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View additional product information for ViraPower™ HiPerform™ Lentiviral TOPO™ Expression Kit - FAQs (K531000)
70 product FAQs found
Possible causes include:
- large volume of viral supernatant used for transduction
- cells sensitive to Polybrene regaent
- too much antibiotic used for selection
- antibiotic used too soon after tranduction
- gene of interest is toxic to cells
Poor expression could result from low transduction efficiency, too low of a MOI, too much antibiotic used for selection, usage of antibiotic too soon after transduction, harveting cells too soon after transduction, having a gene of interest that is toxic to cells, or rerrangement in the LTR regions of the expression construct plasmid DNA.
Here are some possible causes and solutions:
- Promoter silencing; CMV promoter is prone to silencing especially in mouse and rat cells, screen multiple antibiotic resistant clones and select the one with the highest expression levels
- Viral stocks stored incorrectly; aliquot and store at -80 degrees C, do not freeze/thaw more than 3 times
Here are some possible causes and solutions:
- Too little antibotic used for selection
- Selection performed on confluent cells; replate cells
- Viral supernatant not diluted sufficiently; titer lentivus using a wider range of 10-fold serial dilutions
Possible causes include:
- low transfection efficiency; Use a high-quality plasmid prep, 293FT cells under passage 16, ensure removal of Geneticin during transfection, ensure correct DNA:lipid ratio, and that cells are plated at the correct confluency
- transfected cells are not cultured in medium containing sodium pyruvate; this reagent provides an extra energy source for cells
- viral supernatant harvested too early; viral supernatants can generally be collected 48-72 hrs post-transfection
- viral supernatant too dilute; concentrate virus using CsCl purification
- viral supernatant frozen and thawed multiple times; 3 times should be the maximum freeze/thaw
- gene of interest is large; viral titers decrease as size of insert increases, inserts larger than 5.6 kb are not recommended
- rearrangement in the LTR region of the epxression construct plasmid DNA; use Stb3 cells for transformatin of the lentiviral construct
- poor choice of titering cell line; use HT1080 cells or similar cell line
- Polybrene reagent is not included during transduction; transduce lentiviral construct into cells in the presence of Polybrene reagent
- Lipofectamine reagent handled incorrectly; ensure proper storage and mix gently before use
- Use fluorescence micrscopy to check titer with HiPerform FastTiter lentivirus
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If 293FT cells detach shortly after transfection (4 hours to overnight):
- This may be a sign of Lipofectamine 2000 toxicity. Cells may have been plated too sparsely prior to transfections.
- The cells may not have been handled gently enough (these cells have a tendency to lift off easily).
- The cells may have been kept at room temperature for too long.
If cells detach 48 to 72 hours post-transfection:
- If the cells lift off in large sheets, this may be a sign of lentivirus production.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. Hence, we do not recommend using a mini-prep kit for propagation of lentiviral constructs. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or the PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield- basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.
Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low, 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.
We strongly recommend using Stbl3 E. coli for cloning lentiviral constructs. Stbl3 E. coli cells contain the recA13 mutation in their genotype that helps to minimize the likelihood of unwanted recombination between the LTRs. After transforming into Stbl3 E. coli, we recommend picking colonies and validating the lentivirus DNA from mini-preps using Afl II and Xho I digests before proceeding to midi-preps. In all of our lentiviral vectors, Afl II sites are present in both 5' and 3' LTRs, and a Xho I site is present after the 3' end of the MCS. Assuming Afl II cuts only in the LTR sites, and there are no Afl II or Xho I sites in the insert, 3 DNA fragments are expected to be generated from the Afl II + Xho I digest. Any unexpected DNA fragments can be assumed to be a result of LTR recombination. Only clones with the expected pattern of DNA fragments should be chosen for the subsequent midi-prep.
