考文献：Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993) Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors:Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells.Proc.Natl.Acad.Sci.USA 90:8033-8037; Emi N, Friedmann T, Yee JK (1991) Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus.J. Virol.65:1202-1207; Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T (1994) A General Method for the Generation of High-Titer, Pantropic Retroviral Vectors:Highly Efficient Infection of Primary Hepatocytes.Proc.Natl.Acad.Sci.USA 91:9564-9568).
•编码结构及病毒基因组包装所需其它元件的基因被分别独立插入四个质粒中。所有这四个质粒之间经设计，均不含任何同源序列，以避免相互之间发生不良的重组事件而形成具有复制能力的病毒（参考文献：Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System.J. Virol.72:8463-8471).
•尽管三种包装质粒能够在293FT细胞中反向表达生成子代病毒所需的蛋白（如gal，pol，rev，env），但它们都不含有LTR或Ψ包装序列。这意味着包装后的病毒基因组中实际上不会含有任何HIV-1的结构基因，因此，也不会在转导后的靶细胞中表达。转导后的细胞将无法生成具有复制能力的新病毒。
•本系统所生成的慢病毒颗粒不能自主复制，仅携带目的基因序列。本系统也不会生成其它类型的病毒。
•由于gag/pol mRNA转录本中HIV-1的RRE序列，pLP1中gag与pol基因的表达是Rev依赖性的。添加RRE序列能够避免在Rev不存在的情况下发生gag与pol的表达（参考文献：Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System.J. Virol.72:8463-8471).
•在pLenti表达载体中，组成型启动子（RSV启动子）位于5'LTR的上游，以补偿病毒RNA的有效形成过程对tat的依赖（参考文献：Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System.J. Virol.72:8463-8471).
尽管有以上安全特性方面的设计，所生成的慢病毒仍具有某些生物危害方面的风险，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慢病毒液，并严格遵守全部已发表的BL-2准则。此外，在生成包含潜在有毒或有害基因（如激活型致癌基因）的慢病毒过程中，用户应格外小心。
如需了解更多关于BL-2准则以及慢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.htm）。
通过我们所提供的慢病毒系统产生的慢病毒不携带或表达任何病毒基因，因此没有相关的毒性问题。只有LTR位点之间的蛋白编码区会整合进入哺乳动物细胞染色体并实现表达。由于这些慢病毒内部的安全特性设计，其自身不会发生复制。
将慢病毒更久保留在细胞培养体系中的顾虑在于潜在的细胞毒性或影响生长。如果您绝对不能从细胞中去除病毒，我们则建议您将其留在培养体系中，并根据经验对细胞状态进行监控。
注意：这些基于HIV的性质。
形态：病毒颗粒具有复杂的结构，具体由一层包膜，一层核衣壳，一个拟核和一个基质蛋白所组成。病毒颗粒被包膜所包裹，具有球形至多形的形态，直径在80-100 nm。病毒颗粒的表面密集和均匀地覆盖着不明显的小尖峰。表面突起有8 nm长。病毒颗粒核心为棒状或截短的锥形。具有同轴的拟核。
物化与物理性质：病毒颗粒在蔗糖中的浮力密度为1.13–1.18 g cm–3。病毒颗粒对于热、去垢剂和甲醛等处理敏感。其感染性不受辐照影响。
蛋白：蛋白成份占整个粒子质量的60%左右。病毒基因组编码了结构型蛋白与非结构型蛋白。病毒颗粒由5种主要的结构型蛋白和3种非结构型蛋白所组成。病毒基因组编码了一个RNA依赖的DNA聚合酶。
脂类：包膜中存在着脂类。脂质成份占整个粒子质量的35%左右。病毒脂质的组成与宿主细胞膜的组成类似。脂类来源于宿主的细胞膜。
碳水化合物：病毒颗粒约3%的质量来源于碳水化合物。
Polybrene试剂（海地美溴铵）是Sigma-Aldrich提供的阳离子聚合物（货号H9268），能通过中和病毒颗粒与细胞表面之间的电荷斥力来增强转导效率。为了获得最佳结果，我们推荐用户在含有Polybrene试剂的条件下开展转导操作。不过请注意，某些细胞对Polybrene试剂（如原代神经元）比较敏感。因此，在开展转导实验之前，您可能要测试细胞系对Polybrene试剂（0–10 μg/mL）的敏感性；如果细胞表现出毒性效应或表型改变，则应避免使用Polybrene试剂。
