Lentiviral vectors provide the means to yield stable integration of genetic components for the sustained expression or knockdown of genes of interest. Lentiviruses additionally provide the ability to transduce dividing and non-dividing cells with long-term expression. For their utility in producing stable vector integration into a host genome, lentiviral constructs have been used extensively in the functional analysis of genes. Lentiviral gene transfer allows for stable and conditional transgene expression in a wide variety of target cells, including non-dividing hematopoietic stem and neuronal cells. Lentiviral infection has advantages over other gene therapy methods including high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity.
Syno® Lentivirus Production Procedure
Infectious lentiviruses have three main genes coding for the viral proteins in the order: 5´-gag-pol-env-3´. To improve safety, viral accessory genes are deleted, and essential genes are supplied in trans by the packaging and envelope plasmids. We use 3rd generation LV packaging plasmid which includes gag, coding for the virion main structural proteins; pol, responsible for the retrovirus-specific enzymes; and RRE, a binding site for the Rev protein which facilitates export of the RNA from the nucleus. To produce lentivirus particles, the transfer plasmid containing the gene of interest (GOI), the envelope plasmids, and the packaging plasmid are co-transfected into the packaging HEK293T cells. For our lentivirus packaging services, the lentivirus particles are purified using sucrose gradient ultracentrifugation to ensure safe use in vivo. Viral titer is then determined using quantitative real-time PCR.