Lentivirus Packaging FAQ
1. There are currently three generations of lentivirus packaging systems. What is the difference between different generations of lentivirus packaging systems?
In 1998, Miyoshi et al. revealed the minimum elements required for packaging lentivirus through the study of HIV genome (Miyoshi et. al. J. Virology 72:8150, 1998), which they called the first-generation lentivirus; on this basis The second-generation lentiviral packaging system puts these elements on different expression vectors, and at the same time replaces the capsid protein of HIV with the capsid protein of herpes virus, VSVG; the third-generation lentiviral packaging system integrates U3 in the lentiviral LTR. The region is mutated and modified to prepare a "self-inactive" virus, and at the same time, the promoter activity of the wild-type LTR itself is lost, thereby preventing it from expressing some viral structural proteins.
2. Are there cell lines that are not suitable for transfection with lentivirus? What determines the host cell range of a lentivirus?
The tendency of the lentivirus to infect target cells depends on the interaction of the capsid protein of the lentivirus with receptors on the cell membrane of the target cell. VSVG protein is the most widely used lentiviral capsid protein, which can effectively infect a wide range of host cells. Currently known mammalian cell types that are difficult to efficiently infect with lentivirus include cardiomyocytes and mouse B cells. For more information about viral capsid proteins, please refer to Cronin, J., X.-Y. Zhang, et al. (2005). "Altering the tropism of lentiviral vectors through pseudotyping." Current gene therapy 5(4) : 387.
3. What is the difference between recombinant lentivirus and recombinant adenovirus?
VSVG capsid protein
Slow expression speed (2-3 days)
Faster expression speed (~12h)
Unable to expand, use 293T packaging
Can be amplified in HEK293
Each cell can only be infected with a few lentiviral particles
Can prepare high-titer virus
Short preparation time (2-5 days)
Long preparation time (3-4 weeks)
Level 2 (2+) biosafety control
Level 2 biosafety control
The common 293T, HEK293 and 293FT can all be used for lentivirus packaging, but HEK293 and 293FT cells usually produce lower titer lentivirus. 5.
According to NIH's recommendations, there are two main security considerations for operating a lentivirus packaging system. One is the potential for lentiviral replication; it is the potential for tumorigenicity. As a foreign gene transport tool, lentivirus can enter non-dividing or resting mammalian cells very efficiently. Researchers have made a lot of optimizations on the lentivirus packaging system to improve the biosafety without losing the virus packaging titer. The latest generation lentiviral packaging system used by AKD puts the viral capsid protein, packaging elements and target genes into different vectors. The gene transfer vector carries the target gene and also contains the sequence for integrating the target gene into the target cell. Without the capsid protein and other packaging plasmids, the vector alone cannot produce lentiviral particles. After the target cell is infected with the lentivirus produced by Akander, the target cell cannot produce new virus particles. In addition, the lentivirus packaging system of Akander does not contain the tat gene. At the same time, the modification of the 3'-end LTR makes it impossible. The full-length complete virus is transcribed, which further improves biological safety. For more detailed lentivirus biosafety information, please refer to http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html