我们通过研究发现，以大约MOI=1进行慢病毒转导，通常情况下在活跃分裂的细胞系（如HT 1080）中有80-90%成功表达了靶基因。一些非分裂型细胞转导慢病毒的效率较低。举例来说，以大约MOI=1进行转导，人原代成纤维细胞表达靶基因的效率只有50%左右。如果需要向非分裂型细胞中转导慢病毒，您可能需要增加MOI值（如MOI=10）来获得更好的重组蛋白表达水平。如果您首次使用慢病毒转导哺乳动物细胞，我们推荐您尝试一个MOI范围（如0，0.5，1，2，5，10），以帮助在您的应用中确定获得最佳蛋白表达效果所需的MOI值。
慢病毒包膜是由水泡性口膜炎病毒糖蛋白（VSV-G）包被的，这样慢病毒和靶细胞之间就能够以不依赖受体的方式完成相互作用。其结果是，慢病毒就拥有了更广泛的兼容性，进而在理论上适用于任何类型哺乳动物细胞的转导操作。这一不依赖受体而进入靶细胞的过程类似于内吞作用（Espenshade et al.(2002) Proc Natl Acad Sci U S A 99:11694; Aiken (1997) J Virol 71:5871)。
您可通过针对病毒基因的PCR/qPCR分析法来检测慢病毒滴度，但请切记，通过这一方法测得的病毒滴度并非功能性病毒特异性的滴度，因为非活性病毒和活性病毒都将被这一方法检测出来。其结果是，使用这一方法测得的病毒滴度通常比用杀稻瘟菌素筛选等方法测得的功能性病毒滴度高10倍以上。
您可使用p24 ELISA分析法来检测慢病毒滴度，但请切记，通过这一方法测得的病毒滴度并非功能性病毒特异性的滴度，因为非活性病毒和活性病毒都将被这一方法检测出来。其结果是，使用这一方法测得的病毒滴度通常比用杀稻瘟菌素筛选等方法测得的功能性病毒滴度高10倍以上。
我们强烈推荐您使用人纤维瘤细胞系HT1080（ATCC，货号CCL-121）作为慢病毒滴度测定的一个“金标准”。一个主要的原因是该细胞系的转导效率很高，因此所获得的滴度测试结果也很精准稳定。不过，您可能也需要在开展表达实验之前，使用相同的哺乳动物细胞系来测定您的慢病毒储存液。通常情况下，这些细胞应该是非迁移性的贴壁细胞系，倍增时间在18-25小时左右。普通型293细胞也可用于慢病毒的滴度测定，但我们不推荐使用293T或293FT细胞，因为这些细胞中含有SV40大T抗原——在整合的慢病毒表达载体中含有SV40复制起点，它可能会介导不需要的DNA复制。这种DNA复制作用通常会导致细胞死亡或很低的滴度。慢病毒属于慢逆转录病毒属，特点是潜伏期长。
是的，我们的确提供慢病毒生产的定制服务。请向techsupport@thermofisher.com发送相关邮件，以了解更多详情。
使用我们的慢病毒系统产生的是复制缺陷型慢病毒，这是其具有的一个安全特性。如需更多病毒，您必须执行一次全新的转染操作。
我们推荐您立即将所生成的慢病毒母液分装为小体积工作液，之后在–80°C条件下长期保存。慢病毒对于储存温度和冻融操作很敏感，因此需小心操作。每一次冻融都将损失多达5%乃至更多的病毒活性。如保存良好，合适滴度的病毒储存液可保存一年。经长期保存后，我们推荐您在使用前重新测定病毒储存液的滴度。
“F”意味着这一特定的293细胞克隆（称为293F）能够实现高转染效率，“T”表示SV40大T抗原。大T抗原表达质粒已稳定整合进基因组中，并赋予这些细胞对Geneticin抗生素的抗性。SV40大T抗原的存在对于慢病毒高滴度的生成十分重要，其确切机理尚不十分清楚。使用通用型293细胞或其他的293T细胞系来生成病毒是可行的，不过您获得的病毒滴度可能会比较低。
野生型HIV-1基因组的大小在10 kb左右。由于源自pLenti载体的表达所需元件加在一起约有4-4.4 kb，因此目的基因的大小理论上不应超过5.6-6kb，才能有效实现病毒包装过程。病毒滴度通常随着插入片段长度的增加而减小。
ViraPower 慢病毒包装混合系统是三种病毒包装质粒——pLP1、pLP2和pLP VSVG的优化混合物，以1 mg/mL浓度 的TE缓冲液（pH8.0）形式进行供货。这些质粒不提供单品销售。
我们的慢病毒包装系统属于第三代产品，这意味着其中不含有tat基因。如果慢病毒载体属于第三代乃至更新的产品，则病毒生成过程就不依赖于tat基因的参与。
您的第二代慢病毒载体无法与我们提供的包装系统兼容，因为我们的包装系统属于第三代产品，这意味着其中不含tat基因；而你的慢病毒载体需要tat基因参与才能形成病毒。
我们的慢病毒表达载体属于第三代产品，这意味着其中包含了嵌合型的5'LTR——这样病毒的生成就摆脱了对HIV tat反式激活因子的依赖。其结果是，它们能够兼容第二代或第三代的包装混合系统。
我们的慢病毒包装混合系统属于第三代产品，这意味着其中不表达tat基因。而且，gag/pol和rev基因也都做为独立的质粒进行提供，因此消除了重组事件将各个元件重组在一起形成单一载体从而产生可复制慢病毒的风险。
我们的慢病毒表达载体属于第三代，这意味着我们使用了由4个质粒构成的载体系统（1个慢病毒表达载体和3个包装质粒），因此消除了重组事件将各个元件重组在一起形成单一载体从而产生可复制慢病毒的风险。此外，这些载体还包含嵌合型的5'LTR，这样病毒的生成就摆脱了对HIV tat反式激活因子的依赖。同时，LTR（长末端重复）中原始的U3区也被删除，从而形成自失活病毒而不能再复制。
我们的慢病毒表达载体中约包含20%的原始病毒基因组。出于使用安全性的考虑，其余的病毒基因组已从我们的慢病毒表达载体中删除了。
这些系统之间的主要区别在于试剂盒中所包含的慢病毒表达载体。ViraPower慢病毒表达载体的骨架与ViraPower HiPerform慢病毒表达载体的骨架相似，只是后者加入了两个新元件：目的基因下游直接连接了一段源自旱獭肝炎病毒的WPRE（旱獭转录后调控元件）序列，该序列有助于提升转基因的表达水平；源自HIV-1整合酶基因的cPPT（中央多聚嘌呤区）序列能够增加慢病毒整合进宿主基因组的拷贝数，进而能提高病毒滴度达两倍左右。总之，相比不含WPRE和cPPT元件的ViraPower慢病毒表达系统而言，这些元件能够在大多数类型的细胞中将蛋白表达效率提升四倍以上。在ViraPower HiPerform Fast Titer表达系统中，不仅含有WPRE和cPPT元件，慢病毒表达载体中以EmGFP报告基因代替了Bsd基因，这样转导后仅需两天用户就可通过流式细胞术对活性病毒的滴度进行检测。
