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Lentivirus vector construction
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Note: The inserted gene length should be no longer than 3000 bp in Lentivirus vectors to guarantee high Lentivirus titer. If larger gene is extraordinary necessary, please contact us.
Note: If the selection of Lentivirus plasmid elements listed above do not fully meet your requirements, please contact us.
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Introduction to Custom Lentivirus Production Service
Lentivirus vector system and lentivirus production service:lentiviral vector(lentivirus plasmids) cloning, lentivirus packaging, purification,premade lentivirus.
HIV-1 (human immunodeficiency virus type I) based defective lentivirus has been one of the most widely used gene therapy vectors. It is a powerful tool for introduction of exogenous genes. The most advantageous feature of lentivirus vectors is to mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells. The lentivirus system has been widely used in various cell lines for gene overexpression, RNA interference, microRNA research and in vivo animal experiments.
Genemed provides lentiviral vector construction, lentivirus packaging for RNA interference, miRNA, overexpression constructs and stable transfected cell line construction services. Genemed offers high titer, high purified and ready-to use lentivirus in just two weeks. Please refer to the lentivirus packaging options before you order.
|Lentivirus custom packaging|
|Sipping and Storage Guidelines||Shipped by dry ice, stored at -80 ° C, effective for 1 year. Avoid repeatedly freezing and thawing.|
|Titer||> 1*10^8 TU/ml.|
1. Customized cloning for any other gene ORF expression, shRNA/miRNA and CRISPR/Cas9.
2. Ready-to-use, high purified lentivirus.
3. High titer: 108TU/ml or 109TU/ml lentiviral titer for cell line transfection in medium or large scale.
4. A wide range of host: mediate efficient transfection in both dividing and non-dividing cells.
5. Stable expression: mediate long-term expression of exogenous genes.
Applications and Figures
Quality control description
Our optimized production of lentiviral vector and strict quality control systems supply customers with a high titer of functional recombinant lentiviral vectors. Two methods are employed to determine viral titers: physical titer (VP/mL) and functional titer (TU/mL). Physical titer is calculated by the level of protein, such as p24, or viral nucleic acid. The functional titer, a calculation of the active virus that can infect cells, is much less than the physical titer (100-1000 fold lower). The method we adopted is functional titer, which is an accurate solution for testing virus accurate activity and MOI. The physical titer can only reflect the number of virus particles, but not reflect the true viral activity, which will cause a large error in subsequent infection experiments.
1. For further information about lentivirus administration and transduction, please see Lentivirus User Manual.
Frequently Asked Questions(FAQs)
- 1. How are Genemedi lentiviral vectors different from other viral vectors?
Genemedi has a particular know-how in the field of lentiviral vector design and production stemming from years of research. This experience has led us to set up optimized protocols and reach high quality standards, constantly improved. Hence, Genemedi lentiviral vector productions show high efficiency, suited for either in vivo, in vitro or transgenesis applications. Our expertise allows us to take on all challenges. Notably, we provide our clients with lentiviral vectors expressing particularly challenging transgenes, for instance, ones that directly interfere with the lentiviral cycle.
- 2. For what kind of applications can lentiviral vectors be used?
Our lentiviral vectors can be used in large array of purposes. Taking advantage of our products can significantly enhance your research. Here are some examples of in vitro and in vivo applications:
1. Stable expression transgenes of interest in target cells.
2. Expression of reporter systems to characterize a given vector in particular contexts.
3. Transient expression of transgenes of interest in dividing cells.
4. Induction of long term expression in non-dividing cells with reduced genotoxicity.
5. Generation of cellular models, such as cell lines with recombination induced by transiently expressed genome editing tools (eg: CRISPR).
6. Generation of animal models by zygote injection and transient expression of genome editing tools (KO animals).
- 3. Which cell type is transduced by Genemedi?
Lentiviral vectors are very flexible gene transfer tools and can transduce a large variety of cells. Genemedi can mediate ubiquitous or cell specific expression depending on the envelope and the promoter selected for your vector. Indeed the envelope will determine the entry mechanism of the vector into the cell, and the promoter will define the mechanism of transcription of the transgene. Consequently, the combination of these elements generates ubiquitous transduction and transgene expression in a wide range of cell types.
- 4. What are the advantages of lentivirus over retrovirus and adenovirus?
Lentiviruses comprise a subtype of retroviruses. Lentiviruses can stably integrate into the host genome in dividing, non-dividing and post-mitotic mammalian cells, while retroviruses are less active in this scenario. Adenoviruses can also transduce non-dividing cells, but can’t stably integrate into the host cell’s genome. Adenoviruses also take much more time to design and prepare. In addition, lentiviruses are much less immunogenic than the retroviruses and adenoviruses, making lentivirus more suitable for use in most types of cells and animal models.
- 5. What are the storage and handling recommendations?
Upon receipt, the viral vectors must be stored at -80°C.
The vectors should be taken out from the -80°C freezer at the last moment and kept on ice for gentle thawing. Once thawed, they should be used for transduction as soon as possible to avoid degradation. It is essential to avoid heat shock of the viral vectors and cells. If the vectors should be diluted in medium, use a medium at room temperature to minimize the heat shock experienced by both vectors and cells.
We recommend not refreezing the viral vectors. In order to guarantee viral vectors titer, we recommend keeping freeze-thaw cycles to a minimum. In case that more than one freeze-thaw cycle is required according to your application, Genemedi recommends applying a decrease of about 15% on the titer for each freeze-thaw cycle.
- 6. What are the safety issues when using Genemedi lentiviral vectors?
