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查看更多产品信息 ViraPower™ Bsd Lentiviral Support Kit - FAQs (K497000)
33 个常见问题解答
VSV-G on the lentivirus binds to the target cells via a common phospholipid (most likely phosphatidylserine) in a receptor-independent manner, which is what gives it such broad tropism: mammalian cells, fish, etc (Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037).
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.
All of the pLenti vectors have the SV40 poly A downstream of the 3' LTR. There is also a poly A located within the 3' LTR that is derived from HIV-1. So in the pLenti plasmids, there are actually 2 poly A sites. 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.
Currently Thermo Fisher Scientific offers the following reporter vectors:
(1) Vivid Colors pLenti6.2-GW/EmGFP, Cat. No. V369-20. The EmGFP is 719 bp and is flanked by attB sites and thus can be moved to a DEST vector via a pDONR vector (In order to do this, first perform BP reaction with a pDONR vector to create pENTR/EmGFP, followed by an LR reaction with a Gateway destination vector to create an expression vector). Note that the pLenti6.2-GW/EmGFP vector can be packaged into lentivirus without additional manipulation.
(2) miR RNAi lentiviral expression vectors containing EmGFP, Cat. No. K4938-00
(3) Lumio containing lentiviral vectors for creation of N- or C-terminal Lumio fusions with a gene of interest (GOI), Cat. Nos. K370-20 and K371-20.
Please note:
The pLenti6.2-GW/EmGFP vector does not allow you to fuse the EmGFP with a gene of interest (although it is possible to engineer EmGFP fusions to a GOI by PCR and subcloning in another vector, followed by cloning back into this or another lentiviral vector). This lentiviral-based vector serves as a control vector to test the lentiviral system and allows for optimization of target cell transductions as well as 293FT producer cell transfections.
If you desire a pLenti construct containing a gene of interest and EmGFP as a reporter, there are several options available. Please contact Technical Support for possible subcloning strategies for removal of bsd gene and insertion of GFP gene. Alternatively, you may wish to have the Thermo Fisher Scientific Cloning Services group create a custom vector.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Inserts cloned into lentiviral vectors should not have a polyA signal. The native polyA 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 (polyA) signal in the insert, the RNA will be terminated prematurely. There is an SV40polyA 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 polyA 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 polyA 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 polyA signal in the insert.
Size is not usually a problem. The insert size limit of lentiviral is ~5-6 kb (average insert size of the SuperScript II premade libraries is ~1.5 kb).
The proviral construct, that is the viral DNA transfected into the 293FT cells, looks like this:
U3/R/U5---vector backbone---gene of interest---deleted U3/R/U5
In our pLenti vectors, the wild-type U3 at the 5'LTR has been replaced with the RSV promoter. Now when RNA transcripts are being made in the 293FT cells you'll get two RNA molecules. One RNA will just be from the CMV promoter or any other promoter that is on the vector backbone. A second RNA transcript will be made from the RSV promoter and will look like this:
R/U5---vector backbone---gene of interest---deleted U3/R
The above RNA transcript made from the RSV promoter contains the viral packaging signal, but not the 5' U3 region. The CMV promoter is located downstream of the packaging signal. Therefore, the transcript from the CMV promoter will lack a packaging signal and will not be packaged into viral particles. Recall that the U3 region is where the wild type viral promoter would normally be.
Now when the packaged viral RNA gets into the target cell, it is reverse transcribed. During reverse transcription, the U3 at the 3'LTR is used as a template to generate the 5'LTR. Thus after reverse transcription, the proviral DNA will look like this:
deleted U3/R/U5---vector backbone---gene of interest---deleted U3/R/U5
Note there is no RSV promoter at the 5'LTR, because it was lost during viral RNA production as explained above. Also notice the deleted U3 region is now at the 5'LTR, which means there are no promoter functions in the 5'LTR. Thus when this proviral DNA integrates into the target cell's genome, the only active promoters will be the ones given by the vector backbone.