插入慢病毒载体中的片段不应含有poly(A)信号。天然的poly(A)信号（AATAAA或类似序列）会在使用Oligo dT进行cDNA合成的过程中被扩增。因此，它将成为cDNA文库或其克隆的一部分。
由于慢病毒是一种RNA病毒，而RNA基因组在合成过程中还会被包装起来，如果插入序列中含有多聚腺苷酸（poly(A)）信号，则RNA将会被过早终止。载体中包含了一个SV40 poly(A)信号，但这一信号位于第二LTR序列的下游，它的设计也应如此。几乎任何源自Gateway cDNA文库的克隆都很可能含有poly(A)信号，该信号一旦插入慢病毒载体中，就会造成病毒RNA的提前终止。
为了避免慢病毒RNA提前终止的情况发生，请考虑以下建议：
•应先从文库中挑选出所需基因，将其克隆至不含poly(A)信号的入门载体——如pENTR/D-TOPO——之中（即从起始密码子ATG至终止密码子），之后再克隆至慢病毒载体中。
•如果您试图建立一个慢病毒表达文库，您可能需要使用随机六聚体而非oligo dT来建立的文库，因为这样的文库的插入序列中将不太可能含有poly(A)信号。
插入片段的大小通常不是问题。慢病毒插入片段的大小限制为5-6kb左右（SuperScript II预制文库的平均插入片段大小为1.5 kb左右）。
在pLenti6载体中，杀稻瘟菌素（Bsd）抗性标志物基因由SV40启动子来启动；而在pLenti6.2载体中，Bsd抗性标志物则由磷酸甘油酸激酶-1（PGK）启动子所启动。
这一差别在操作干细胞的过程中变得至关重要；PGK启动子为天然的哺乳动物启动子，能够耐受沉默效应并在干细胞中表现出长期的持续表达，而SV40启动子通常在原代细胞和干细胞中会随着时间推移而被逐渐沉默。
完整的试剂盒组成信息列举于产品手册中的“试剂盒内容与保存方式”部分中。请在我们的网站（www.thermofisher.com）使用货号进行搜索，以查寻您感兴趣的产品。一旦进入产品页面，您就可使用手册链接来浏览和下载产品手册了。我们提供各类载体/试剂盒来满足不同研究者多种多样的克隆和表达策略的需要。
我们提供Vivid Colors pLenti6.3/V5-GW/EmGFP表达对照载体（货号V37006）和Vivid Colors pLenti6.2-GW/EmGFP表达对照载体（货号V36920)，这两种产品均为包含祖母绿荧光蛋白（EmGFP）的慢病毒载体。它们针对ViraPower慢病毒表达系统中的阳性对照品应用进行了专门设计，在转染293FT细胞之后用户可实现EmGFP荧光的检测。这些载体可作为滴度对照品，生成表达EmGFP的慢病毒，也可在分裂与非分裂的哺乳动物细胞的转导过程中作为转导对照品。这两类载体都不是克隆载体。Vivid Colors pLenti6.3/V5-GW/EmGFP表达对照载体拥有能够驱动EmGFP持续表达的CMV启动子，以及驱动杀稻瘟菌素——稳转筛选标志物长期、持续表达的PGK启动子，而Vivid Colors pLenti6.2-GW/EmGFP表达对照载体则拥有驱动EmGFP持续表达的CMV启动子和驱动杀稻瘟菌素——稳转筛选标志物表达的SV40启动子。此外，Vivid Colors pLenti6.3/V5-GW/EmGFP表达对照载体是一种HiPerform载体，这意味着它包含有两种关键的遗传元件：旱獭转录后调控元件（WPRE）和源自HIV-1整合酶基因的中央多聚嘌呤区（cPPT）序列，进而能够在大多数类型的细胞中将蛋白表达水平提升4倍以上（相比不含这些元件的慢病毒载体而言）。
Lumio标签非常小；只有6个氨基酸（600 Da左右），而GFP标签平均在27 kDa左右。由于慢病毒滴度在融合基因>6 kb的条件下会发生显著下降，带有Lumio小标签的慢病毒表达载体就能够有助于用户插入更大的目的基因（GOI）。如果使用GFP标签，留给GOI的长度就减少了719 bp的长度。这是EmGFP的碱基对长度（bp）。此外，带Lumio-标签的慢病毒载体能够帮助用户方便地在活细胞中对蛋白进行检测和定位。
我们提供C或N端带有Lumio标签的慢病毒表达载体，这些载体是我们所推出表达系统的一部分，分别是货号K37020和货号K37120。这些载体可帮助用户在活细胞中方便地对蛋白进行检测和定位。
我们所推出的全部pLenti表达载体均含有2个poly(A)位点：一个poly(A)位于HIV-1来源的3'LTR中，一个SV40 poly(A)位于3'LTR的下游。包含两个poly(A)序列的原因是为了降低转录受干扰的几率（举例来说，如果这里有大量转录持续通过RSV启动子区域，就可能会对RSV启动子自身的转录过程造成潜在干扰，而后一转录过程对于病毒RNA的合成至关重要。一旦慢病毒整合进靶细胞，SV40 poly(A)就不存在了（由于病毒序列只从5'LTR延伸至3'LTR），但3'LTR区域内的poly(A)区域仍会存在并发挥功能。
HIV-1基因组由两条相同的单链RNA拷贝所组成。双链RNA在上述情况下可能会形成，由于dsRNA将干扰基因组的包装，会降低病毒滴度。因此，从LTR的反向插入表达阅读框将会降低病毒滴度。
参考文献：Mautino et al.(2000) Human Gene Therapy 11:895.
