Lentiviral Gene Delivery for Mammalian Expression—Getting Started

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.

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.

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.

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.

Lentivirus is a genus of slow retroviruses, characterized by a long incubation period.

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.

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.

We recommend storing lentiviral expression vectors at –20°C. Due to their relatively large size, we do not recommend storing these vectors at –80°C, as the vector solution will completely freeze and too many freeze thaws from –80°C will affect the cloning efficiency.

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.

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.

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.

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 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.

We do offer lentiviral expression vectors with C- or N-terminal Lumio™ tags, which are part of the expression systems, Cat. No. K37020 and Cat. No. K37120, respectively). These vectors allow for convenient detection and localization of proteins in live cells.

The Lumio™ tag is very small; it is only 6 amino acids (~600 Da), whereas the GFP tag is on average ~27 kDa. Since the lentivirus titer drops significantly if the fusion gene is >6 kb, lentivirus expression vectors with the small Lumio™ tag allow insertion of a larger gene of interest (GOI). With GFP, the GOI must be shortened by 719 bp. This is the length of EmGFP in base pairs (bp). Further, Lumio™-tagged lentiviral vectors allow for convenient detection and localization of proteins in live cells. 

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.

The complete kit composition is listed in the product manual under “Kit Contents and Storage.” Search our website (www.lifetechnologies.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.

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.

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 ~5–6 kb (average insert size of the SuperScript® II premade libraries is ~1.5 kb).

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. 

We offer the ViraPower™ HiPerform™ T-REx™ Gateway® Expression System (Cat. No. A11141) that combines ViraPower™ HiPerform™ Lentiviral and T-REx™ technologies to facilitate lentiviral-based, regulated, high-level expression of a target gene in dividing or non-dividing mammalian cells. The ViraPower™ HiPerform™ T-REx™ Gateway® Vector Kit is available separately (Cat. No. A11144). For more information about the T-REx™ system, please refer to Mammalian Expression Support.

Yes, you can use one of our T-REx™ cell lines as the host for your pLenti6.3/TO/V5-DEST lentiviral construct. However, please note that while the pLenti6.3/TO/V5-DEST vector is a HiPerform™ lentiviral vector containing the genetic elements WPRE and cPPT for enhancing viral titer and expression; the T-REx™ cell lines we offer do not contain these elements. Further, you can use these T-REx™ cell lines only for transient expression, because the Lenti6.3/TO/V5-DEST lentiviral expression construct, and the Tet repressor plasmid (pcDNA™6/TR) that is stably integrated within the T-REx™ cells, both contain the blasticidin selection marker, making stable cell line development not possible.

We offer the ViraPower™ HiPerform™ Promoterless Gateway® Expression System (Cat. No. A11145) that combines ViraPower™ HiPerform™ Lentiviral and MultiSite Gateway® technologies to facilitate easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The ViraPower™ HiPerform™ Promoterless Gateway®Vector Kit is available separately (Cat. No. A11146).

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.

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. 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 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 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.

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 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. 

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. 

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 “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. 

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 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. 

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. Please see the image below, showing syncytia formation in 293FT cells after transfection with a GFP-expressing lentiviral expression vector. 

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.

We recommend aliquoting lentiviral stocks immediately after production into small working volumes, and storing at –80°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.

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.

Yes, we do offer a lentivirus production custom service. Please send an email to techsupport@lifetech.com for details.

We strongly recommend the human fibrosarcoma cell line HT1080 (ATCC, Cat. No. 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.

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.

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.

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). 

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 ~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 ~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.

Polybrene® reagent (hexadimethrine bromide) is a cationic polymer from Sigma-Aldrich (Cat. No. H9268) 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® reagent. Note, however, that some cells are sensitive to Polybrene® reagent (e.g.,primary neurons). Hence, before performing a transduction experiment, you may want to test your cell line for sensitivity to Polybrene® reagent at a range of 0–10 μg/mL and avoid using Polybrene® reagent if the cells exhibit toxicity or phenotypic changes.

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.

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.

The proviral construct, which is the viral DNA transfected into 293FT cells, looks like this: 
U3/R/U5---vector backbone---gene of interest---deleted U3/R/U5 

In our pLenti expression vectors, the wild-type U3 at the 5'LTR has been replaced with the RSV promoter. When RNA transcripts are made in 293FT cells, you will 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. 

