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?

Lentivirus 

Adenovirus 

RNA virus 

DNA virus 

~9Kb(HIV1) 

~36Kb (AD5) 

Integrated virus 

Non-integrated 

VSVG capsid protein 

Non-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 

Multiple infections 

Lower titer 

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 

4.  Which cells can be used for lentivirus packaging? 

The common 293T, HEK293 and 293FT can all be used for lentivirus packaging, but HEK293 and 293FT cells usually produce lower titer lentivirus. 5.  What factors affect the titer of lentivirus? 

1)  The state and generation of packaging cells: During the culture process, try to avoid 293T cells with high confluence. 293T cells at a very high confluence state have low transfection efficiency and produce less virus. Try to use lower passage 293T cells. After 20 consecutive passages, resuscitate lower passage cells.

2)  Target gene: The titer of the lentivirus decreases as the length of the inserted target gene increases; the cytotoxic gene produces a lower titer when the lentivirus is packaged. It is recommended to use an induced expression system to design the project. 

3)  The influence of culture medium and serum: The composition of culture medium and the source of serum have a greater influence on the packaging titer of lentivirus. Usually adding sodium pyruvate to the medium can increase the production of lentivirus; different sources of FBS can also cause different production of lentivirus. It is recommended that after testing different batches of FBS, a large amount of FBS of the same batch number should be stored for lentivirus production. 

4)  Concentration method: The currently commonly used methods for lentivirus concentration include ultracentrifugation, PEG8000, and column purification. Due to the large centrifugal force of ultracentrifugation, part of the lentivirus may lose its infectious viability during the concentration process; the use of PEG8000 to concentrate lentivirus is due to The concentration factor is small, which is not suitable for mass production of lentivirus. The column purification method has low lentivirus recovery efficiency. 

5)  Storage method: Lentivirus is more sensitive to temperature changes, and repeated freezing and thawing must be avoided; under normal circumstances, storage at -80°C for half a year will not cause a significant drop in titer; storage at 4°C for a week will not cause infection The difference. 

6)  Titer determination method: At present, there are two commonly used lentivirus titer determination methods, one is ELISA method and the other is realtime  qPCR method. The former measures its physical titer, while the latter measures its activity titer. The titer determined by the ELISA method is usually higher than the titer obtained by the qPCR method. In the actual application process, it is recommended to use GFP-expressing lentivirus as a preliminary experiment to determine the best MOI.

6.  Are there any special biological safety considerations for operating the lentivirus packaging system? 

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