可以，慢病毒表达载体自身可作为表达载体，通过适当的抗生素实现稳转筛选。请注意这一载体的大小大约是一般常用载体的两倍。因此，您可能需要增加载体的转染量，以获得近似的摩尔量水平。
我们不推荐使用小量制备试剂盒来制备慢病毒载体，因为慢病毒小量制备DNA的得率通常很低，这是由于载体骨架中存在的LTR所致。我们推荐用户使用S.N.A.P.中量制备试剂盒（MidiPrep Kit，货号K191001）或PureLink HiPure质粒中量制备试剂盒（货号K210004）来制备慢病毒载体DNA，这两款试剂盒的重悬缓冲液中均包括10 mM EDTA。由于慢病毒DNA的中量制备通常得率也较低，我们推荐用户按照专用实验方案进行操作，以提高得率——简言之，缓慢培养细胞；每个抽提柱加入少量细胞；每次DNA中量制备使用100 mL慢病毒培养物。
注意：如果您希望在克隆操作/克隆筛选过程中通过小量制备慢病毒质粒，我们推荐您使用PureLink HQ小提试剂盒（Mini Kit，货号K210001），并按照说明书来开展实验，只是有一处需要优化：使用50 mL TE，pH 8.0缓冲液洗脱一次。通常情况下，此方法的得率很低：100–150 ng/mL（即一共获得5–7 mg）。其OD 260/280通常介于1.8和2.1之间。
我们推荐用户在–20°C条件下储存慢病毒表达载体。由于它们相对较大，我们并不推荐在–80°C保存这些载体，因为在后一温度条件下，载体溶液将彻底冻结，–80°C条件下的过多次反复冻融将会影响克隆效率。
TOPO克隆方法是简单易用的5分钟PCR克隆法，我们基于此种技术开发了许多试剂盒。如果您只需构建一个载体，就可选择这一方法。如果您希望将目的基因克隆进多个不同的表达系统，可考虑Gateway克隆技术——这一技术也应用于我们推出的多个表达试剂盒。挑选克隆方法的另一个考量因素是插入序列的大小。如果您的插入序列>4kb，我们推荐您选择Gateway克隆法，对于长片段插入序列，TOPO克隆法的效率较低。
我们的慢病毒载体基于HIV-1骨架。不过，我们对此骨架进行了一些修改，使它们只作为基因转入载体而不会发生后续的病毒复制或导致疾病。我们删除了HIV-1特异性的基因以提升其安全性。HIV-1基因仅表达于生产细胞（293FT），而且不会被装入病毒基因组，因此不会在转导的靶向细胞中表达。
慢病毒是慢作用逆转录病毒属的成员，其特点是潜伏期较长。
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。
Lumio试剂具有疏水性，因此很容易穿过细胞膜。无需通过透化处理来帮助该试剂进入细胞内部。
哺乳动物细胞培养基中的血清蛋白（如BSA，66KD）可能会与Lumio试剂发生交叉反应，从而形成非特异性的条带。吸去细胞培养基并在收获细胞后使用PBS润洗哺乳动物细胞3-4遍，有助于减少BSA的非特异性结合。
我们尚未在细胞中发现用于蛋白检测浓度的Lumio试剂具有任何不良效应。我们也未在使用Lumio Green之后发现细胞形态出现任何不良改变。加入Lumio Red后，我们确实发现细胞发生了某些微小的形态改变，不过这一变化在加入试剂24小时之后恢复。
Lumio染色技术的优势在于可同时兼容体内和体外的蛋白标记操作。在体内标记实验中，向细胞中加入Lumio试剂后即可在荧光显微镜下观察到细胞/蛋白。这一效果与GeneBLAzer检测步骤相似，只是GeneBLAzer是基于扩增报告基因信号的酶促反应。GFP的荧光信号只能在细胞（体内）中检测到，因为需要GFP蛋白的正确折叠。相比GeneBLAzer检测法中的bla蛋白（264个氨基酸，29 kDa）和GFP蛋白（27 kDa）而言，Lumio标签非常之小（6个氨基酸，585Da），因此在很大程度上不会对所融合蛋白的功能造成影响。GFP的劣势在于需要融合一个很大的标签蛋白，而且该检测法并非是基于酶法的报告系统。不同于GeneBLAzer检测法和GFP标签，Lumio-标签蛋白可在Lumio试剂处理细胞裂解液或蛋白之后通过凝胶电泳变得可视化。与Lumio和GFP相比，GeneBLAzer检测法在活体细胞的应用中更为灵敏。GeneBLAzer检测法也不同于Lumio和GFP，这一方法能够实现比率化的读数，因此有助于减少样本间的差异。
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.
