Viral Vectors Technology

Adeno-associated viruses (AAVs) are replication-deficient parvoviruses, which have traditionally required co-infection with a helper adenovirus or herpes virus for productive infection. AAV has proven especially valuable for long-term gene expression due to the ability of AAV to replicate epichromosomally under replicationpermissive conditions. Slowly dividing or non-dividing cells can maintain the epichromosomal AAV genome over time, and gene expression will be stable while the AAV genome is maintained. Rapidly dividing cells will lose epichromosomal genome after cell replication and will, therefore, lose gene expression. Viral integration into the genome can occur, however, integration events are rare. We routinely produce AAV vectors of 1, 2 or 5 serotypes or as well mosaics.

HIV-based recombinant lentiviral vectors are able to transduce dividing and also non-dividing cells, therefore they are extensively used in the scientific and medical approach.

Their ability to integrate into genomic DNA ensures a stable and constant expression of the incorporated transgene. Lentiviral vectors are used as an alternative for the low-efficient transfection of in vitro cultured cells (infection efficacy is almost 100%), as well as the tools enabling transgene delivery to adult animal tissues and embryos. At LAM we routinely use the 2nd generation lentiviral system. In order to obtain LV vectors we cotransfect HEK 293T cells with a set of vector plasmids (the envelope plasmid, the packaging plasmid and expressing plasmid – carrying the modified gene of interest). 48 h and/or 72h after after transfection, the culture medium is collected and ultracentrifuged. Pellet containing viral particles is dissolved in PBS, aliquoted and stored at -80°C.

Collection of vectors can take place 48-72 hrs after transfection. You can choose one or two time points. The latter will provide twice as much aliquots of viral suspension of the same/comparable concentration for your use.
The qPCR reaction enables to estimate the concentration of LV particles. We also offer titering of vectors produced in our facility, as well as any other vector provided by you.

The quality of plasmid DNA is critical for proper transfection and further viral vector packaging. For high quality, plasmid DNA should be propagated in an endonuclease negative E. coli strain such as NEB stable or STBL3. To minimise the bacterial recombination of the plasmid, bacteria should be grown in 30ºC (instead of regular 37ºC).

Endotoxins can inhibit transfection, therefore, plasmid DNA purification should include an endotoxin removal step.

Submission form (Viral_vector_order_form.pdf) can be posted to LAM together with plasmid construct, as well as scanned and e-mailed in advance.