FAQ

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 (Multiplicity of Infection) represents the ratio of the number of infectious units of virus vector per target cell.  

For example, an MOI of 10 refers to 10 infectious units of vector per target cell. Therefore, if you need to infect 100,000 cells, you need to add 1,000,000 viral particles.

For LV vector titration, genomic DNA isolated from target cells transduced with LV vector, is subjected to digital 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 and/or digital PCR using primers specific to vector promoter or primers to gene of interest. AAV vector titers are reported as GC/mL, where GC is genome copies and generally represents the number of viral particles with a genome. 

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

PBS pH 7.4 and 0.001% pluronic acid F68.

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 1x10¹³ and 1x10¹⁴ 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.

Choose AAV vectors for their low immunogenicity and long-lasting expression, making them suitable for transducing various tissues, such as neurons and retinal cells.

Choose LV vectors for when a larger genetic payload, stable integration into the host genome and long term expression is required. Commonly used for targeting hematopoetic stem cells and fibroblast. Contact us if you have any questions.