Possible causes include:
- large volume of viral supernatant used for transduction
- cells sensitive to Polybrene regaent
- too much antibiotic used for selection
- antibiotic used too soon after tranduction
- gene of interest is toxic to cells
Poor expression could result from low transduction efficiency, too low of a MOI, too much antibiotic used for selection, usage of antibiotic too soon after transduction, harveting cells too soon after transduction, having a gene of interest that is toxic to cells, or rerrangement in the LTR regions of the expression construct plasmid DNA.
Here are some possible causes and solutions:
- Promoter silencing; CMV promoter is prone to silencing especially in mouse and rat cells, screen multiple antibiotic resistant clones and select the one with the highest expression levels
- Viral stocks stored incorrectly; aliquot and store at -80 degrees C, do not freeze/thaw more than 3 times
Here are some possible causes and solutions:
- Too little antibotic used for selection
- Selection performed on confluent cells; replate cells
- Viral supernatant not diluted sufficiently; titer lentivus using a wider range of 10-fold serial dilutions
Possible causes include:
- low transfection efficiency; Use a high-quality plasmid prep, 293FT cells under passage 16, ensure removal of Geneticin during transfection, ensure correct DNA:lipid ratio, and that cells are plated at the correct confluency
- transfected cells are not cultured in medium containing sodium pyruvate; this reagent provides an extra energy source for cells
- viral supernatant harvested too early; viral supernatants can generally be collected 48-72 hrs post-transfection
- viral supernatant too dilute; concentrate virus using CsCl purification
- viral supernatant frozen and thawed multiple times; 3 times should be the maximum freeze/thaw
- gene of interest is large; viral titers decrease as size of insert increases, inserts larger than 5.6 kb are not recommended
- rearrangement in the LTR region of the epxression construct plasmid DNA; use Stb3 cells for transformatin of the lentiviral construct
- poor choice of titering cell line; use HT1080 cells or similar cell line
- Polybrene reagent is not included during transduction; transduce lentiviral construct into cells in the presence of Polybrene reagent
- Lipofectamine reagent handled incorrectly; ensure proper storage and mix gently before use
- Use fluorescence micrscopy to check titer with HiPerform FastTiter lentivirus
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If 293FT cells detach shortly after transfection (4 hours to overnight):
- This may be a sign of Lipofectamine 2000 toxicity. Cells may have been plated too sparsely prior to transfections.
- The cells may not have been handled gently enough (these cells have a tendency to lift off easily).
- The cells may have been kept at room temperature for too long.
If cells detach 48 to 72 hours post-transfection:
- If the cells lift off in large sheets, this may be a sign of lentivirus production.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. Hence, we do not recommend using a mini-prep kit for propagation of lentiviral constructs. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or the PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield- basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.
Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low, 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.
We strongly recommend using Stbl3 E. coli for cloning lentiviral constructs. Stbl3 E. coli cells contain the recA13 mutation in their genotype that helps to minimize the likelihood of unwanted recombination between the LTRs. After transforming into Stbl3 E. coli, we recommend picking colonies and validating the lentivirus DNA from mini-preps using Afl II and Xho I digests before proceeding to midi-preps. In all of our lentiviral vectors, Afl II sites are present in both 5' and 3' LTRs, and a Xho I site is present after the 3' end of the MCS. Assuming Afl II cuts only in the LTR sites, and there are no Afl II or Xho I sites in the insert, 3 DNA fragments are expected to be generated from the Afl II + Xho I digest. Any unexpected DNA fragments can be assumed to be a result of LTR recombination. Only clones with the expected pattern of DNA fragments should be chosen for the subsequent midi-prep.
The ViraPower Lentiviral Expression System includes the following features designed to enhance its biosafety:
The pLenti expression vector contains a deletion in the 3' LTR (deltaU3) that does not affect the generation of the viral genome in the producer cell line, but results in “self-inactivation” of the lentivirus after transduction of the target cell (References: Yee JK, Moores JC, Jolly DJ, Wolff JA, Respess JG, Friedmann T (1987) Gene Expression from Transcriptionally Disabled Retroviral Vectors. Proc. Natl. Acad. Sci. USA 84: 5197-5201; Yu SF, Ruden Tv, Kantoff PW, Garber C, Seiberg M, Ruther U, Anderson WF, Wagner EF, Gilboa E (1986) Self-Inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells. Proc. Natl. Acad. Sci. USA 83: 3194-3198; Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Selfinactivating Lentivirus Vector for Safe and Efficient in vivo Gene Delivery. J. Virol. 72: 9873-9880). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome.
- The number of genes from HIV-1 used in the system has been reduced to three (i.e., gag, pol, and rev).
- The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (References: Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993) Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 90: 8033-8037; Emi N, Friedmann T, Yee JK (1991) Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus. J. Virol. 65: 1202-1207; Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T (1994) A General Method for the Generation of High-Titer, Pantropic Retroviral Vectors: Highly Efficient Infection of Primary Hepatocytes. Proc. Natl. Acad. Sci. USA 91: 9564-9568).
- Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids. All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication-competent virus (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
- Although the three packaging plasmids allow in trans expression of proteins required to produce viral progeny (e.g., gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the psi packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced.
- The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. No other viral species are produced.'
- Expression of the gag and pol genes from pLP1 has been rendered Rev-dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
- A constitutive promoter (RSV promoter) has been placed upstream of the 5' LTR in the pLenti expression vector to offset the requirement for Tat in the efficient production of viral RNA (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
Despite the presence of the above safety features, the lentivirus produced can still pose some biohazardous risk, since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral 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 lentivirus carrying potential harmful or toxic genes (e.g., activated oncogenes).
