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 construction 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 coronavirus spike (S) proteins
  • Bald lentivirus lacking viral envelope protein (this can be used as negative control in viral transduction)

Service Details

Price and turnaround
Scale Application Typical Titer Minimum Titer Volume Price (USD) Turnaround
Pilot Cell culture >4x10TU/ml >10TU/ml 250 ul (10x25 ul) $399 8-16 days
Medium >3x10TU/ml 1 ml (10x100 ul) $599
Large >2x10TU/ml >10TU/ml 1 ml (10x100 ul) $999
Ultra-purified medium Cell culture & in vivo >2x10TU/ml >10TU/ml 500 ul (10x50 ul) $1,199
Ultra-purified large 1 ml (10x100 ul) $1,499

TU = Transduction units (also known as infectious unit)

Deliverables
For non-ultra-purified scales For ultra-purified scales
Your custom lentivirus Your custom lentivirus

Free: control virus

  • Pilot: >10TU/ml, 100 ul
  • Medium: >10TU/ml, 2x100 ul
  • Large: >10TU/ml, 2x100 ul

  Add-on purchase (optional): ultra-purified control virus

  • Ultra-purified medium: >10TU/ml, 10x50 ul, at $400
  • Ultra-purified large: >10TU/ml, 10x100 ul, at $500

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:

Vector System Control Virus Vector Name Control Virus Vector ID
Lentivirus gene expression system pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry VB160109-10005
Lentivirus U6-based shRNA knockdown system pLV[shRNA]-EGFP:T2A:Puro-U6>Scramble_shRNA VB151023-10034
Lentivirus miR30-based shRNA knockdown system pLV[miR30]-EGFP/Puro-EF1A>mCherry:Scramble_miR30-shRNA VB180124-1174nde
Lentivirus CRISPR system pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry VB160109-10005
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

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 cushion ultracentrifugation and concentrated. 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.

Technical documents
User Instructions Material Safety Data Sheet (MSDS) Certificate of Analysis (COA)

How to Order

Order both vector construction and virus packaging
Order virus packaging of your own vector

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.

FAQ

What features does lentivirus have comparing to other viral vectors?
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 Very High 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
How is viral titer determined in VectorBuilder?

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.

What is VectorBuilder's guarantee for virus titer and turnaround?

Our titer guarantee applies to 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.