CaMKII-GCaMP6f Lentivirus

Lentiviruses (LVs) belong to the same family as gamma-retroviruses: Retroviridae. They all integrate their double-stranded DNA into the host genome and then use the host machinery to transcribe their genes back into RNA. Once replication is complete and they return to the cytoplasm, the viral RNA is translated and packaged into a new viral particle, which then buds out of the cell to complete the life cycle. LVs have a more complex genome than gamma-retroviruses. The essential genes required for survival and function are the gag, pol, and env genes, where gag encodes structural proteins, pol encodes enzymes required for reverse transcription and integration into the host cell genome, and env encodes the viral envelope glycoprotein. The first generation LV vectors contained most of the HIV genome, including the gag, pol, and env (most commonly VSV-G) genes. The env gene was encoded on a plasmid separate from the other lentiviral genes. In addition, the accessory genes vif, vpr, vpu, and nef, as well as the regulatory genes tat and rev, were included in the first generation LV vectors. Vif, vpr, vpu, and nef provide survival/fitness advantages for lentiviral replication in vivo, but they are not essential for viral growth in vitro. Tat and rev are required for viral replication. The second-generation LV vector removes the auxiliary virulence factors vif, vpr, vpu, and nef, which does not affect the transfer of genetic material to host cells. The gag and pol genes of the third-generation LV vector are encoded on a different plasmid from the rev or env genes, dividing the viral genome into separate plasmids, and introducing a deletion in the 3'LTR of the viral genome to produce a self-inactivating (SIN) lentiviral vector, destroying the promoter/enhancer activity of the LTR. This vector made of three independent plasmids contains both the essential viral sequences for packaging and further improves safety. In addition, the third-generation lentiviral vector also removes the unnecessary tat gene and engineers a constitutively active promoter into the upstream LTR of the transgenic construct.