Lentivirus Packaging
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.
VectorBuilder offers lentivirus packaging services with superior quality. We have developed a series of proprietary technologies and reagents that have greatly improved recombinant lentivirus production protocols in terms of titer, purity, viability and consistency, especially for the third-generation lentiviral vector systems used in our vector cloning services. As a result, we have a growing base of highly satisfied customers who come back to us time after time for their lentiviral vector cloning and lentivirus packaging needs. In addition to research-grade lentivirus, we also offer GMP-grade lentivirus manufacturing for clinical applications.
Types of lentivirus offered
- VSV-G pseudotyped third-generation lentivirus (this is the default virus type)
- VSV-G pseudotyped second-generation lentivirus
- Lentivirus pseudotyped with other envelope proteins as
requested, such as GaLV, RD114, BaEV, and coronavirus spike (S) proteins - Bald lentivirus lacking viral envelope protein (this can be used as negative control in viral transduction)
- Integrase-deficient lentivirus (IDLV)
Service Details
Price and turnaround Price Match
| Scale | Application | Typical Titer | Minimum Titer | Volume | Price (USD) | Turnaround |
|---|---|---|---|---|---|---|
| Mini | Cell culture | >2x108 TU/ml | >108 TU/ml | 100 ul (4x25 ul) | $239 |
6-12 days
|
| Pilot | >4x108 TU/ml | 250 ul (10x25 ul) | $449 | |||
| Medium | >3x108 TU/ml | 1 ml (10x100 ul) | $649 | |||
| Large | >2x109 TU/ml | >109 TU/ml | 1 ml (10x100 ul) | $1,099 | ||
| Ultra-purified medium | Cell culture & in vivo | >2x109 TU/ml | >109 TU/ml | 500 ul (10x50 ul) | $1,399 | |
| Ultra-purified large | 1 ml (10x100 ul) | $1,699 | ||||
| High-throughputNew | Cell culture | >5x106 TU/ml | >2x106 TU/ml | 100 ul/well | $99/well | 14-21 days |
Note:
1. The above table applies to VSV-G pseudotyped 2nd- and 3rd-generation lentivirus (integrase-deficient lentivirus (IDLV) included).
2. Click to view service details of lentivirus pseudotyped with coronavirus S protein.
3. TU = Transduction units (also known as infectious units).
4. The high-throughput packaging service is performed using 96-well plates, with a minimum order of 50 wells. Please contact us to place an order.
Deliverables
| For non-ultra-purified scales | For ultra-purified scales |
|---|---|
| Your custom lentivirus | Your custom lentivirus |
|
Free: control virus
|
Add-on purchase (optional): ultra-purified control virus
|
|
Free: Polybrene (5 mg/ml, 200 ul) |
Free: Polybrene (5 mg/ml, 200 ul) |
Control virus
The control lentivirus is designed to match the biological application of the custom virus and to be used for testing lentiviral transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control lentivirus (lentivirus overexpressing EGFP), and if the custom virus expresses an shRNA against a gene, then the control virus provided will express a scramble shRNA. Detailed information on the control virus is shown below:
Shipping and storage
Our lentivirus is stored in HBSS buffer and is shipped on dry ice. Upon receiving, it should be stored at -80°C for long term (stable for at least 6 months), or -20°C 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.
Technical Information
Lentivirus production and quality control (QC)
For our third-generation lentivirus packaging, transfer plasmid carrying the gene of interest (GOI) is co-transfected with our proprietary envelope plasmid encoding VSV-G and packaging plasmids encoding Gag/Pol and Rev into HEK293T packaging cells. After 48 hours of incubation, the supernatant is collected and centrifuged to remove cell debris and then filtered. Lentiviral particles are subsequently concentrated with PEG. For ultra-purified lentivirus (in vivo grade), viral particles are further purified by sucrose gradient ultracentrifugation and concentrated by ultrafiltration. We use the p24 ELISA method to measure lentivirus titer.
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.
| Additional QC Service | Method |
|---|---|
| Physical titer determination | qRT-PCR |
| Functional titer determination | qPCR in HEK293T cells |
| Flow cytometry | |
| Endotoxin testing | LAL |
Experimental validation
We have developed a number of proprietary techniques to optimize our lentiviral packaging protocols, and our virus has been validated to exhibit high transduction efficiency in vitro and for a broad range of applications.
Expression
CAR
CRISPR
shRNA knockdown
Figure 1. HEK293T cells were transduced with recombinant lentivirus packaged from the lentiviral gene overexpression vector, guide RNA expression vector, or short hairpin RNA expression vector with either EGFP or mCherry as a reporter. Cells were imaged at 48 hrs post-transduction.
Figure 2. High-throughput lentiviral gRNA library displays efficient transduction in vitro. (A) Individual titers (dots) of arrayed lentiviral libraries in 96-well plates as quantified by p24 ELISA. (B) Transduction of HEK293T cells with arrayed lentiviral libraries. HEK293T cells were cultured in a 96-well plate containing an arrayed lentiviral gRNA library at MOI 10, and GFP expression was imaged 48 hrs post-transduction.