The ViraPower Lentiviral Expression System includes the following features designed to enhance its biosafety:
The pLenti expression vector contains a deletion in the 3' LTR (deltaU3) that does not affect the generation of the viral genome in the producer cell line, but results in “self-inactivation” of the lentivirus after transduction of the target cell (References: Yee JK, Moores JC, Jolly DJ, Wolff JA, Respess JG, Friedmann T (1987) Gene Expression from Transcriptionally Disabled Retroviral Vectors. Proc. Natl. Acad. Sci. USA 84: 5197-5201; Yu SF, Ruden Tv, Kantoff PW, Garber C, Seiberg M, Ruther U, Anderson WF, Wagner EF, Gilboa E (1986) Self-Inactivating Retroviral Vectors Designed for Transfer of Whole Genes into Mammalian Cells. Proc. Natl. Acad. Sci. USA 83: 3194-3198; Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Selfinactivating Lentivirus Vector for Safe and Efficient in vivo Gene Delivery. J. Virol. 72: 9873-9880). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome.
- The number of genes from HIV-1 used in the system has been reduced to three (i.e., gag, pol, and rev).
- The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (References: Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK (1993) Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 90: 8033-8037; Emi N, Friedmann T, Yee JK (1991) Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus. J. Virol. 65: 1202-1207; Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T (1994) A General Method for the Generation of High-Titer, Pantropic Retroviral Vectors: Highly Efficient Infection of Primary Hepatocytes. Proc. Natl. Acad. Sci. USA 91: 9564-9568).
- Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids. All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication-competent virus (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
- Although the three packaging plasmids allow in trans expression of proteins required to produce viral progeny (e.g., gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the psi packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced.
- The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. No other viral species are produced.'
- Expression of the gag and pol genes from pLP1 has been rendered Rev-dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
- A constitutive promoter (RSV promoter) has been placed upstream of the 5' LTR in the pLenti expression vector to offset the requirement for Tat in the efficient production of viral RNA (Reference: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72: 8463-8471).
Despite the presence of the above safety features, the lentivirus produced can still pose some biohazardous risk, since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral stocks generated using this system as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating lentivirus carrying potential harmful or toxic genes (e.g., activated oncogenes).
For more information about the BL-2 guidelines and lentivirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories”, 4th Edition, published by the Centers for Disease Control (CDC) (www.cdc.gov/biosafety/publications/index.htm).
Lentiviruses produced with our system do not carry or express any viral genes, and therefore have no associated toxicity issues. Only the protein expressed from the coding region between the LTR sites is incorporated into the mammalian cell chromosome and expressed. The lentivirus itself cannot replicate because of the built-in safety features.
The concern with leaving the lentivirus on the cells longer would be potential toxicity or growth effects. If you absolutely cannot remove the virus from the cells, we suggest to leave it on the cells and empirically monitor the cells.
Note: These are based on the characteristics of HIV.
Morphology: Virions have a complex construction and consist of an envelope, a nucleocapsid, a nucleoid, and a matrix protein. Virions are enveloped, spherical to pleomorphic in shape, and have a size of 80-100 nm in diameter. The surface projections are small or inconspicuous spikes that are densely dispersed, evenly covering the surface. Surface projections are 8 nm long. The core is rod-shaped, or is truncated cone-shaped. The nucleoid is concentric.
Physicochemical and Physical Properties: Virions have a buoyant density in sucrose of 1.13-1.18 g cm-3. Virions are sensitive to treatment with heat, detergents, and formaldehyde. The infectivity is not affected by irradiation.
Proteins: Proteins constitute about 60% of the particle weight. The viral genome encodes structural proteins and non-structural proteins. Virions consist of 5 major structural and 3 non-structural proteins. The virus codes for an RNA-dependent DNA polymerase.
Lipids: Lipids are present and located in the envelope. Virions are composed of 35% lipids by weight. The composition of viral lipids and host cell membranes are similar. The lipids are of host origin, derived from plasma membranes.
Carbohydrates: Three percent of the particle weight is attributed to carbohydrates.
Polybrene (hexadimethrine bromide) is a cationic polymer(Cat. No. H9268 from Sigma Aldrich) that increases transduction efficiency by neutralizing the charge repulsion between virus particles and the cell surface. For best results, we recommend performing transduction in the presence of Polybrene.
Note: Some cells (e.g., primary neurons) are sensitive to Polybrene. Hence, before performing a transduction experiment, we recommend testing your cell line for sensitivity to Polybrene at a range of 0-10 µg/mL and avoiding it if the cells exhibit toxicity or phenotypic changes.