For more information about the BL-2 guidelines and lentivirus 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).
Lentiviruses produced with our system do not carry or express any viral genes, and therefore have no associated toxicity issues. Only the protein expressed from the coding region between the LTR sites is incorporated into the mammalian cell chromosome and expressed. The lentivirus itself cannot replicate because of the built-in safety features.
The concern with leaving the lentivirus on the cells longer would be potential toxicity or growth effects. If you absolutely cannot remove the virus from the cells, we suggest to leave it on the cells and empirically monitor the cells.
Note: These are based on the characteristics of HIV.
Morphology: Virions have a complex construction and consist of an envelope, a nucleocapsid, a nucleoid, and a matrix protein. Virions are enveloped, spherical to pleomorphic in shape, and have a size of 80-100 nm in diameter. The surface projections are small or inconspicuous spikes that are densely dispersed, evenly covering the surface. Surface projections are 8 nm long. The core is rod-shaped, or is truncated cone-shaped. The nucleoid is concentric.
Physicochemical and Physical Properties: Virions have a buoyant density in sucrose of 1.13-1.18 g cm-3. Virions are sensitive to treatment with heat, detergents, and formaldehyde. The infectivity is not affected by irradiation.
Proteins: Proteins constitute about 60% of the particle weight. The viral genome encodes structural proteins and non-structural proteins. Virions consist of 5 major structural and 3 non-structural proteins. The virus codes for an RNA-dependent DNA polymerase.
Lipids: Lipids are present and located in the envelope. Virions are composed of 35% lipids by weight. The composition of viral lipids and host cell membranes are similar. The lipids are of host origin, derived from plasma membranes.
Carbohydrates: Three percent of the particle weight is attributed to carbohydrates.
Polybrene (hexadimethrine bromide) is a cationic polymer(Cat. No. H9268 from Sigma Aldrich) that increases transduction efficiency by neutralizing the charge repulsion between virus particles and the cell surface. For best results, we recommend performing transduction in the presence of Polybrene.
Note: Some cells (e.g., primary neurons) are sensitive to Polybrene. Hence, before performing a transduction experiment, we recommend testing your cell line for sensitivity to Polybrene at a range of 0-10 µg/mL and avoiding it if the cells exhibit toxicity or phenotypic changes.
We have found that, in general, 80-90% of the cells in an actively dividing cell line (e.g., ht1080) express a target gene when transduced with lentivirus at an MOI of approximately 1. Some non-dividing cell types transduce lentiviral constructs less efficiently. For example, only about 50% of the cells in a culture of primary human fibroblasts express a target gene when transduced at an MOI of approximately 1. If you are transducing your lentiviral construct into a non-dividing cell type, you may need to increase the MOI (e.g., MOI = 10) to achieve optimal expression levels for your recombinant protein. If you are transducing your lentiviral construct into your mammalian cell for the first time, we recommend using a range of MOIs (e.g., 0, 0.5, 1, 2, 5, 10) to determine the MOI required to obtain the optimal protein expression for your application.
The lentiviral envelope is pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), which allows the lentivirus to interact with its target cell in a receptor-independent manner. As a result, lentivirus has broad tropism and can, in theory, transduce any mammalian cell type. This receptor-independent entry into the target cell likely involves endocytosis (Espenshade et al. (2002) Proc Natl Acad Sci U S A 99:11694; Aiken (1997) J Virol 71:5871).
You can perform PCR/qPCR analysis of viral genes for lentivirus titering, but keep in mind that the titer will not be a measure of functional virus since it will measure both inactive as well as active virus. As a result, titers obtained using this method are usually about 10-fold higher than with methods that measure functional virus, such as blasticidin selection.
You can use the p24 ELISA assay for lentivirus titering, but keep in mind that the titer will not be a measure of functional virus since it will measure both inactive as well as active virus. As a result, titers obtained using this method are usually about 10-fold higher than with methods that measure functional virus, such as blasticidin selection.
We strongly recommend the human fibrosarcoma cell line ht1080 Cells (ATCC# CCL-121) as the “gold standard” for titering lentivirus. The primary reason is that transduction efficiency is high in these cells, and titering results are very accurate and reproducible. However, you may also use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies. In general, this should be an adherent, non-migratory cell line, and exhibit a doubling time in the range of 18-25 hours. Regular 293 cells may be used for lentivirus titering, but we do not recommend using 293T or 293FT cells because these cells contain the SV40 large T antigen that will induce unwanted DNA replication at the SV40 ori contained within the integrated lentiviral expression vector. This often leads to cell death and results in very low titers.Lentivirus is a genus of slow retroviruses, characterized by a long incubation period.
Yes, we do offer a lentivirus production custom service. Please send an email to techsupport@thermofisher.com for details.
Lentivirus produced using our system is replication-incompetent, and this is a safety feature. You must perform a fresh transfection each time you need more virus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend aliquoting lentiviral stocks immediately after production into small working volumes, and storing at -80 degrees C for long-term storage. Lentivirus is sensitive to storage temperature and to freeze/thaw and should be handled with care. It can lose up to 5% or more activity with each freeze/thaw. 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.
The “F” stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the “T” stands for the SV40 large T antigen. The large T antigen expression plasmid is stably integrated in the genome and confers resistance to Geneticin antibiotic in these cells. The presence of the SV40 large T antigen is important for high-titer lentivirus production and the mechanism is not known. If regular 293 cells or another 293T cell line is used as the producer cell line, you will be able to produce virus, but the titers will be lower.
