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View additional product information for pLenti6/V5-DEST™ Gateway™ Vector - FAQs (V49610)
91 product FAQs found
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：
•所用检测法可能不适当或不够灵敏： ◦我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
•筛选到的克隆数不够：至少筛选出20个克隆。
•在稳转筛选中使用了不适当的抗生素浓度：请确保正确获取了抗生素的杀死曲线。由于某一既定抗生素的效力依赖于细胞类型，血清，培养基和培养技术，因此必须在每次进行稳定筛选的时候确定抗生素的用量。如果采用的培养基或血清条件明显不同，则即使是我们所提供的稳转细胞系对于我们推荐的剂量也可能出现更敏感或更不敏感的情况。
•基因产物（即使低水平）的表达可能与该细胞系的生长不相容（如毒性基因）：使用一个可诱导的表达系统。
•阴性克隆可能由基因表达的关键载体位点处优先发生了线性化所致：在一个不影响表达的位点实施载体线性化，如在细菌抗药性标志物序列中。
这里列举了一些可能的原因与解决方案：
•尝试试剂盒自带的表达对照。
•可能的检测问题：
◦检测瞬转的表达蛋白可能有难度，因为转染效率可能过低，以致用于整个转染群体的评估手段无法成功实现检测。我们推荐您通过稳转筛选或采用能够逐个检测单一细胞的技术手段来优化您的转染操作。您也可尝试通过改变启动子或细胞类型来提高表达水平。
◦细胞中的蛋白表达水平对于所选择的检测方法来说可能过低。我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
◾蛋白可能降解或截短了：使用Northern杂交进行检测。
◾可能的时程问题：由于蛋白表达随时间延长而发生的变化依赖该蛋白的天然属性，我们一般推荐您先获取一份表达的时程曲线。尝试进行一次时程分析将帮助您确定最优的表达时间窗。
◾可能的克隆问题：通过限制性酶切和/或测序来验证克隆。
不可以；新霉素对哺乳动物细胞有毒性。我们推荐您使用Geneticin（又称 G418硫酸盐)，这一产品的毒性较低，是在哺乳动物细胞中进行有效筛选的新霉素的替代品。
即使缺乏Kozak序列，翻译也还是会在核糖体遇到的第一个ATG处启始，不过启始效率可能相对较低。只要处于最初ATG的阅读框内，任何下游的插入序列都可能表达为融合蛋白，不过如果这里没有Kozak保守序列，则蛋白的表达水平预期会比较低。如果载体中包含一个非Kozak型的保守ATG，我们则推荐您将基因克隆至该ATG上游，再包含一个Kozak序列来优化表达效果。
我们提供pJTI R4 Exp CMV EmGFP pA载体，货号A14146，您可使用这一产品来监控转染和表达情况。
我们推荐使用One Shot ccdB Survival 2 T1^R 感受态细胞，货号A10460。该菌株能够耐受ccdB基因的毒性效应。
注意： 请勿使用常规的大肠杆菌克隆株 - 包括TOP10或DH5α - 来进行扩增和培养，因为这些菌株均对ccdB的效应很敏感。
在小鼠细胞系中，人们已知CMV启动子的效率会随时间延长而逐渐下降。因此，我们推荐您使用一款非CMV型的载体，如EF1α或UbC启动子，以在小鼠细胞系中长时间表达蛋白。
保守的Kozak序列为A/G NNATGG，其中的ATG表示起始密码子。ATG周围的核苷酸点突变会影响翻译效率。尽管我们通常情况下都推荐加入一段Kozak保守序列，不过这一操作的必要性还是基于具体的目的基因，一般只需ATG就足以高效地启始翻译过程。最佳的建议是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有具有N-融合表达的表达载体都已经包含了一个翻译起始位点。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
理论上，pDONR载体在BP反应时对插入片段没有大小的限制。我们自己测试过的最大片段是12 kb。TOPO载体对插入片段大小更敏感一些，要获得较高的克隆效率其插入片段长度的上限是3-5 kb。
在得到attB-PCR产物之后，我们建议对产物进行纯化以去除PCR缓冲液，残留的dNTP，attB引物，以及attB引物二聚体。引物和引物二聚体在BP反应中会高效的与供体载体重组，因而会增加转化E. coli时的背景，而残留的PCR缓冲液可能会抑制BP反应。使用酚/氯仿抽提，加醋酸铵和乙醇或异丙醇沉淀的标准PCR产物纯化方案不适合对attB-PCR产物进行纯化，因为这些实验方案通常仅能去除小于100 bp的杂质，而在去除较大的引物二聚体时效果不佳。我们推荐一种PEG纯化方案（请参见使用Clonase II的Gateway技术手册第17页）。如果使用上述实验方案您的attB-PCR产物仍然不够纯，您可以进一步对其进行凝胶纯化。我们推荐使用Purelink Quick 凝胶纯化试剂盒。
请检查您所用的菌株的基因型。我们的Gateway目的载体通常含有一个ccdB基因元件，该元件如果不被破坏，则E. Coli生长将受到抑制。因此，未进行克隆的载体应该在ccdB survival菌株如我们的ccdB Survival 2 T1R感受态细胞中扩增。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
目的基因必须两端带有合适的att位点，或者是入门克隆中的attL (100 bp)位点，或者是PCR产物中的 attB (25 bp)位点。对于入门克隆而言，所有位于attL位点之间的部分都将被转移到含有attR位点的Gateway目的载体中，而两端带有attB位点的PCR产物需被转移到一个含有attP位点的供体载体，例如pDONR221。
翻译起始位点的位置，终止子，或者用于表达的融合标签必须在最开始的克隆设计中考虑到。例如，如果您的目的载体包含一个N末端标记而非C末端标记，则该载体应当已经带有合适的翻译起始位点，但是终止子应当被包含在插入片段当中。
小抽（碱裂解）纯化的DNA即适用在Gateway克隆反应中。重要的一点是要将RNA污染去除干净以便得到精确的定量。推荐使用通过我们的S.N.A.P. 核酸纯化试剂盒，ChargeSwitch试剂盒，或PureLink试剂盒纯化的质粒DNA。
理论上没有片段大小限制。长度在100 bp到11 kb之间的PCR产物可以被直接克隆到pDONR Gateway载体中。其它DNA片段如带有att位点的150 kb DNA片段可以成功和一个Gateway兼容载体发生重组。对于大的插入片段，推荐进行过夜孵育反应。
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
Here are possible causes and solutions:
Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.
Here are possible causes and solutions:
- Try the control expression that is included in the kit
Possible detection problem:
- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
Protein might be degraded or truncated: Check on a Northern.
Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
Possible cloning issues: Verify clones by restriction digestion and/or sequencing.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.
Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.
We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.
We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.
The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.
Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.
LR Clonase II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway LR reactions. LR Clonase and LR Clonase II enzyme mixes are not recommended for MultiSite Gateway LR recombination reactions, but LR Clonase II Plus is compatible with both multi-site and single-site LR recombination reactions.
When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.
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.
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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.
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No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.
For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST vector: 6 kb
pLenti6/V5-DEST vector: 6 kb
pLenti6/V5/D-TOPO vector: 6 kb
pLenti6/UbC/V5-DEST vector: 5.6 kb
For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST vector: 6 kb
pAd/PL-DEST vector: 7.5 kb
In most cases, there will not be enough pENTR vector DNA present to go directly from TOPO cloning into an LR reaction. You need between 100-300 ng of pENTR vector for an efficient LR reaction, and miniprep of a colony from the TOPO transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO reaction using pENTR/D, or SD TOPO, or pCR8/GW/TOPO vectors:
1. Heat inactivate the topoisomerase after the TOPO cloning reaction by incubating the reaction at 85 degrees C for 15 minutes.
2. Use the entire reaction (6 µL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 µL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO reaction is very inefficient and will result in a very low colony number, if any at all.
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO adapted Gateway entry vectors. All TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
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No, not directly. The attB-PCR product must first be cloned, via a BP Clonase reaction, into a pDONR vector which creates an "Entry Clone" with attL sites. This clone can then be recombined, via an LR Clonase reaction, with a Destination vector containing attR sites. However, It is possible to perform both of these reactions in one step using the "One-Tube Protocol" described in the manual entitled "Gateway Technology with Clonase II".