We have found that, in general, 80-90% of the cells in an actively dividing cell line (e.g., ht1080) express a target gene when transduced with lentivirus at an MOI of approximately 1. Some non-dividing cell types transduce lentiviral constructs less efficiently. For example, only about 50% of the cells in a culture of primary human fibroblasts express a target gene when transduced at an MOI of approximately 1. If you are transducing your lentiviral construct into a non-dividing cell type, you may need to increase the MOI (e.g., MOI = 10) to achieve optimal expression levels for your recombinant protein. If you are transducing your lentiviral construct into your mammalian cell for the first time, we recommend using a range of MOIs (e.g., 0, 0.5, 1, 2, 5, 10) to determine the MOI required to obtain the optimal protein expression for your application.
The lentiviral envelope is pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), which allows the lentivirus to interact with its target cell in a receptor-independent manner. As a result, lentivirus has broad tropism and can, in theory, transduce any mammalian cell type. This receptor-independent entry into the target cell likely involves endocytosis (Espenshade et al. (2002) Proc Natl Acad Sci U S A 99:11694; Aiken (1997) J Virol 71:5871).
You can perform PCR/qPCR analysis of viral genes for lentivirus titering, but keep in mind that the titer will not be a measure of functional virus since it will measure both inactive as well as active virus. As a result, titers obtained using this method are usually about 10-fold higher than with methods that measure functional virus, such as blasticidin selection.
You can use the p24 ELISA assay for lentivirus titering, but keep in mind that the titer will not be a measure of functional virus since it will measure both inactive as well as active virus. As a result, titers obtained using this method are usually about 10-fold higher than with methods that measure functional virus, such as blasticidin selection.
We strongly recommend the human fibrosarcoma cell line ht1080 Cells (ATCC# CCL-121) as the “gold standard” for titering lentivirus. The primary reason is that transduction efficiency is high in these cells, and titering results are very accurate and reproducible. However, you may also use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies. In general, this should be an adherent, non-migratory cell line, and exhibit a doubling time in the range of 18-25 hours. Regular 293 cells may be used for lentivirus titering, but we do not recommend using 293T or 293FT cells because these cells contain the SV40 large T antigen that will induce unwanted DNA replication at the SV40 ori contained within the integrated lentiviral expression vector. This often leads to cell death and results in very low titers.Lentivirus is a genus of slow retroviruses, characterized by a long incubation period.
Yes, we do offer a lentivirus production custom service. Please send an email to techsupport@thermofisher.com for details.
Lentivirus produced using our system is replication-incompetent, and this is a safety feature. You must perform a fresh transfection each time you need more virus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend aliquoting lentiviral stocks immediately after production into small working volumes, and storing at -80 degrees C for long-term storage. Lentivirus is sensitive to storage temperature and to freeze/thaw and should be handled with care. It can lose up to 5% or more activity with each freeze/thaw. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
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 transfection.
- 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.
Syncytia are large multi-nucleated cells that result from VSV-G-induced fusion with neighboring 293FT producer cells. Syncytia production is indicative of high transfection efficiency and lentivirus production. Keep in mind, though, that the absence of syncytia does not mean that virus will not be produced.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The 293FT cell line stably expresses the SV40 large T antigen from the pCMVSPORT6Tag.neo plasmid that contains the neomycin resistance marker. In order to maintain the plasmid/phenotype, the cells have to be routinely cultured in medium containing Geneticin (G418) antibiotic at a concentration of 500 µg/mL.
We use Mycoplasma-tested Gibco FBS (Cat. No. 16000-044). 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 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.
The “F” stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the “T” stands for the SV40 large T antigen. The large T antigen expression plasmid is stably integrated in the genome and confers resistance to Geneticin antibiotic in these cells. The presence of the SV40 large T antigen is important for high-titer lentivirus production and the mechanism is not known. If regular 293 cells or another 293T cell line is used as the producer cell line, you will be able to produce virus, but the titers will be lower.
The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors adds up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging. Titers will generally decrease as the size of the insert increases.