Figure 3. Lentiviral transduction of Jurkat cells for efficient CAR expression. (A) Jurkat cells were transduced with lentivirus encoding CD19-targeting CAR and co-cultured with CD19-expressing Ramos cells. Upon binding of CD19, Jurkat cells are activated, as shown by production of CD69 surface antigen. (B) Following incubation, flow cytometry analysis was performed on CAR Jurkat cells, detecting a drastic increase in the surface expression of CD69 upon co-culturing with CD19+ Ramos cells (blue), signifying robust CAR-mediated activation of Jurkat cells.
Figure 4. High-efficiency gene editing with the all-in-one lentivirus-based CRISPR system. (A) Lentiviral vectors expressing Cas9:T2A:Puro and EGFP-targeting or scramble gRNA were packaged into lentiviral particles and transduced into HEK293T cells stably expressing EGFP at MOI 10 or 50. Antibiotic selection with puromycin was performed to isolate positively transduced cells and EGFP expression detected by microscopy (100X). (B) The gRNA-targeted region was PCR amplified from the genomic DNA of non-transduced cells (NC) and cells transduced with scramble gRNA or EGFP-targeting gRNA (EGFP-1 and EGFP-2). Gene editing was confirmed using the T7E1 assay. (C) EGFP-positive cells were quantified using flow cytometry. (D) The ratios of EGFP-positive cells were decreased to 28-32% or 8-14% after editing using lentivirus at MOI 10 or 50, respectively. Statistical analysis was performed using ANOVA followed by Dunnett’s post hoc test. ***P<0.001 vs gRNA scramble control.
Figure 5. Up-regulation of gene expression achieved by lentivirus-based CRISPRa. NIH3T3 cells stably expressing SAM complex, dCas9/VP64, and MS2/P65/HSF1 were transduced with msgRNA expression lentivirus followed by antibiotic selection. (A) Illustration of SAM system regulated transcriptional activation. (B) Diagram of msgRNA design targeting the promoter region of the mouse Brn2 gene. (C) Relative gene expression of Brn2 in NIH3T3 cells transduced with scramble or targeting msgRNA or no treatment control (NC), measured by qRT-PCR. Mean±SD, *P<0.05, ANOVA with Tukey’s post hoc test.
Figure 6. Down-regulation of gene expression achieved by lentivirus-based CRISPRi. Jurkat cells stably expressing the dCas9/KRAB/MeCP2 transcriptional repressor complex were transduced with gRNA expression lentivirus followed by antibiotic selection. (A) Diagram of gRNA design targeting the promoter region of the human CXCR4 gene. (B) CXCR4 protein levels in Jurkat cells transduced with scramble or targeting gRNA or no treatment control (NC), measured by western blot. (C) Relative CXCR4 gene expression in Jurkat cells transduced with scramble or targeting gRNA or no treatment control (NC), measured by qRT-PCR. Data are presented as mean±SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. ***P<0.001, ****P<0.0001. (D) Surface expression levels of CXCR4 in Jurkat cells transduced with scramble or targeting gRNA were quantified by flow cytometry. CXCR4 expression was detected by labelling with a monoclonal primary antibody, followed by incubation with a fluorophore-conjugated secondary antibody. Unlabeled Jurkat cells and cells incubated with secondary Ab only were included as controls. Data shown are mean±SD.
Figure 7. U6 versus miR30-based shRNA lentiviral knockdown systems. (A) Lentiviral vectors carrying U6-based shRNA, CMV-driven miR30-based single shRNA, and CMV-driven miR30-based quad shRNA were separately packaged into lentiviral particles. HEK293T cells stably expressing EGFP were transduced with the shRNA lentivirus, and EGFP expression was measured by flow cytometry before (B) and after (C) drug selection using the appropriate antibiotics. Relative EGFP expression was calculated by dividing the median fluorescence intensities of the transduced cells by those of the non-transduced cells. Technical replicates were performed (n=3) and data shown are mean±SD. Statistical analysis was performed using Tukey’s test. ***P<0.001.
How to Order
Customer-supplied vectors
If customer-supplied lentiviral plasmids are used in packaging, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.