Usually our lentiviral vectors have two active promoters, a CMV and a SV40 promoter. The RNA transcript made from the CMV promoter will be quite long, and will include the gene of interest, the SV40 sequence and the antibiotic-resistance gene. There is no transcription stop codon in front of the SV40 promoter. A second RNA transcript will be made from the SV40 promoter and will only code for the antibiotic resistance. The polyA tailing of these two RNA molecules is performed by the polyA signal located in the R region of the 3'LTR.
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CDC guidelines should be followed, please see the Safety information section of the Lentivirus manual or CDC guidelines (www.cdc.gov). Our R&D scientists use 70% ethanol or 10% bleach to inactivate the virus.
The lentivirus is sensitive to temperature (65 degrees C or higher), hypo-osmolarity, 10% bleach, 70% ethanol, and detergents (Triton X-100, etc.). The lentivirus should be stored at -80 degrees C. It should be stable at 4 degrees C overnight, but longer time is not recommended.
The lentiviral (plasmid) vectors should not be stored at -80 degrees C, as too many freeze thaws from -80 degrees C will affect the vector. It is better to store the vector at -20 degrees C, as the vector will not freeze at this temperature.
Our lentivirus is a VSV-G pseudotyped virus. The VSV-G envelope allows transduction of a wide range of cells. However, the pseudotyped virus cannot be transmitted between cells or between individuals since the virus is incapable of producing infectious offspring.
The virus does not replicate in the target cells; it only inserts into the target cell genome to express heterologous genes or hairpins. Since the transduced virus does not produce infectious offspring after transduction, it has no life cycle per se.
Lentiviral packaging proteins (gag/pol and rev) and the vesicular stomatitis virus envelope protein (VSV-G) are provided in trans on three plasmids as a pre-optimized ViraPower Packaging Mix (supplied in the ViraPower Support Kit). For optimal packaging, the ViraPower Packaging Mix is co-transfected with the pLenti6 Expression Vector containing the gene of interest into the 293FT Cell Line using Lipofectamine 2000 reagent, also included in the Support Kit.
In the 293FT Cell Line, the gag/pol, rev, and VSV-G envelope proteins are expressed. The expression of the gag and pol genes is dependent on the expression of rev, and rev expression is also required to transport the viral genome RNA containing the gene of interest out of the nucleus. The packaging site on the viral genome RNA is recognized by the gag protein, and the gag protein packages the pol gene product, reverse transcriptase, and the viral RNA containing the gene of interest. The viral capsids bud off the 293FT cells, and, in a process known as pseudotyping, they pick up the VSV-G envelope protein that once expressed studs the 293FT cell membrane. The packaging proteins are provided in trans and are not part of the final viral particles. These replication-incompetent viral particles are harvested from the media, titered, and can then be used to transduce any mammalian target cells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Adenovirus: For cells that have sufficient expression of the CAR receptors and are actively dividing, it should be possible to get adenovirus transduction efficiencies in the range of 80-90%, as long as an adequate MOI is used (for instance in HT1080 cells, which are readily transducible with adenovirus, transduction efficiencies are around 90% with an MOI of 1).
Lentivirus: Similar transduction efficiencies are possible with lentivirus in certain cell types (for instance, in HT1080 cells, which are readily transducible with lentivirus as well as adenovirus, an MOI of 1 gives transduction efficiencies of around 90%). There is definitely variability in the transduction efficiencies, based on cell type, for both adenovirus and lentivirus. For instance, in some cell types, you may need to use a 10-fold higher MOI to get the same transduction efficiency.