Yes, this can be done using the Multisite Gateway Technology. MultiSite Gateway Pro Technology enables you to efficiently and conveniently assemble multiple DNA fragments - including genes of interest, promoters, and IRES sequences - in the desired order and orientation into a Gateway Expression vector. Using specifically designed att sites for recombinational cloning, you can clone two, three, or four DNA fragments into any Gateway Destination vector containing attR1 and attR2 sites. The resulting expression clone is ready for downstream expression and analysis applications.
For the BP reaction, approximately 5-10% of the starting material is converted into product. For the LR reaction, approximately 30% of the starting material is converted into product.
The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.
We do have an alternative method called the "attB Adapter PCR" Protocol in which you make your gene specific primer with only 12 additional attB bases and use attB universal adapter primers. This protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. You can find the sequence of these primers in the protocol on page 45 of the "Gateway Technology with Clonase II" manual.
There is a protocol in which all 4 primers mentioned above are in a single PCR reaction. You can find this protocol at in the following article: Quest vol. 1, Issue 2, 2004. https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/quest-archive.reg.in.html. The best ratio of the first gene-specific and the second attB primers was 1:10.
We do not offer pre-made primers, but we can recommend the following sequences that can be ordered as custom primers for sequencing of pDONR201:
Forward primer, proximal to attL1: 5'- TCGCGTTAACGCTAGCATGGATCTC
Reverse primer, proximal to attL2: 5'-GTAACATCAGAGATTTTGAGACAC
1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.
2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.
3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).
4. High-throughput Yeast Two-Hybrid Assays for Large-Scale Protein Interaction mapping. Methods: A Companion to Methods in Enzymology 24(3), pp.297-306
5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.
6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.
7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.
8. Protein-over expression and crystallography Evdokimov, A.G., Anderson, D.E., Routzahn, K.M. & Waugh, D.S.
9. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis. Acta Crystallography (2000) D56, 1676-1679.
10. Evdokimov, et al. Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallographica D57, 793-799 (2001).
11. Lao, G. et al. Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells. Cryobiology 43(2):106-113 (2001).
12. High-throughput cloning and expression Albertha J. M. Walhout, Gary F. Temple, Michael A. Brasch, James L. Hartley, Monique A. Lorson, Sander Van Den Huevel, and Marc Vidal.
13. Gateway Recombinational Cloning: Application to the Cloning of Large Numbers of Open Reading Frames or ORFeomes. Methods in Enzymology, Vol. 328, 575-592.
14. Wiemann, S. et.al., Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs, Genome Research (March 2001) Vol. 11, Issue 3, pp.422-435
15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination
16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)
17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs
18. Loftus S K et al. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 31;8(5):221 (2001).
19. James L. Hartley, Gary F. Temple and Michael A. Brasch. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research (2000) 10(11), pp. 1788-1795.
20. Reboul et al. Open-reading frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nature Genetics 27(3):332-226 (2001).
21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).
The attP1 sequence (pDONR) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]
The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.
The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN
attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
The sequence in brackets comes from attB, and N is your gene-specific sequence.
Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway Entry vectors. The backbone in the case of PCR BP cloning is pDONR201.
There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO adapted Gateway Entry vector is approximately 5 kb. A Gateway recombination reaction can occur between DNA fragments that are as large as 150 kb.
Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag), the N-terminus of an expressed protein will contain an additional 9 amino acids from the attB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.
Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.
Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case, the attB sites do not interfere with transcription or translation.
Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.
Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.
Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence
No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
The Gateway nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.
The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway compatible TOPO Cloning vectors such as pCR8/GW/TOPO and pENTR/D-TOPO. The final option is to use restriction enzymes to clone into a pENTR Dual Selection vector.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.
BP Clonase II and LR Clonase II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.
These enzymes are more stable than the original BP and LR Clonase and can be stored at -20 degrees C for 6 months.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P. nucleic acid purification kits, ChargeSwitch kits, or PureLink kits are recommended.
A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.
This depends on whether you are expressing a fusion or a native protein in the Gateway destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.
The Gateway attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).
Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.
No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.
The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.
There is no theoretical size limitation. PCR products between 100 bp and 11 Kb have been readily cloned into a pDONR Gateway vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway-compatible vector. Overnight incubation is recommended for large inserts.
Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway cloning (about 50 bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.
Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.