Genemedi vectors are biosafety level 2 products. The use of retroviral derived vectors implies laboratory biosafety procedures and practices in accordance with regulations applicable in your country.
Genemedi vectors are suited for research applications, either in vitro or in vivo, providing that you respect local laws regarding GMO class 2 storage and handling. All Genemedi vectors are distributed for laboratory research use only and cannot under any circumstances be used for diagnostic or treatment applications. For more information on biosafety levels please visit http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
- 7. What is virus titer? How is lentiviral stock titer determined?
In strict sense, the definition of lentiviral vector titer is the number of lentivirus particles required to infect a cell, present in a volume. Titration of lentiviral preparation is important to consistent and reliable experiment result. Two classes of titration methods have been described for evaluation of lentiviral vector titers, including functional and physical titration methods. Functional assay includes determination of transducing units following transducing cells with limiting dilution of lentiviral preparation, assessment of the number of colony forming units following antibiotic selection, as well as FACS when fluorescent reporters are expressed. The latter includes determination of the quantity of p24 antigen by ELISA, reverse transcriptase activity and the genomic RNA concentration in vector preparation. The physical methods provide the information of the number of physical lentivirus particles in lentiviral preparation. Functional assay generates valuable data on the quality of these lentiviral vector particles.
- 8. What's the optimal concentration of viruses that I should use for infection?
It depends on the purpose of the experiment. Higher titer of lentivirus should be used for in vivo experiment compared to in vitro experiment. Though lentiviral vector is highly efficient in transduction, the transduction efficiency of lentivirus depends heavily on the cell type to be transduced. Pilot experiment is highly recommended to determine how efficient the lentivirus on your target cells.
- 9. How much culture media should I use during infection?
For your reference, we recommend the following amount of virus-containing media for infection:
1. 10-cm plate: 8-10 ml/plate.
2. 6-well plate: 1 ml/well.
3. 12-well plate: 0.5 ml/well.
4. 24-well plate: 0.2 ml/well.
- 10. How do I determine the correct amount of lentivirus to add to the target cells?
One of the key factors of a successful transduction is the cell type. For example, transduction efficiency is much higher in actively dividing cells than in non-dividing cells. In addition, transduction of cells works better at lower MOI (multiplicity of infection) than at higher MOI. MOI is the ratio of the number of lentivirus particles to the number of cells. For some cell types, the higher the MOI , the larger the volume and higher the titer of lentivirus is required in order for the experiment to succeed. You can adjust the cell number and add the appropriate amount of lentivirus according to what has been reported in the scientific literature. If there is no adequate information in the scientific literature, we recommend performing a preliminary experiment using gradient dilutions of lentivirus, such as 0.1 μl, 0.3 μl, 0.5 μl, 0.7 μl, 0.9 μl for Genemedi purified particles. Another important consideration for getting good transduction efficiency is the cell status. Transduction efficiency varies greatly between healthy cells and unhealthy cells. Therefore, it is essential to keep the cells as healthy as possible. For some cells with high MOI, you could also include additives such as polybrene to enhance the transduction efficiency. However, the overall health of the cells itself is always the most essential element.
- 11. Does freeze-thaw cycle infect the titer of lentiviruses?
Yes, multiple freeze-thaw cycles may reduce the functional titer of the virus stock by up to 2-4 folds. However, first one freeze-thaw cycle does not lower the lentiviral titer. Genemedi recommends our customer not to use lentivirus that has gone through more than two freeze-thaw cycles for titer reason.
- 12. Why do my cells die in quantity after adding lentivirus to the culture medium?
Lentivirus has some level of toxicity to cells. It may cause damage to your cell of interest with either superfluous amounts of lentivirus, or if the infection were allowed to go on for too long a period of time. In these cases, you can adjust the multiplicity of infection (MOI) to a lower range. We recommend replacing the old culture medium with fresh complete medium 4-8 hours post transduction (no later than 12 hours post transduction).
- 13. What is the capacity of lentiviral vector as an expression system?
The cloning capacity for the transgene is approximately 3-4 kb for most vector formats.
- 14. How do I create a stable cell line using lentivirus?
Lentiviruses can stably integrate into the host cell’s genome and obtain a consistent level of expression. With a selectable marker in the lentiviral gene transfer vector plasmid, it is easy to generate a stable cell line using drug selection. You can use qRT-PCR, western blot or other detection methods to estimate the expression level of your gene.
- 15. How custom lentivirus production service proceeds
If lentiviral construct is ready, please provide us:
1. The construct map or sequence.
2. 1~5 µg of your lentiviral construct plasmid DNA or bacterial culture.
We will prepare Endotoxin-free plasmid and transfect our packaging cell line for your lentivirus production.
Lentiviral particles will be harvested followed by concentration and titration.
For 200 ul of lentivirus production, you will receive eight 25 µl aliquots.
If you need us to generate lentiviral transfer vector for your target gene, please:
1. Choose lentiviral vector format from our vector collection and specify the gene of interest.
2. Please send us 1~5 µg plasmid or bacterial culture for your gene if it is available, otherwise please send us cDNA sequence.
We will subclone the gene of interest or shRNA into the chosen lentiviral vector and confirm its accuracy by sequencing.
- 16.How long does the service take? Is my project too big (or too small)?
Get ready-to-transduce, high-quality, high titer lentiviral preparations at the production scale your project needs—even at large scales of up to 10 mL. Use your own lentivector construct or take advantage of our Custom Construct services and we’ll handle vector construction as well. We even have an ultra-high titer offering for demanding applications such as in vivo and stem cell transductions.
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