FAQ
Shipping to the U.S.
An import permit from Centers For Disease Control And Prevention (CDC) is required for shipment of viral vectors to the U.S. An import permit is a document issued by the national government authorizing the importation of infectious biological materials that could potentially cause disease in humans and in order to prevent their introduction and spread into the U.S.
It is the customer’s responsibility to provide the relevant import permit and/or supporting documentation in order to facilitate shipping biological materials into the country. There may also be duties applied by local customs upon receipt. Incorrect or invalid contact information could delay your delivery. Users should consult their official permitting office as these forms are subject to change.
Use the following link to get the import permit from the CDC: https://eipp.cdc.gov/.
A step-by-step guide to walk you through obtaining your permit: https://www.cdc.gov/cpr/ipp/docs/eIPP_agents_training_508.pdf
General
We predominantly manufacture 3rd Generation LV vectors. These 3rd generation vectors are manufactured by transfection of producer cells with 4 expression plasmids, a LV gag-pol expression plasmid, a LV Rev expression plasmid, a VSV-G envelope expression plasmid and an expression plasmid containing the gene of interest.
We can also manufacture 2nd Generation lentivirus vector if requested. The 2nd generation vectors are produced by transfection of producer cells with 3 expression plasmids; a LV gag-pol-rev-tat expression plasmid, a VSV-G envelope expression plasmid, and an expression plasmid containing the gene of interest.
The MOI or the multiplicity of infection represents the ratio of the number of infectious units of virus vector per target cell. Therefore, a MOI of 10 refers to 10 infectious units of vector per target cell.
Vector titers are determined by Q-PCR.
For LV vector titration, genomic DNA isolated from target cells transduced with LV vector, is subjected to Q-PCR to determine the number of integrated LV vector genome using primer and probe specific to the vector sequence. LV vector titers are reported as IU/mL, where IU is infectious units and represents the number of infective viral particles. Since the delivered LV vector is stored at -80°C, the LV vector titer is performed on vector preparations that have been stored at -80°C and thawed.
For AAV vector titration, a small aliquot of AAV vector preparation is subjected to Q-PCR using primers specific to vector promoter. AAV vector titers are reported as GC/mL, where GC is genome copies and generally represents the number of viral particles with a genome. As with LV vector titering, AAV titering is also performed on vector preparations that have been stored at -80°C and thawed.
LV vector kit must be kept frozen and stored at -20°C. Use immediately after thawing and avoid multiple freeze-thaw cycles as it reduces the efficiency of the kit.
LV and AAV vectors should be stored immediately at -80°C. All our vectors have shelf-life of one year without loss in titer. In our tests we have observed a 10% loss in virus titer when vectors are stored for two years.
We recommend not to freeze-thaw your viral vector preparations multiple times. In our test studies we have found that there is an approximate 15% loss in vector titer from a single freeze-thaw. If you plan to use the virus for multiple experiments, thaw the viral preparation on ice, remove the desired volume for use in experiment, aliquot the remaining viral vector preparation into smaller aliquots, and snap-freeze aliquots in dry ice before storing at -80°C.
The maximum gene capacity between the two ITRs of an AAV vector is 4.5kb.
The maximum size of sequence that can be accommodated between LTRs is 9.0kb. Taking into consideration the LV packaging sequence, RRE and cPPT required in the transgene expression vector, only 7.5kb of promoter-transgene sequences can be added to the vector. There is an inverse relationship between the size of the transgene vector and LV vector titer (Systematic Determination of the Packaging Limit of Lentiviral Vectors Mukesh Kumar, Brian Keller, Ndeye Makalou, Richard E. Sutton Human Gene Therapy. 2001, 12(15): 1893-1905).
LV vectors
Dulbecco’s Modified Eagle Medium.
AAV vectors
Sterile PBS with 35 mM NaCl and 5% glycerol.
Lentivirus (LV) vectors
We provide functional titers ie number of infectious units (IU) per mL which are more relevant than genomic copy/mL or total virus particles/mL. We provide 1 mL of LV vector with titers of 108 IU/mL based on most transgene plasmids. Virus titers are dependent on the size and nature of the transgene expression construct. The infectious titer of each viral preparation is determined by Q-PCR or by flow cytometry if fluorescent gene is expressed and titers are reported in the Certificate of Analysis sent with each shipment
Adeno-associated virus (AAV) vectors
We provide AAV vector with titers ranging from 1×1012 – 1×1013 genomic copy/mL. Titers are determined by Q-PCR and is based on genomic copies in vector particles. Where vectors have fluorescent marker, we can also determine titers as infectious units/mL.