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查看更多产品信息 ViraPower™ HiPerform™ Lentiviral FastTiter™ TOPO™ Expression Kit - FAQs (K532520)
15 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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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.