The ViraPower Lentiviral Packaging Mix consists of an optimized mixture of three viral packaging plasmids, pLP1, pLP2, and pLP VSVG, supplied at 1 mg/mL in TE buffer, pH 8.0. The individual plasmids are not available as standalones.
Our lentiviral packaging mix belongs to the third generation, meaning that it does not express the tat gene. It can be used with lentiviral vectors that belong to the third generation or higher, where virus production is independent of the tat gene.
Your second-generation lentiviral vector will not be compatible with our packaging mix because our packaging mix belongs to the third generation, meaning that it does not express the tat gene; whereas your lentiviral vector will need the tat gene for virus production.
Our lentiviral expression vectors belong to the third generation, meaning that they contain a chimeric 5' LTR, by means of which virus production is not dependent on the HIV tat transactivator. As a result, they are compatible with a second- or third-generation packaging mix.
Our lentiviral packaging mix belongs to the third generation, meaning that it does not express the tat gene. Further, gag/pol and rev genes are supplied as independent plasmids, thus eliminating concerns about recombination events bringing components together as a single vector to produce replication-competent lentivirus.
Our lentiviral expression vectors belong to the third generation, meaning that we use a four-plasmid vector system (1 lentiviral expression vector and 3 packaging plasmids), thus eliminating concerns about recombination events bringing components together as a single vector to produce replication-competent lentivirus. Gag/pol and rev genes are supplied as independent plasmids. Further, these vectors contain a chimeric 5' LTR, by means of which virus production is not dependent on the HIV tat transactivator. Also, the original U3 region of the LTR (long terminal repeat) is deleted to make the virus self-inactivating and thus replication-incompetent.
Our lentiviral expression vectors contain approximately 20% of the original viral genome. The rest of the viral genome is deleted from our lentiviral expression vectors for safety reasons.
The main difference between these systems is in the lentiviral expression vector contained within the kits. The ViraPower Lentiviral Expression Vector backbone is similar to the ViraPower HiPerform Lentiviral Expression Vector backbone except that the latter contains two new elements: WPRE (Woodchuck Posttranscriptional Regulatory Element) from the woodchuck hepatitis virus that is placed directly downstream of the gene of interest, allowing for increased transgene expression and the cPPT (central Polypurine Tract) from the HIV-1 integrase gene, which increases the copy number of lentivirus integrating into the host genome and thus allowing for a two-fold increase in viral titer. Together, WPRE and cPPT produce at least a four-fold increase in protein expression in most cell types, compared to the vectors in the ViraPower Lentiviral Expression Systems that do not contain these elements. In the ViraPower HiPerform Fast Titer Expression System, in addition to the WPRE and cPPT elements, the lentiviral expression vector contains the EmGFP reporter gene instead of Bsd, which allows titer of active virus by flow cytometry in just two days post-transduction.
Inserts cloned into lentiviral vectors should not have a poly(A) signal. The native poly(A) signal (AATAAA or something similar) will be amplified when using the oligo dT during cDNA synthesis. Thus, it will then become part of the cDNA library or its clones.
Since lentivirus is an RNA virus, during the synthesis of the RNA genome to be packaged, if there is a polyadenylation (poly(A)) signal in the insert, the RNA will be terminated prematurely. There is a SV40 poly(A) signal in the vector, but it is after the second LTR, and it is supposed to be there. Almost any clone transferred from a Gateway cDNA library will probably have a poly(A) signal, which, if inserted into a lentiviral vector, would end up terminating the viral RNA prematurely.
In order to circumvent premature termination of the lentiviral RNA, consider these recommendations:
- The desired gene should first be isolated from the library, cloned into an entry vector such as pENTR/D-TOPO without the poly(A) signal (i.e., ATG to Stop), and then transferred into the lentiviral vector.
- If you are trying to establish a lentiviral expression library, you will probably have to go with a library that was amplified using random hexamers rather than an oligo dT, since such a library would be less likely to include a poly(A) signal in the insert.
Size is not usually a problem. The insert size limit of the lentivirus is approximately 5-6 kb (average insert size of the SuperScript II premade libraries is approximately 1.5 kb).