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 adds 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. Titers will generally decrease as the size of the insert increases.
The ViraPower Lentiviral Packaging Mix consists of an optimized mixture of three viral packaging plasmids, pLP1, pLP2, and pLP VSVG, supplied at 1 mg/mL in TE buffer, pH 8.0. The individual plasmids are not available as standalones.
Our lentiviral packaging mix belongs to the third generation, meaning that it does not express the tat gene. It can be used with lentiviral vectors that belong to the third generation or higher, where virus production is independent of the tat gene.
Your second-generation lentiviral vector will not be compatible with our packaging mix because our packaging mix belongs to the third generation, meaning that it does not express the tat gene; whereas your lentiviral vector will need the tat gene for virus production.
Our lentiviral expression vectors belong to the third generation, meaning that they contain a chimeric 5' LTR, by means of which virus production is not dependent on the HIV tat transactivator. As a result, they are compatible with a second- or third-generation packaging mix.
Our lentiviral packaging mix belongs to the third generation, meaning that it does not express the tat gene. Further, gag/pol and rev genes are supplied as independent plasmids, thus eliminating concerns about recombination events bringing components together as a single vector to produce replication-competent lentivirus.
Our lentiviral expression vectors belong to the third generation, meaning that we use a four-plasmid vector system (1 lentiviral expression vector and 3 packaging plasmids), thus eliminating concerns about recombination events bringing components together as a single vector to produce replication-competent lentivirus. Gag/pol and rev genes are supplied as independent plasmids. Further, these vectors contain a chimeric 5' LTR, by means of which virus production is not dependent on the HIV tat transactivator. Also, the original U3 region of the LTR (long terminal repeat) is deleted to make the virus self-inactivating and thus replication-incompetent.
Our lentiviral expression vectors contain approximately 20% of the original viral genome. The rest of the viral genome is deleted from our lentiviral expression vectors for safety reasons.
The main difference between these systems is in the lentiviral expression vector contained within the kits. The ViraPower Lentiviral Expression Vector backbone is similar to the ViraPower HiPerform Lentiviral Expression Vector backbone except that the latter contains two new elements: WPRE (Woodchuck Posttranscriptional Regulatory Element) from the woodchuck hepatitis virus that is placed directly downstream of the gene of interest, allowing for increased transgene expression and the cPPT (central Polypurine Tract) from the HIV-1 integrase gene, which increases the copy number of lentivirus integrating into the host genome and thus allowing for a two-fold increase in viral titer. Together, WPRE and cPPT produce at least a four-fold increase in protein expression in most cell types, compared to the vectors in the ViraPower Lentiviral Expression Systems that do not contain these elements. In the ViraPower HiPerform Fast Titer Expression System, in addition to the WPRE and cPPT elements, the lentiviral expression vector contains the EmGFP reporter gene instead of Bsd, which allows titer of active virus by flow cytometry in just two days post-transduction.
Inserts cloned into lentiviral vectors should not have a poly(A) signal. The native poly(A) signal (AATAAA or something similar) will be amplified when using the oligo dT during cDNA synthesis. Thus, it will then become part of the cDNA library or its clones.
Since lentivirus is an RNA virus, during the synthesis of the RNA genome to be packaged, if there is a polyadenylation (poly(A)) signal in the insert, the RNA will be terminated prematurely. There is a SV40 poly(A) signal in the vector, but it is after the second LTR, and it is supposed to be there. Almost any clone transferred from a Gateway cDNA library will probably have a poly(A) signal, which, if inserted into a lentiviral vector, would end up terminating the viral RNA prematurely.
In order to circumvent premature termination of the lentiviral RNA, consider these recommendations:
- The desired gene should first be isolated from the library, cloned into an entry vector such as pENTR/D-TOPO without the poly(A) signal (i.e., ATG to Stop), and then transferred into the lentiviral vector.
- If you are trying to establish a lentiviral expression library, you will probably have to go with a library that was amplified using random hexamers rather than an oligo dT, since such a library would be less likely to include a poly(A) signal in the insert.
Size is not usually a problem. The insert size limit of the lentivirus is approximately 5-6 kb (average insert size of the SuperScript II premade libraries is approximately 1.5 kb).
In pLenti6 vectors, the blasticidin (Bsd) resistance marker is driven by the SV40 promoter whereas in pLenti6.2 vectors, the Bsd resistance marker is driven by the phosphoglycerate kinase-1 (PGK) promoter.
This difference is important when working with stem cells; the PGK promoter is a native mammalian promoter that is resistant to silencing and shows long-term, persistent expression in stem cells, whereas the SV40 promoter is often silenced over time in primary cells and stem cells.
The complete kit composition is listed in the product manual under “Kit Contents and Storage.” Search our website (www.thermofisher.com) using the catalog number to find the product you're interested in. Once you are on the product page, the manual can be viewed and downloaded using the Manuals link. We provide a variety of vectors/kits to suit the various cloning and expression strategies used by different researchers.