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 provided 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 poly(A) tailing of these two RNA molecules is performed by the poly(A) signal located in the R region of the 3'LTR.

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 ViraPower™ Lentiviral Expression System includes the following features designed to enhance its biosafety:

• The pLenti expression vector contains a deletion in the 3′ LTR (ΔU3) 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 Ψ 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).

Yes, the protocol we provide in the user guide can be linearly scaled-up to 1 L in a 2 L shaker flask. For larger scale needs, we have also developed a bioreactor protocol for up to 2 L. If you are interested, please email us at cts@thermofisher.com for details.

LV-MAX Transfection Reagent, enhancer, and supplement are specifically designed to work synergistically with the LV-MAX Production Medium and Viral Production Cells to provide maximal lentiviral titer. Mixing and matching reagents is not recommended, as this will decrease the overall performance.

Viral Production Cells can be frozen directly in cryogenic medium consisting of 90% LV-MAX Production Medium plus 10% DMSO. Here is the protocol:

- Bank cells at passage 3 when cell density reaches ˜3.0-4.5 x 10E6 viable cells/mL at >95% viability.
- Centrifuge the cells at 100 x g (low speed) for 5 mins to pellet. Discard the spent medium.
- Prepare freezing medium: e.g., 10 mL of freezing medium is 9 mL of LV-MAX Production Medium + 1 mL DMSO; mix well and sterilize by 0.2 µm filtration.
- Resuspend the cell pellet in the prepared freezing medium.
- Dilute the cells to a final density of 10 x10E6 viable cells/mL and aliquot 1 mL per cryovial.
- Freeze the cells at -80 degrees C for 1 day.
- Transfer frozen vials to liquid nitrogen for long-term storage.

Several factors are known to affect viral yield:

- Production cell density: We suggest a cell density of 4 x 10E6 viable cells/mL in our protocol, but since different cell counting methods may give different counting results, we recommend that you optimize your production cell density based on your cell counting system. We recommend trying 3 different cell densities, i.e., 3 x 10E6, 4 x 10E6, and 4.5 x 10E6 cells/mL to determine the optimal cell density.
- Quality of the plasmid: Make sure that your plasmids are endotoxin-free and of high purity.
- Protocol: Following our cell culture protocol to establish your culture is the key to successfully use our LV-MAX System.
- Size of the gene: The size of the gene you are interested in will impact viral particle yield; the larger the size of the gene, the lower will be the viral particle yield.

Note: While viral titer yields are highly dependent on production volume, the specific gene being expressed, and its length, our R&D team as well as external collaborators have consistently seen an increase in viral titer that is equivalent to or 10-fold higher when using LV-MAX Lentiviral Production System as compared to adherent PEI, or suspension PEI methods.

While we currently don't have data for prior generation lentiviral packaging systems, the LV-MAX Lentiviral Production System should be compatible with them but may require further optimization.

You may use an alternative shaker, but it must be CO2 resistant. If you use a non-digital shaker, you will need to optimize the shaker speed based on general observation. You must ensure that the cells do not settle but maintain suspended. You will need to generate your own cell growth curve to ensure you exceed 90% cell viability.

No. In addition to the LV-MAX System, you will need Opti-MEM I Reduced Serum Medium (Cat. No.31985088) as the DNA-LV-MAX Transfection Reagent complexation medium. You will also need a pLenti transfer plasmid and pLenti packaging mix. Please see the user guide for a complete list of recommended materials and equipment.

No, we've done the work for you. You will need to determine the best production cell density under your cell counting system. We recommend trying 3 different cell densities, i.e., 3 x 10E6, 4 x 10E6, and 4.5 x 10E6 cells/mL to determine the optimal cell density.

We have identified 3 key protocols that will address most customers' needs. In the user guide, you will find protocols for 1 mL in a 2 mL 96-well deep block, 5-30 mL in 50 mL conical tubes, and 30 mL-1 L in various shaker flask sizes. For larger-scale needs, we have developed a bioreactor protocol for up to 2 L. if you are interested, please email us at cts@thermofisher.com for details.

We have not optimized the LV-MAX Production System for AAV production. However, the Viral Production Cells contain the right genetic elements for producing AAV.

We understand the importance of this classification and currently have an active GMP-development program in place, and plan to offer a GMP version in the future.