In pLenti6 vectors, the blasticidin (Bsd) resistance marker is driven by the SV40 promoter whereas in pLenti6.2 vectors, the Bsd resistance marker is driven by the phosphoglycerate kinase-1 (PGK) promoter.
This difference is important when working with stem cells; the PGK promoter is a native mammalian promoter that is resistant to silencing and shows long-term, persistent expression in stem cells, whereas the SV40 promoter is often silenced over time in primary cells and stem cells.
The complete kit composition is listed in the product manual under “Kit Contents and Storage.” Search our website (www.thermofisher.com) using the catalog number to find the product you're interested in. Once you are on the product page, the manual can be viewed and downloaded using the Manuals link. We provide a variety of vectors/kits to suit the various cloning and expression strategies used by different researchers.
We carry the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector (Cat. No. V37006) and Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector (Cat. No. V36920), both of which are lentiviral vectors containing Emerald Green Fluorescent Protein (EmGFP). They are designed for use with the ViraPower Lentiviral Expression Systems as positive controls to enable the detection of EmGFP fluorescence following transfection in 293FT cells. These vectors serve as titer controls to produce an EmGFP-expressing lentivirus stock and as a transduction control following transduction in both dividing and non-dividing mammalian cells. Both of these vectors are not cloning vectors. The Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long-term, persistent expression of the blasticidin-stable selection marker, whereas the Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the SV40 promoter for driving expression of the blasticidin-stable selection marker. In addition, the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector is a HiPerform vector, meaning that it is equipped with two key genetic elements: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in increase in protein expression in most cell types, compared to lentiviral vectors that do not contain these elements.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
All our pLenti expression vectors have 2 poly(A) sites-a poly(A) located within the 3' LTR that is derived from HIV-1 and the SV40 poly(A) downstream of the 3'LTR. The reason for having both is to reduce the chances of transcriptional interference (for instance, if there were a significant amount of transcriptional read-through that continued through the RSV promoter region, this could potentially interfere with transcription from the RSV promoter, which is critical for production of the viral RNA). Once the lentivirus has integrated in the target cells, the SV40 poly(A) will not be present (since the virus just extends from the 5' to 3' LTR), but the poly(A) within the 3' LTR region will still be present and functional.
The HIV-1 genome consists of two identical copies of single-stranded RNA. Generating dsRNA, as could happen in this instance, will reduce titers since the dsRNA will interfere with genome packaging. Hence, reversing the orientation of the expression cassette with respect to the LTRs will decrease virus titers. Reference: Mautino et al. (2000) Human Gene Therapy 11:895.
Yes, the lentiviral expression vector will work as an expression vector by itself and can be stably selected with the appropriate antibiotic. Please note that the vector will be about twice the size of most regular vectors. Therefore, you may need to increase the amount of transfected vector to approximate molar equivalents.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We do not recommend using a mini-prep kit for propagation of lentiviral constructs because the DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield-basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.
Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low: 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.
We recommend storing lentiviral expression vectors at -20 degrees C. Due to their relatively large size, we do not recommend storing these vectors at -80 degrees C, as the vector solution will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency.
The TOPO cloning method is an easy-to-use, 5-minute benchtop PCR cloning method, and we have developed many kits based on this technology. You may want to choose this method if you have only one construct to make. If you plan to place your gene of interest into several different expression systems, you may want to consider Gateway cloning technology, also used in many of our expression kits. Another consideration in choosing the cloning method would be the size of your insert. If your insert is >4 kb, we recommend choosing Gateway cloning, as TOPO cloning will not work efficiently for these large inserts.
Our lentiviral vectors are based on the HIV-1 backbone. However, several alterations have been made so they function solely as a gene delivery vehicle without subsequent viral replication or disease. Specific HIV-1 genes have been deleted to enhance safety. The HIV-1 genes are only expressed in the producer cells (293FT) and none of them are packaged into the viral genome, and thus are never expressed in the transduced target cell.
Lentivirus is a genus of slow retroviruses, characterized by a long incubation period.
If you are interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO (D-TOPO) and Gateway version of the kit to provide flexibility in the cloning of the gene of interest. If you are looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have th CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.