We carry the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector (Cat. No. V37006) and Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector (Cat. No. V36920), both of which are lentiviral vectors containing Emerald Green Fluorescent Protein (EmGFP). They are designed for use with the ViraPower Lentiviral Expression Systems as positive controls to enable the detection of EmGFP fluorescence following transfection in 293FT cells. These vectors serve as titer controls to produce an EmGFP-expressing lentivirus stock and as a transduction control following transduction in both dividing and non-dividing mammalian cells. Both of these vectors are not cloning vectors. The Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long-term, persistent expression of the blasticidin-stable selection marker, whereas the Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the SV40 promoter for driving expression of the blasticidin-stable selection marker. In addition, the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector is a HiPerform vector, meaning that it is equipped with two key genetic elements: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in increase in protein expression in most cell types, compared to lentiviral vectors that do not contain these elements.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The Lumio tag is very small; it is only 6 amino acids (approximately 600 Da), whereas the GFP tag is on average 27 kDa. Since the lentivirus titer drops significantly if the fusion gene is >6 kb, lentivirus expression vectors with the small Lumio tag allow insertion of a larger gene of interest (GOI). With GFP, the GOI must be shortened by 719 bp. This is the length of EmGFP in base pairs (bp). Further, Lumio-tagged lentiviral vectors allow for convenient detection and localization of proteins in live cells.
We do offer lentiviral expression vectors with C- or N-terminal Lumio tags, which are part of the expression systems, Cat. No. K37020 and Cat. No. K37120, respectively). These vectors allow for convenient detection and localization of proteins in live cells.
All our pLenti expression vectors have 2 poly(A) sites-a poly(A) located within the 3' LTR that is derived from HIV-1 and the SV40 poly(A) downstream of the 3'LTR. The reason for having both is to reduce the chances of transcriptional interference (for instance, if there were a significant amount of transcriptional read-through that continued through the RSV promoter region, this could potentially interfere with transcription from the RSV promoter, which is critical for production of the viral RNA). Once the lentivirus has integrated in the target cells, the SV40 poly(A) will not be present (since the virus just extends from the 5' to 3' LTR), but the poly(A) within the 3' LTR region will still be present and functional.
The HIV-1 genome consists of two identical copies of single-stranded RNA. Generating dsRNA, as could happen in this instance, will reduce titers since the dsRNA will interfere with genome packaging. Hence, reversing the orientation of the expression cassette with respect to the LTRs will decrease virus titers. Reference: Mautino et al. (2000) Human Gene Therapy 11:895.
Yes, the lentiviral expression vector will work as an expression vector by itself and can be stably selected with the appropriate antibiotic. Please note that the vector will be about twice the size of most regular vectors. Therefore, you may need to increase the amount of transfected vector to approximate molar equivalents.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We do not recommend using a mini-prep kit for propagation of lentiviral constructs because the DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield-basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.
Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low: 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.
We recommend storing lentiviral 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 TOPO cloning method is an easy-to-use, 5-minute benchtop PCR cloning method, and we have developed many kits based on this technology. You may want to choose this method if you have only one construct to make. If you plan to place your gene of interest into several different expression systems, you may want to consider Gateway cloning technology, also used in many of our expression kits. Another consideration in choosing the cloning method would be the size of your insert. If your insert is >4 kb, we recommend choosing Gateway cloning, as TOPO cloning will not work efficiently for these large inserts.
Our lentiviral vectors are based on the HIV-1 backbone. However, several alterations have been made so they function solely as a gene delivery vehicle without subsequent viral replication or disease. Specific HIV-1 genes have been deleted to enhance safety. The HIV-1 genes are only expressed in the producer cells (293FT) and none of them are packaged into the viral genome, and thus are never expressed in the transduced target cell.
Lentivirus is a genus of slow retroviruses, characterized by a long incubation period.
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.
The Lumio reagent is hydrophobic and can easily pass through the membrane. There is no need to permeabilize the membrane in order to get this reagent into cells.
Serum proteins such as BSA (66 kDa) from the mammalian cell culture medium may cross-react with the Lumio reagent, producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3-4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA.
We have not experienced negative effects with Lumio reagents at the concentrations used to detect protein in the cells. We also do not see any change in cell morphology when using Lumio Green. After application of the Lumio Red, we do see some minor morphological changes in the cells that are reversed after 24 hours of application of the reagent.
The advantage of Lumio staining is that one can do both in vivo and in vitro protein labeling. For in vivo labeling, load the cells with the Lumio reagent and then visualize the cells/proteins under a fluorescence microscope. This is similar to the GeneBLAzer detection procedure except that GeneBLAzer detection is based on an enzymatic reaction that amplifies the reporter signal. GFP fluorescence can only be detected within the cell (in vivo) because proper protein folding is needed. The Lumio tag is very small (6 amino acids, 585 Da), in contrast to the bla protein in GeneBLAzer detection (264 amino acids, 29 kDa) and the GFP protein (27 kDa), and therefore most likely will not interfere with the function of the protein it is fused to. GFP has the disadvantage of being a large fusion tag and is not an enzymatic-based reporter system. Unlike GeneBLAzer detection and GFP, a Lumio-tagged protein can be visualized on a gel after treating the cell lysate or protein with the Lumio reagent. Compared to Lumio and GFP, GeneBLAzer detection is a more sensitive detection method for use in live cells. Also unlike Lumio and GFP, the GeneBLAzer detection method allows for ratiometric read-outs and thus eliminates sample-to-sample variation.
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.
Yes, fluorescent protein-expressing cells can be fixed using 4% paraformaldehyde in PBS for 10 min followed by one quick PBS rinse and 3 x 5 min washes with 1 mL PBS.
Yes, all of the fluorescent proteins offered by (EmGFP, YFP, CFP, BFP and Cycle 3 GFP) have been humanized for optimal mammalian expression.
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.
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.
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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