The anti-CD19 CAR lentiviruses are replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that are ready to infect almost all types of mammalian cells, including primary and non-dividing cells. These viruses transduce the ScFv portion of anti-CD19 (clone FMC63) linked to 2nd generation CAR (Chimeric Antigen Receptor) containing CD8 hinge, 4-1BB and CD3ζ signaling domains (Figure 1).
BiosafetyNone of the HIV genes (gag, pol, rev) will be expressed in the transduced cells, as they are expressed from packaging plasmids lacking the packing signal. Although the pseudotyped lentiviruses are replication-incompetent, they require the use of a Biosafety Level 2 facility. BPS recommends following all local federal, state, and institutional regulations and using all appropriate safety precautions.
We are offering ready-to-use viral preparations of several popular lentiviral plasmids and pooled libraries in our repository. With viral service, Addgene provides high-quality and high-titer viral preparations. We perform quality control experiments, including using our lentiviruses for stable cell line generation, colony formation titering assay, or fluorescence titering assay. You can see the results of our quality control experiments on the material pages for viral catalog items. For descriptions of Addgene's viral production protocols and quality controls, visit our viral production page. For protocols on how to use virus from Addgene or how to make your own virus, see our list of viral protocols with tips and sample data.
Lentiviruses, a subset of retroviruses, are some of the most common and useful types of viruses used in research. Lentivirus can transduce both dividing and non-dividing cells without a significant immune response. These viruses also integrate stably into the host genome, enabling long term transgene expression. There are some safety considerations to evaluate when working with lentivirus: these viruses are based on HIV-1, which may require additional lab biosafety procedures. Additionally, because this virus integrates randomly into the host genome, it has the potential for insertional mutagensis. The 2nd and 3rd generation lentiviral systems available at Addgene have been designed for increased researcher safety.
One safety feature of lentivirus used in research is that the components necessary to produce an infectious viral particle (a virion) are generally divided among multiple plasmids. Some plasmids (typically called packaging and envelope plasmids) encode components of the viral capsid and envelope and are used in conjunction with the plasmid that encodes the viral genome (typically called the transfer plasmid). This table lists common lentiviral envelope and packaging plasmids that can be used with 2nd and 3rd generation lentivirus technologies.
The lentiviral genome (i.e., the genetic material enclosed in the viral particle) is delivered to a target cell upon infection. When lentivirus is used for research, it is the lentiviral genome that encodes genetic material that the researcher wants delivered to specific target cells. This genome is encoded by plasmids called "transfer plasmids," which can be modified to encode a wide range of gene products. The main restrictions on a lentiviral transfer plasmid are size (i.e., a limited number of basepairs of nucleic acid can fit into a viral particle) and toxicity of the gene product (i.e., if the gene encoded by the transfer plasmid is toxic to mammalian cells, it can be difficult to efficiently produce viral particles). This table lists lentiviral transfer plasmids that can be used with 2nd and 3rd generation lentivirus technologies.
Our custom lentivirus product service provides customers with maximal flexibility and top quality. Our services range from construct design, to lentivral vector production, concentration and titration.
We will subclone the gene of interest or shRNA into chosen lentiviral vector and confirm by sequencing, prepare high quality plasmid DNA and transfect packaging cell line, harvest lentivirus particle followed by concentration and titration.
Transduction of neural progenitor cells by Lenti-X lentivirus. Recombinant lentivirus for expressing ZsGreen1 was produced using Lenti-X virus and used to transduce normal human neural progenitor cells. A single transduced cell is shown under phase contrast microscopy (Panel A) and fluorescence microscopy (Panel B).
High-titer lentivirus production. Lenti-X 293T cells were transduced with the indicated volumes (µl) of lentiviral packaging supernatant generated with the Lenti-X Expression System and then selected with puromycin for 9 days to allow the formation of the resistant colonies, which were then stained with crystal violet.
Recombinant lentivirus is the most commonly used viral vector for efficient gene delivery into mammalian cells. Unlike plasmid DNA vector which only allows transient and episomal expression of the foreign gene in the host cell, lentiviral vector can achieve permanent expression of the foreign gene through integration into the host cell genome. Lentivirus is also efficient for in vivo gene delivery.
Our lentivirus is stored in HBSS buffer and is shipped on dry ice. Upon receiving, it should be stored at -80C for long term (stable for at least 6 months), or -20C for use within one week. The shelf life for lentivirus is approximately one year. Please avoid repeated freeze-thaw cycles of lentivirus, as this can result in a large titer drop.
For each recombinant lentivirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the transfer vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. If the transfer vector encodes a drug-selectable marker, we would perform transduction test followed by corresponding drug selection. Additionally, for ultra-purified lentivirus, we routinely perform endotoxin assay to check the endotoxin level. To include the endotoxin results in your COA, an extra cost is required. Additional QC services can be provided upon request.
These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce a recombinant lentivirus, containing the gene of interest
For use with ViraPower Lentiviral Expression System
Lentivirus production platforms are often limited by insufficient viral titers which require concentration before use. The TransIT Lentivirus System was developed for enhanced delivery of the essential transfer and packaging vectors required for higher-titer lentivirus production. With the TransIT Lentivirus System, greater than 2-fold higher functional titers are achieved over competing lentivirus production systems.
Q1. What is lentivirus?Q2. How is lentivirus produced?Q3. What components are included in the TransIT Lentivirus System?Q4. What is the composition of the TransIT-Lenti Transfection Reagent?Q5. What vectors/elements are included in the Lentivirus Packing Mix Powered by MISSION Genomics?Q6. What is the shelf life of the TransIT Lentivirus System?Q7. How should the TransIT Lentivirus System components be stored?Q8. Can the TransIT-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension cells?Q9. What titers are expected when using the TransIT Lentivirus System?Q10. How does TransIT Lentivirus System compare to other commercially available lentiviral packaging systems?
Q1. What is lentivirus?Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family commonly used as a gene delivery tool for robust and stable transgene expression in both dividing and non-dividing cells. Stable transgene expression is achieved through the random integration of the viral genome into the host cell genome.
Q2. How is lentivirus produced?To produce lentivirus, cells are co-transfected with packaging plasmids encoding the required gag, pol, and rev structural and regulatory genes and a transfer vector encoding the gene of interest (GOI). Essential components are combined with the vesicular stomatitis virus (VSV) envelope protein G for a broad virus tropism and increased stability during purification procedures. The lentivirus particles are secreted into the culture medium where they are collected, filtered and frozen into aliquots for subsequent transduction into target cells.
Q8. Can the TransIT-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension HEK 293 cells?No. TransIT-Lenti Transfection Reagent is only recommended for adherent cell transfections (e.g. HEK 293T/17) and is not recommended for lentivirus generation in suspension cell types. For highest lentivirus titers in suspension 293 cell types, we recommend using our TransIT-PRO Transfection Reagent.
Q13. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation? The TransIT-Lenti Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.
Q15. Is a media change required post-transfection?No. A media change to remove TransIT-Lenti:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus titers. 041b061a72