Adenovirus Packaging

Recombinant adenoviral vectors are used for achieving high levels of transgene expression in a wide variety of mammalian cell types, in which the vector remains as episomal DNA without integration into the host genome. High transduction efficiency and high levels of short-term gene expression make adenoviral vector a preferred tool for in vivo gene delivery and they are often used in gene therapy and vaccination.

VectorBuilder has developed a series of proprietary technologies and reagents that have greatly improved recombinant adenovirus production protocols in terms of titer, purity, viability and consistency, especially for the adenoviral 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 adenoviral vector cloning and adenovirus packaging needs.

Types of adenovirus offered
  • Human Ad5 adenovirus
  • Chimeric Ad5/F35 adenovirus

Service Details

Price and turnaround
Scale Application Typical Titer Minimum Titer Volume Price (USD) Turnaround
Pilot Cell culture >2x1010 PFU/ml >1010 PFU/ml 250 ul (10x25 ul) $399 20-32 days
Medium 1 ml (10x100 ul) $599
Large >2x1011 PFU/ml >1011 PFU/ml 1 ml (10x100 ul) $999
Ultra-purified large Cell culture & in vivo >2x1012 VP/ml >1012 VP/ml 1 ml (10x100 ul) $1,499 22-36 days

PFU = Plaque forming units; VP = Virus particles

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

Free: control virus

  • Pilot: >1010 PFU/ml, 100 ul
  • Medium: >1010 PFU/ml, 2x100 ul
  • Large: >1010 PFU/ml, 2x100 ul

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

  • Ultra-purified large: >1012 VP/ml, 10x100 ul, at $500
Control virus

The control adenovirus vector is designed to match the biological application of the custom virus and to be used for testing adenovirus transduction. For example, if the custom virus overexpresses a gene, then the control virus provided will be EGFP control adenovirus (adenovirus 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 for the various control virus is shown below:

Vector System Control Virus Vector Name Control Virus Vector ID
Adenovirus gene expression system pAV[Exp]-CMV>EGFP VB150925-10024
Adenovirus U6-based shRNA knockdown system pAV[shRNA]-EGFP-U6>Scramble_shRNA VB161026-1127kyf
Adenovirus miR30-based shRNA knockdown system pAV[miR30]-CMV>EGFP:Scramble_miR30-shRNA VB180124-1101mjs
Adenovirus CRISPR system pAV[Exp]-CMV>EGFP VB150925-10024
Shipping and storage

Our non-ultra-purified adenovirus is stored in HBSS buffer and our ultra-purified adenovirus is stored in GTS buffer. All our adenovirus are shipped on dry ice. Upon receiving, adenovirus should be stored at -80°C for long term (stable for at least 1 year), or -20°C for short term, e.g., 2-3 weeks. Although adenovirus is more stable than many other virus (e.g. lentivirus) and can be frozen and thawed several times with minimal loss of virus activity, it is best to avoid repeated freeze-thaw cycles in practice.

Technical Information

Adenovirus production and QC

For our adenovirus manufacturing, the adenoviral vector carrying the gene of interest (GOI) is first linearized by restriction digestion with PacI. The linearized vector DNA is transfected into packaging cells expressing the adenovirus gene E1 to produce recombinant adenovirus, and adenoviral particles are released into tissue culture medium where they are collected. For ultra-purified adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the titer using an immunocytochemistry-based approach to detect the adenovirus-specific hexon protein. For ultra-purified adenovirus, we measure the optical density (using OD260) of the viral particles to estimate the titer.

For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the adenoviral vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. Additionally, for ultra-purified adenovirus, 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 cloning and virus packaging
Order virus packaging of your own vector

Customer-supplied vectors

If customer-supplied adenovirus 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 adenovirus 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?

For adenovirus, we measure the functional titer. After transducing serially-diluted adenovirus into 293A cells, we use an immunocytochemistry-based approach to count the number of cells being successfully transduced via the detection of adenovirus-specific hexon protein, and each immunostained cell is considered as one infectious unit. Cells are infected at very low multiplicity of infection (MOI) to ensure that most transduced cells are each infected by a single viral particle. This assay shows good correlation with conventional plaque assay. For ultra-purified adenovirus, we directly measure the optical density (using OD260) of the viral particles to estimate titer, because there is a tight correlation between the optical density of ultra-purified adenovirus and functional titer. Adenovirus has very good stability. In our preparation, the viral particles are essentially all alive and can remain functional at room temperature for many days.

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

We guarantee the minimum titer when the region of the vector being packaged into virus (from 5’ ITR to 3’ ITR) is below the adenovirus cargo limit (38.7 kb). For sizes above this, the adenoviral genome may be unstable, and rearrangement may occur during packaging. 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 adenoviral functional elements (e.g. ITR) that may introduce uncertainties to packaging efficiency.

The 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.