The lentivirus vector contains the SV40 early promoter/ori. Both the 293T and 293FT cell line contain the large T antigen; hence the letter T. (The letter F stands for the high transfection efficiency). The presence of the large T antigen and the SV40 promoter/ori allows for attempted DNA replication at the site of integration. This often leads to cell death because of illegitimate/uneven DNA replication and results in low titers. You may use regular 293 to titer the virus but ideally you should titer with HT1080 cells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There are several steps you can take to help ensure that the viral titer is optimized. The most common reason for low lentiviral titers is poor transfection efficiency. Only in rare instances do our own research scientists obtain a titer of less than 10e5, and in those cases, it has been narrowed down to the transfection efficiency, more specifically, because of issues with the health of the 293FT cells.
(1) Do not vortex the Lipofectamine 2000 reagent. Mix gently by inversion, and make sure it's stored properly.
(2) Plasmid DNA must be sufficiently clean for these transfections.
(3) Adhere to the recommended DNA:lipid ratio of 3:1 (9 µg packaging mix + 3 µg vector, then 36 µL of Lipofectamine 2000 reagent). If the above recommended amounts do not yield acceptable titers, you may also try the following: for each 100 mm plate, use 4.5 µg of the pLenti vector + 18 µg of the packaging mix + 67.5 µL of Lipofectamine 2000 reagent.
(4) Do not pipet up and down the lipid:DNA complexes; mix very gently.
(5) Use low-passage cells. Do not use 293FT cells over passage 20.
(6) Plate cells at 5 x 10e6 per 100 mm dish; density is very important.
(7) Make sure the cells are growing well before re-plating prior to the day of transfection. Avoid overgrowth of 293FT cells when passaging.
(8) G418 has been in the media to this point. When plating for transfection the next day, omit G418 from the media.
(9) On the morning of transfection, replace media on cells with fresh Opti-MEM + 10% FBS (about 1 hour prior to transfection, no G418). Instead of using fresh Opti-MEM + 10% FBS, you can also use complete media without antibiotics. Because the cells seem to be a little healthier, it is therefore favored by some of our researchers in R&D over the fresh Opt-MEM + 10% FBS.
(10) 24 hours post transfection, change the media. Aspirate off the media, add fresh media gently to the side of the dish, not directly to cells (serum-containing media can contain pen/strep but NOT G418).
(11) After another 24 hours (48 hours posttransfection), you can harvest the medium containing your virus. Transfected cells may be detached from the plate, or if attached should look very "plump". If most are on the plate and the cells still look healthy at this point, you can wait an additional 24 hours (72 hours post transfection) before harvesting the media (virus).
(12) To spin down the cells and harvest virus, you can centrifuge virus-containing medium at 1,500 rpm at room temperature or 4 degrees C for 5 min.
(13) Titer the virus on HT1080 cells if available. The titer can be 10-fold less if using HeLa or NIH3T3 cells.
(14) Add "non-essential" amino acids to media when working with cells at high densities.
These suggestions have helped most (if not all) customers to produce good viral titers in their own labs. We are very confident that the materials in our kits are fully functional. The components of the kits are thoroughly tested with the control lacZ vector, where we produce lacZ-expressing virus and titer it on HT1080 cells. The cut-off for the manufactured lot to pass our quality control is a titer of at least 1 x 10e5 cfu/mL. Typically the lots pass with higher titers, and we would never ship a kit that did not meet this virus production criterion. There are many things that must happen for your cells to produce high titers of virus. Cell density, health, cell cycle, lipid:DNA complexes, incubator temps, CO2 levels, etc. all contribute to good virus production. Some variables are easy to control, and some not. If you do observe low titers, it could be due to less-than-optimal conditions for some of these variables on that day for that particular experiment; and in a subsequent experiment, it is possible to achieve a higher titer.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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
Generating stable lines in cells expressing the large T antigen (e.g., COS, 293T) is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. Interaction between the large T antigen and the SV40 origin of replication induces replication of the plasmid. If the plasmid has integrated into the host genome, there will be attempted DNA replication at the site of integration. This often leads to cell death because of illegitimate/uneven DNA replication. Deleting the origin could be an option, however, this will probably affect the promoter activity.
In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".
Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.