Currently, only the LV-MAX Production Medium is manufactured according to cGMP guidelines. We have an active GMP-development program in place and plan to offer a complete GMP system in the future.

Yes, all components of the LV-MAX Production System are serum-free.

Yes, all components of the LV-MAX Production System are animal origin-free. However, if Opti-MEM I Reduced Serum Medium is used as the DNA-LV-MAX Transfection Reagent complexation medium, it will no longer be animal origin-free.

No, these cells are derived from Freestyle 293F, and have been adapted to our LV-MAX Production Medium.

Yes, that should be fine.

The critical incubation time is the one for the diluted LV-MAX Transfection Reagent. Prior to combining with diluted DNA, the diluted LV-MAX Transfection Reagent should be incubated for only 1 min, and not more than 4 mins.

In the user guide, we have provided recommendations based on our development of the kit. If you need to optimize further for your specific need, we recommend a weight-to-weight ratio of transfer plasmid:packaging vectors at 1:3, 2:3, and 3:3.

We recommend a weight-to-weight ratio of 2:3 transfer plasmid:packaging vectors; if needed use a 3:3 weight-to-weight ratio.

Spinoculation increases lentiviral transfection efficiency and provides higher viral titer than without the spin step. If you don't spin, you can expect to get 5- to 10-fold less lentiviral titer.

We strongly recommend the human fibrosarcoma line HT1080 (ATCC, Cat. No. CCL-121) as the “gold standard” for reproducibly titering lentivirus. However, you may wish to use the same mammalian cell line to titer your lentiviral stocks as you will use to perform your expression studies (e.g., if you are performing expression studies in a dividing cell line or a non-primary cell line). If you have more than one lentiviral construct, we recommend that you titer all of the lentiviral constructs using the same mammalian cell line.

Lentiviral particles packaged in our LV-MAX Production Medium will be stable at 4 degrees C for 24 hours and will then begin to lose activity. For long-term storage, we recommend storing at –80 degrees C.

Lentiviral particles packaged in our LV-MAX Production Medium will be stable at 4 degrees C for 24 hours and will then begin to lose activity. For long-term storage, we recommend storing at –80 degrees C.

Yes. Please provide your details here and mention your interest in our lentivirus production services. A member of our services team will contact you shortly.

The LV-MAX and CTS LV- MAX products have equivalent performance. In contrast to the research use only LV-MAX products, CTS LV-MAX products have an additional intended use for cell and gene therapy applications, and are complemented with a more robust regulatory support package to enable clinical therapeutic applications. This means that to the best of our ability, we keep our CTS product specifications and release requirements up-to-date in accordance with the latest regulatory guidance specific for this intended use.

The intended use, validation, and protocol optimization varies among the different cell types. Expi293F Cells and FreeStyle 293-F Cells were specifically developed and validated for transient protein production while Viral Production Cells and CTS Viral Production Cells were designed and validated for lentiviral vector manufacturing. The commonality between the Expi293F Cells, Viral Production Cells, and CTS Viral Production Cells is that they were originally derived from FreeStyle 293-F lineage. Both Expi293F and Freestyle 293F cells come with regulatory support intended for bio-production applications whereas CTS Viral Production Cells (cGMP banked) are intended for cell and gene therapy applications, and complemented with a more robust regulatory support package to enable clinical gene therapeutic applications. This means that to the best of our ability, we keep our CTS product specifications and release requirements up-to-date in accordance with the latest regulatory guidance specific for this intended use.

We can provide protocol guidance for how to detect transfection reagent components under CDA. Please contact our outlicensing@thermofisher.com for information.

We have tried other media and have seen reduced performance. For maximal lentiviral vector yield, we recommend using the CTS LV-MAX Production Medium.

Depending on whether your culture will be 3 or 4 days, we have outlined the differences in seeding densities in the product manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018450_CTS_LV-MAX_LentiviralProdSys_UG.pdf). We also have specific guidance for seeding densities to be used for lentiviral vector production.

The passages should be counted upon thawing, and the cells should be discarded when they reach passage 20 post-thaw.

Our system has been optimized to provide maximal lentiviral production at 48 hr post transfection. Waiting until 72 hr is not recommended since existing lentiviral particles within the media will infect the production cells and can reduce the titer.

We recommend using the lentiviral packaging plasmids and transfer plasmids at a ratio of 3:2.