CAG-Citrine Lentivirus

Lentiviral vectors derived from human immunodeficiency virus (HIV-1) have become the main tool for gene delivery in mammalian cells. Lentiviral vectors can mediate efficient transduction and stable expression in dividing and non-dividing cells in vitro and in vivo. The HIV-1 genome contains nine open reading frames encoding at least 15 different proteins involved in the infection cycle, including structural and regulatory proteins. In addition, several cis-acting elements are required at various stages of the viral life cycle, including long terminal repeats (LTRs), TAT activation regions (TARs), splice donor and acceptor sites, packaging and dimerization signals (Ψ), Rev response elements (RREs), and central and terminal polypurine tracts (PPTs). The earliest lentiviral vectors were replication-competent viruses carrying transgenes. To make these vectors safer, HIV vectors evolved through a series of modifications to separate the viral sequences required for packaging and production from those encoding viral proteins. The first generation of HIV-1-based lentiviral vectors separated the vector components into three plasmids to increase safety: (i) packaging plasmid; (ii) Env plasmid (envelope plasmid) encoding viral glycoproteins; and (iii) transfer plasmid. Genetic engineering was used to specifically delete the packaging signal or LTRs of the packaging plasmid to avoid their transfer to the vector particles and reduce the production of replicative infectious lentiviruses (RCLs) in the vector preparation. The second generation vector was developed by modifying the four accessory protein encoding genes (Vif, Vpu, Vpr and Nef) in the system. That is, all the accessory genes of HIV in the packaging plasmid of the first generation lentiviral vector were deleted. The removal of these accessory genes did not affect the titer and transfection ability of the virus, while increasing the safety of the vector. The third generation lentiviral vector system consists of four plasmids. The packaging vector is divided into two plasmids: one encoding Rev and one encoding Gag and Pol. The tat-independent plasmid was constructed by replacing the U3 promoter region of the 5'LTR of the transfer plasmid with a strong viral promoter from CMV or RSV. The third-generation system effectively reduced the generation of RCLs and improved biosafety.