Search
Search
查看更多产品信息 ViraPower™ T-REx™ Lentiviral Gateway™ Vector Kit - FAQs (K496600)
38 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。
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。
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。
“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。
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）。
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。
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。
当执行共转染时，用户无法在稳转细胞系中同时完成功能性TetR或GeneSwitch蛋白的双重测试。另一方面，如果执行连续转染，用户就可对所生成的T-REx或GeneSwitch细胞系进行功能性测试，他们可将LacZ对照表达质粒瞬转进入细胞，并挑取那些在诱导剂缺乏条件下表达LacZ的本底水平最低，而在含诱导剂条件下LacZ表达水平最高的克隆。之后可对这一克隆进行扩增，并按需用于T-REx或GeneSwitch表达载体的转染实验。
使用GeneSwitch系统能够确保目的基因处于极低的基础表达水平，而T-REx系统可能会有少量渗漏表达，因为FBS中不可避免的存在着一些四环素。GeneSwitch系统的诱导表达水平可能甚至高于CMV启动子。GeneSwitch系统的劣势在于，尽管该系统能够在转基因条件下以优异的性能工作，但在培养系统中关闭表达的操作不是很容易实现。而另一方面，T-REx系统可通过加入和去除诱导剂来切换开关状态。
Flp-In T-REx系统将Flp-In系统的靶向整合与T-REx系统的强大诱导表达能力整合在一起。该系统能够生成同基因，可诱导的稳定表达细胞系，并能够针对这些细胞系实行多克隆筛选。一旦建立了包含整合FRT位点的Flp-In T-REx宿主细胞系，后续建立表达目的基因的Flp-In T-REx细胞系就变得迅速高效了。
强力霉素可作为T-REx系统中诱导剂的代替品。它的作用机理与四环素相近，并在T-REx系统中表现出与四环素相似的剂量效应和诱导性质。强力霉素显示出比四环素更长的半衰期（分别为48小时和24小时）。我们未提供强力霉素，但用户可从Sigma（货号D9891）进行购买。
我们提供三类独特的哺乳动物表达系统来帮助用户实现目的基因可诱导/调控性的表达。
•T-REx系统
•Flp-In T-REx系统
•GeneSwitch系统
请参见下表中的比较结果：
系统 --基础表达水平--诱导表达水平--表达最大化的响应时间--转基因应用
T-REx系统--低--最高--高--合适
Flp-In T-REx系统--较低--高--24-48小时--合适
GeneSwitch系统--最低--高--24-48小时--合适
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:
- 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.
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.
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.
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.
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.
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.
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.
When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.
With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.
The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.
Doxycycline may be used as an alternative inducing agent in the T-REx system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).
We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:
- T-REx system
- Flp-In T-REx system
- GeneSwitch system
Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable