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查看更多产品信息 ViraPower™ Lentiviral Gateway™ Expression Kit - FAQs (K496000)
128 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）。
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。
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.
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 ViraPower lentiviral packaging mix is an optimized mix of three packaging plasmids: pLP1, pLP2 and pLP/VSVG, supplied in TE buffer, pH 8.0, at a concentration of 1 µg/µL. These plasmids supply the helper functions as well as structural and replication proteins in trans that are required to produce lentivirus using our ViraPower lentiviral expression vectors.
Polybrene (hexadimethrine bromide) is a cationic polymer used to increase the efficiency of infection of certain cells with virus particles in cell culture. Polybrene acts by neutralizing the charge repulsion between the virions and cell surface.
The plasmid expressing the SV40 large T antigen, pCMVSPORT6TAg.neo, is stably integrated in 293FT cells. Hence, we recommend culturing 293FT cells in the presence of 500 µg/mL Geneticin (G418) antibiotic.
Here is a summary of the safety features that have been built into the ViraPower lentiviral expression system.
• The system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild-type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead.
• The system is a four-plasmid system, and the packaging functions are supplied in the packaging mix in trans, eliminating concerns about recombination events bringing components together as a single vector.
• The lentivirus particles that are generated carry only the gene of interest, and the packaging mix vectors never become part of the packaged virus.
• The lentivirus has a self-inactivation feature, meaning that once it is transduced into cells and has integrated into the mammalian cell host, it is no longer capable of producing a packageable viral genome.
• Expression of gag/pol from pLP1 is rev dependent, so both plasmids (pLP1 and pLP2) must be co-expressed to package the gene of interest into virions.
There are many safety features that we have built into our ViraPower lentiviral expression system. We recommend strict adherence to all CDC guidelines for Biosafety Level 2 (BL-2), as well as institutional guidelines. BL-2 guidelines may be found in “Biosafety in Microbiological and Biomedical Laboratories” 4th edition, Centers for Disease Control (www.cdc.gov).
Lentivirus should be aliquoted immediately after production and stored at -80 degrees C. Lentivirus can lose up to 5% of titer with each freeze/thaw. When stored properly, viral stocks should be suitable for use for up to one year. After long-term storage, we recommend re-titering the viral stocks before use.
As the size of the insert increases, lentiviral titers decrease. The size of the wild-type HIV genome is approximately 10 kb. Since the size of the elements required for expression from ViraPower lentiviral vectors totals approximately 3–4.5 kb, the size of the insert should not exceed 5.6–6 kb. In-house data shows that viral titer drops approximately 2-fold for every kb of insert size over 4 kb. If you wish to produce lentivirus with an insert of > 4 kb, you will need to concentrate the virus to obtain a suitable titer.
a. Try different MOIs. All cells are not created equal.
b. Try Polybrene. Test in the range 0–10 µg/mL (manual recommends 6 µg/mL).
c. If Polybrene does not help, you might try using DEAE-dextran. We have not tried it in-house, but it has been used in the literature [Reiser et al. (1996) PNAS 93, 15271].
ViraPower lentiviral expression kits contain a control lentiviral expression vector (the lacZ- expression vector or GFP- expression vector). When packaged into virions and transduced into a mammalian cell line, these vectors express either ß-galactosidase or GFP, respectively. If you use the lacZ control virus, you can use the ß-galactosidase staining kit (Cat. No. K1455-01) to determine transduction efficiency. If you use the GFP control virus, you can use a fluorescent microscope or perform FACS analysis.
The lentiviruses produced in this system will not replicate under any conditions. 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.
Yes, it will work as an expression vector by itself and can be stably selected with blasticidin. 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.
Lentiviruses produced with this 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.
For routine maintenance of 293FT cells, you need to add Geneticin (G418) antibiotic at a concentration of 500 µg/mL to maintain the Large T antigen plasmid/phenotype.
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. If you want to use regular 293 cells or another 293T cell line, you will be able to produce virus, but the titers will be lower. The large T antigen expression plasmid is stably integrated in the 293FT cell and confers resistance to Geneticin antibiotic in these cells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
For HT1080 cells we typically use 10 µg/mL, but we strongly recommend that you generate a kill-curve for each antibiotic and cell line before proceeding. Most cell types respond to between 1 µg/mL and 10 µg/mL of blasticidin. For HT1080 cells, we typically use 100 µg/mL of Zeocin for Zeocin-containing lentiviral vectors. But again, generation of a kill-curve is strongly suggested.
We strongly recommend titering on HT1080 cells to determine the absolute titer of infectious virus in your supernatant. The primary reason is that it's a way to standardize titers obtained in different labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible, making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.Accurate titer, however, can be obtained in essentially any mammalian cell line, but 3T3 and HeLa cells have a lower transduction efficiency than HT1080 cells (for reasons unknown). Do not use 293FT cells for titering.
Yes, you can use restriction enzymes Cla I (cutting at 1796) and BamH I (cutting at 2401) to remove the CMV promoter from the pLent6/V5-D-TOPO vector. Use Cla I and Spe I for the pLenti6/V5-DEST vector. Alternatively, we offer promoter-less lentiviral vector, pLenti6.4/R4R2/V5-DEST (Cat. No. A11145).
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.
Titers between 1 x 10e5 and 3 x 10e5 cfu/mL (unconcentrated) are typical. If the titer is lower than 1x 10e5 cfu/mL, virus production was not optimal (arising for various reasons). Titers for the LacZ virus are typically in this low to mid 10e5 range. The sample lentiviral titer experiment shown in the ViraPower instruction manual shows lacZ lentivirus with a titer of 4.8 x 10e6 cfu/mL.
We strongly suggest that you titer your lentivirus on HT1080 cells, which allows you to compare titers from day-to-day within your lab and also with external labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible--making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.
The ViraPower Lentiviral System:
(1) effectively transduces both dividing and non-dividing cells
(2) efficiently delivers the gene of interest to mammalian cells in culture or in vivo
(3) produces a pseudotyped virus with a broadened host range
(4) includes multiple features designed to enhance the biosafety of the system
Clone your gene of interest into one of our lentiviral expression vectors. We have a Directional TOPO version (pLenti6/V5/D-TOPO) and a Gateway version (pLenti6/V5-DEST vector). Co-transfect your recombinant vector along with the optimized ViraPower packaging mix into the 293FT producer cell line using Lipofectamine 2000 reagent (if using a different transfection reagent, follow the manufacturer's recommendations). Harvest the viral supernatant and determine the titer of the virus. Add the viral supernatant to your mammalian cell line of interest at the appropriate MOI. Assay for "transient" expression of your recombinant protein or select for stably transduced cells using the appropriate selection antibiotic, if desired, then examine expression of your protein of interest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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