1.Is recombinant lentivirus safe to use?
Lentivirus particles provided by BrainVTA are pseudoviruses whose toxic genes have been removed and replaced by exogenous target genes. They are capable of infection but not replication, so they are safe to use. Nevertheless, but all work with lentiviruses should be performed in a BSL-2.
2.What is the difference between 2nd and 3rd generation lentiviral systems?
Briefly, 3rd generation lentiviral systems are considered safer than second generation systems. The 3rd generation lentiviral systems require transfection with four separate vectors in order to create functional lentiviral particles.
3.What plasmids are included in the 3rd generation lentiviral system?
The third generation system is widely used because of its high security. It consists of one core plasmid and three packaging plasmids. The core plasmid carry the long terminal repeat sequence (LTRs), packaging signal (Ψ), which is necessary for virus packaging, and WPRE, cPPT/CTS , RRE, which can promote the expression of exogenous gene, improve titer of virus. Three packaging plasmids provided Gag/Pol, Rev, and VSVG, respectively, which is necessary for lentivirus packaging.
4.What's the cloning capacity for rLV?
rLV has a packaging capacity of 8.5Kb. After removing the promoter and the resistance/fluorescence genes, the lentivirus can contain no more than 4KB of exogenous gene, and the longer the exogenous gene, the more difficult it is to obtain a higher titer.
5.What is the recommended storage temperature?
rLV should be stored at -80℃. However if the virus is repeated freeze-and-thaw, it will cause significant decrease of titer.
6.What are the precautions for lentivirus dilution and use?
It can be diluted with PBS or serum-free medium, mixed and divided, and it is recommended to use up the diluted sample at one time. The lentivirus should be thawed in 37℃water after being taken out at -80°C with the volume is more than 1mL, while it can be thawed at room temperature if the volume is 100-1ml. Bur if the virus is less than 100ul, it should be thawed in 4°C ice.
7.How can lentiviruses be inactivated?
The virus can be inactivated by 1 % 1%SDS solution, 84 Disinfectant ( 1:20）or high temperature and high pressure. If the lentivirus accidentally touches the skin, wash it immediately with plenty of water and disinfect it with alcohol. After handling the lentivirus, wash your hands with soap in time.
8.What's the difference between the different unit?
TU/ml: Transducing Units/ml, which represents the number of bioactive virus particles per milliliter of the virus venom, that is, the number of viral genomes that can infect and enter target cells. This is a common unit of measure for lentiviruses.
VG/ml : Vector Genomes/ml, which refers to the genome copies per milliliter of the virus venom.
PFU/ml:Plaque Forming Units/ml, it was used to calibrate the virus titer by measuring the number of plaques after virus infection.
9.When do I need to select the lentivirus? What is the advantages and disadvantages?
Lentiviruses have the ability to infect both dividing and non-dividing cells, but it can cause immune response. Lentiviruses are often used in hard-to-infect cells such as primary cells and neural stem cells. It is also a common tool for building stable cell lines. Of course, it can also be used in vivo and the signal can be detected 7 days after injection. BrainVTA have developed random integration and non-integrated lentivirus vectors, which can meet the needs of the vast number of researchers.
10.How can lentiviral vectors be used to make stable cell lines?
Many lentiviral vectors insert some selectable markers, such as the puromycin resistance gene, conferring resistance to antibiotics. If these antibiotics are added to the growth medium, they kill off any cells that have not incorporated the vector and those cells that survive can be expanded to create stable cell lines. Apart from that kind of lentiviral vectors, some lentiviral vectors have some other makers such as GFP. Cells which have incorporated the vector can be separated using FACS.
11.When does gene expression peak appearing after lentivirus infection?
Lentivirus expression is relatively slow. Most cells have the expression peak of the fluorescence or target gene within 72 to 96 hours after infection, but for slow-growing cells, the time may be longer.
12.The efficiency of lentivirus infection to target cells is very low, how to improve the efficiency?
The first is to ensure that the cells are in a good state of growth before infection, and the second is to detect if there is a significant drop in the titer of lentivirus.The MOI of lentivirus infection can be appropriately increased and the infection time can be prolonged during the infection process after being ensured to be correct. Meanwhile, reagents promoting infection can be added.
13.What causes changes in cell morphology and even cell death when the virus infects the cell? What should I do?
Step1: Determine if the virus is contaminated.
1) Bacterial pollution
Millipore 0.22um filter was used to filter and remove the bacteria.
2) Fungal pollution
Virus drop directly.
3) Mycoplasma contamination
There are black spots in the intercellular stroma, which are motile.
4) Cell debris
Step2: Change the medium several times.
Step3: Determine whether the MOI is too high, and find the appropriate MOI after gradient dilution.
Step4: Consider that the target cell may be sensitive and use other cell infections to determine.
Step5: If the cell state is still bad after all the above are excluded, try to increase the serum level and see if the cell state improves.
14.Why does the overexpressed lentiviral vector have darker fluorescent than the control?
The vector capacity of the virus is limited, if the inserted gene is too large, the expression of genes will be affected. If this happens, the amount of virus infection can be appropriately increased.
15.How to determine the drug concentration of stable cell line screening?
Before using G418, hygromycin B, or purinomycin to screen stable cell lines, the optimum drug concentration suitable for target cells should be determined by gradient experiments. For most cell lines, recommended drug concentrations can usually be found in the literature, etc. With G418 or hydamycin B, the concentration of mass cell death at about 5 days and total death at 2 weeks was selected as the screening concentration. In the case of puromycin, concentrations that kill all cells are usually used at 3-4 days. The drug activity of different batches was different to some extent. Therefore, the optimum concentration needs to be determined again when new batches of drugs are used.
16.Why does the overexpression (interference) effect disappear after a period of culture after establishing the lentivirus stable cell lines?
After the stable cell lines is obtained by puro screening, the cells should be incubated with puro medium all the time to avoid gene loss.It is suggested that the culture concentration can be as 1/10-1/2 of the screening concentration.
17.Why does it take a long time to see the fluorescence?
It takes a long time for the fluorescence to be observed, which indicates that the fluorescence is relatively weak. Possible reasons include:
1.Microscopical mercury lamps have long service life. The problem can be ruled out by observing the control samples.
2.Low pH results in green fluorescence quenching. Observe whether the medium is yellow or not.
3.Large target gene fragment results in weak fluorescence expression. The exposure time can be slightly longer than the control.