ChAT-GFP Lentivirus

With the in-depth study of lentiviral vectors, the optimization of lentiviral vectors is also being explored to improve the safety of lentivirus in clinical use. The development of lentiviral vectors has gone through three stages. The first generation of lentiviral vector system is represented by the three-plasmid system. During the construction, the cis-acting elements required for packaging, reverse transcription and integration in the HIV-1 genome are separated from the sequences encoding trans-acting proteins and constructed on three plasmid expression systems, namely packaging plasmid, envelope plasmid and vector plasmid. Under the action of the cytomegalovirus (CMV) promoter, the packaging plasmid controls the expression of all viral structural genes except env; the envelope plasmid encodes the G glycoprotein of vesicular stomatitis virus (VSV); and the vector plasmid contains the target gene. These three plasmids are co-transfected into packaging cells such as human embryonic kidney 293T cells, and lentiviral particles with only one infection ability but no replication ability can be harvested in the cell supernatant. The first generation of lentiviral vector system is characterized by the fact that when constructing three packaging plasmids, in order to reduce the possibility of producing replication-competent viruses, the homologous sequences between the three plasmids are reduced as much as possible, but the packaging plasmids still retain the accessory genes of HIV. The second generation of lentiviral vector system is an improvement on the first generation, and all the accessory genes of HIV are deleted in the packaging plasmid. The removal of these accessory genes does not affect the titer and infectivity of the virus, and at the same time increases the safety of the vector. The third generation of lentiviral vector system adds two more safety features: one is to construct a self-inactivated lentiviral vector, that is, the 3′LTR of the U3 region is deleted, so that the vector loses the HIV-1 enhancer and promoter sequence, and RNA cannot be transcribed even if all viral proteins are present; the second is to remove the tat gene and replace it with a heterologous promoter sequence, so that only 3 of the 9 lentiviral vectors in the original HIV genome are retained (gag, pol and rev). Therefore, the third generation of lentiviral vector system is safer.