It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
MOI stands for multiplicity of infection. Theoretically, an MOI of 1 will provide 1 virus particle for each cell on a plate, while an MOI of 10 represents ten virus particles per cell. However, several factors can influence the optimal MOI including the nature of your mammalian cell line, (non-dividing vs. dividing), transduction efficiency, your application of interest, and your protein of interest.
When transducing your adenoviral or lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOIs (0, 0.5, 1, 2, 5, 10, 50) to determine the MOI required to obtain optimal gene expression. MOIs greater than 50, such as MOI 100, are common for the transduction of neurons with lentivirus. After you determine the MOI that gives optimal gene expression, subsequent transductions can be performed at the optimal MOI.
Adenoviral expression is used for transient expression, whereas lentiviral expression is used for longer-term expression. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have the CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.
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.
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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.
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The ViraPower lentiviral packaging mix is an optimized mix of three packaging plasmids: pLP1, pLP2 and pLP/VSVG, supplied in TE buffer, pH 8.0, at a concentration of 1 µg/µL. These plasmids supply the helper functions as well as structural and replication proteins in trans that are required to produce lentivirus using our ViraPower lentiviral expression vectors.
Polybrene (hexadimethrine bromide) is a cationic polymer used to increase the efficiency of infection of certain cells with virus particles in cell culture. Polybrene acts by neutralizing the charge repulsion between the virions and cell surface.
The plasmid expressing the SV40 large T antigen, pCMVSPORT6TAg.neo, is stably integrated in 293FT cells. Hence, we recommend culturing 293FT cells in the presence of 500 µg/mL Geneticin (G418) antibiotic.
Here is a summary of the safety features that have been built into the ViraPower lentiviral expression system.
• The system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild-type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead.
• The system is a four-plasmid system, and the packaging functions are supplied in the packaging mix in trans, eliminating concerns about recombination events bringing components together as a single vector.
• The lentivirus particles that are generated carry only the gene of interest, and the packaging mix vectors never become part of the packaged virus.
• The lentivirus has a self-inactivation feature, meaning that once it is transduced into cells and has integrated into the mammalian cell host, it is no longer capable of producing a packageable viral genome.
• Expression of gag/pol from pLP1 is rev dependent, so both plasmids (pLP1 and pLP2) must be co-expressed to package the gene of interest into virions.
There are many safety features that we have built into our ViraPower lentiviral expression system. We recommend strict adherence to all CDC guidelines for Biosafety Level 2 (BL-2), as well as institutional guidelines. BL-2 guidelines may be found in “Biosafety in Microbiological and Biomedical Laboratories” 4th edition, Centers for Disease Control (www.cdc.gov).
Lentivirus should be aliquoted immediately after production and stored at -80 degrees C. Lentivirus can lose up to 5% of titer with each freeze/thaw. When stored properly, viral stocks should be suitable for use for up to one year. After long-term storage, we recommend re-titering the viral stocks before use.
As the size of the insert increases, lentiviral titers decrease. The size of the wild-type HIV genome is approximately 10 kb. Since the size of the elements required for expression from ViraPower lentiviral vectors totals approximately 3–4.5 kb, the size of the insert should not exceed 5.6–6 kb. In-house data shows that viral titer drops approximately 2-fold for every kb of insert size over 4 kb. If you wish to produce lentivirus with an insert of > 4 kb, you will need to concentrate the virus to obtain a suitable titer.
a. Try different MOIs. All cells are not created equal.
b. Try Polybrene. Test in the range 0–10 µg/mL (manual recommends 6 µg/mL).
c. If Polybrene does not help, you might try using DEAE-dextran. We have not tried it in-house, but it has been used in the literature [Reiser et al. (1996) PNAS 93, 15271].
ViraPower lentiviral expression kits contain a control lentiviral expression vector (the lacZ- expression vector or GFP- expression vector). When packaged into virions and transduced into a mammalian cell line, these vectors express either ß-galactosidase or GFP, respectively. If you use the lacZ control virus, you can use the ß-galactosidase staining kit (Cat. No. K1455-01) to determine transduction efficiency. If you use the GFP control virus, you can use a fluorescent microscope or perform FACS analysis.
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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.