Resources
FAQ
| Lentivirus | MMLV | Adenovirus | AAV | |
|---|---|---|---|---|
| Tropism | Broad | Broad | Ineffective for some cells | Depending on viral serotype |
| Can infect non-dividing cells? | Yes | No | Yes | Yes |
| Stable integration or transient | Stable integration | Stable integration | Transient, episomal | Transient, episomal |
| Maximum titer | High | Moderate | High | Very high |
| Promoter customization | Yes | No | Yes | Yes |
| Primary use | Cell culture and in vivo | Cell culture and in vivo | In vivo | In vivo |
| Immune response in vivo | Low | Low | High | Very low |
We use the p24 ELISA for measuring lentivirus titer. This method employs a sandwich immunoassay to measure the levels of the HIV-1 p24 core protein in lentiviral supernatants. The lentivirus samples are first added to a microtiter plate, the wells of which are coated with an anti-HIV-1 p24 capture antibody, to bind the p24 in the lentivirus samples. This is followed by the addition of a biotinylated anti-p24 secondary antibody, which in turn binds to the p24 captured by the first antibody on the plate. A streptavidin-HRP conjugate is then added for binding the biotinylated anti-p24 antibody due to the interaction between streptavidin and biotin. A substrate solution is ultimately added to the samples which produces color upon interaction with HRP. The intensity of the colored product is proportional to the amount of p24 present in each lentivirus sample, which is measured by the use of a spectrophotometer and is then precisely quantified by comparing against a recombinant HIV-1 p24 standard curve. The p24 value is then correlated with the viral titer of the corresponding lentivirus sample.
Our titer guarantee applies to lentiviral vectors for which the region being packaged into virus (from Δ5’ LTR to ΔU3/3’ LTR) is below the lentivirus cargo limit (9.2 kb). For sizes above this, it may still be possible to package the vector into virus, but the titer may be reduced. Additionally, we are not able to guarantee titer for the following vectors:
- Vectors containing sequences that could adversely affect the packaging process such as toxic genes (e.g. proapoptotic genes), genes that compromise the integrity of packaging cells or virus (e.g. membrane proteins that cause cell aggregation), and sequences prone to rearrangements or secondary structures (e.g. repetitive or highly GC-rich sequences);
- Customer-supplied plasmids containing undisclosed sequences or atypical lentiviral functional elements (e.g. LTR) that may introduce uncertainties to packaging efficiency.
Our estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA or viral vectors), QC of such materials, and transit time for shipping final deliverables to the customer.
Featured Citations
Mammalian ubiquitous promoter isolated from proximal regulatory region of bovine MSTN gene
Eom, et al.
Nature Scientific Reports, 2024, doi: 10.1038/s41598-024-76937-2
Products used:
- Lentivirus Packaging
- Vector Cloning
Abstract: Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.
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Itaconate transporter SLC13A3 impairs tumor immunity via endowing ferroptosis resistance
Lin, et al.
Cancer Cell, 2024, doi: 10.1016/j.ccell.2024.10.010
Products used:
- Lentivirus Packaging
- Vector Cloning
Abstract: Immune checkpoint blockade (ICB) triggers tumor ferroptosis. However, most patients are unresponsive to ICB. Tumors might evade ferroptosis in the tumor microenvironment (TME). Here, we discover SLC13A3 is an itaconate transporter in tumor cells and endows tumor ferroptosis resistance, diminishing tumor immunity and ICB efficacy. Mechanistically, tumor cells uptake itaconate via SLC13A3 from tumor-associated macrophages (TAMs), thereby activating the NRF2-SLC7A11 pathway and escaping from immune-mediated ferroptosis. Structural modeling and molecular docking analysis identify a functional inhibitor for SLC13A3(SLC13A3i). Deletion of ACOD1 (an essential enzyme for itaconate synthesis) in macrophages, genetic ablation of SLC13A3 in tumors, or treatment with SLC13A3i sensitize tumors to ferroptosis, curb tumor progression, and bolster ICB effectiveness. Thus, we identify the interplay between tumors and TAMs via the SLC13A3-itaconate-NRF2-SLC7A11 axis as a previously unknown immune ferroptosis resistant mechanism in the TME and SLC13A3 as a promising immunometabolic target for treating SLC13A3+ cancer.
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Glioblastoma remodelling of human neural circuits decreases survival
Saritha Krishna, et al.
Nature, 2023, doi: 10.1038/s41586-023-06036-1
Products used:
- Lentivirus Packaging
- Vector Cloning
Abstract: Gliomas synaptically integrate into neural circuits. Previous research has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth and gliomas increasing neuronal excitability. Here we sought to determine how glioma-induced neuronal changes infuence neural circuits underlying cognition and whether these interactions infuence patient survival. Using intracranial brain recordings during lexical retrieval language tasks in awake humans together with site-specifc tumour tissue biopsies and cell biology experiments, we fnd that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumour-infltrated cortex well beyond the cortical regions that are normally recruited in the healthy brain. Site-directed biopsies from regions within the tumour that exhibit high functional connectivity between the tumour and the rest of the brain are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumour cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the diferential neuron–glioma interactions observed in functionally connected tumour regions compared with tumour regions with less functional connectivity. Pharmacological inhibition of thrombospondin-1 using the FDA-approved drug gabapentin decreases glioblastoma proliferation. The degree of functional connectivity between glioblastoma and the normal brain negatively afects both patient survival and performance in language tasks. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumour progression and impairs cognition.
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