First, operational safety
(1) The operation of lentivirus can be carried out in the biological safety cabinet and the conventional ultra-clean workbench (without opening the exhaust fan);
(2) The operator must wear a lab coat, wear a mask, disposable gloves, and guard against venom directly contacting the eyes, ears, or skin wounds;
(3) Pay attention to avoid scatter and dripping of venom during operation, and be careful not to scratch the skin with sharp equipment or needles;
(4) Inadvertently contact the venom station and the equipment to be recycled, the virus can be inactivated by a solution such as 0.25% SDS, 70% ethanol, 1% sodium hypochlorite;
(5) Used work surface, microscope stage, etc., wiped with 70% ethanol;
(6) Waste materials such as culture vessels and tips should be immersed in 84 disinfectant (1:9 dilution) or sterilized at 121 °C before disposal.
Second, the preservation of lentivirus
(1) The lentiviral vector is transported on dry ice and stored at -80 °C for 6 months. When stored for more than 6 months, the working titer needs to be re-explored.
(2) The frozen venom is placed on ice or melted at 4 °C before use. It is best to use it on the same day. The unused venom can be temporarily stored at 4 ° C for no more than 2 days.
(3) Repeated freezing and thawing will seriously reduce the lentivirus titer, and each freeze-thaw can be reduced by more than 20%. It is recommended to use a small amount of cryopreservation according to the dosage at the initial use.
Lentiviral packaging
Third, slow virus packaging common problems and prevention
1. Common cell lines that are not suitable for polybrene
Polybrene can increase the efficiency of lentiviral infection by neutralizing the charge on the cell surface glycoprotein sialic acid residues. However, it has obvious cytotoxicity to some cells, and if it is used, it will affect the experimental effect. These cells are, for example, HSC-T6 (rat hepatic stellate cells), Raw264.7 (mouse mononuclear macrophage leukemia cells), MCF-7 (human breast cancer cells), H929 (human multiple myeloma cells) ), GBC-SD (human gallbladder cancer cell line), NB4 (human leukemia cell line), kasumi (human leukemia cell line), Jurkat (human leukemia cell line), and the like. It is recommended to consult the corresponding literature reports before starting the experiment, as well as the reference polybrene working concentration.
2 , possible reasons for over-successful expression
(1) Plasmid construction has certain problems.
(2) Due to the special structure of the sequence, which makes transcription difficult and the mRNA is very unstable, the mRNA level cannot be significantly overexpressed. After excluding the plasmid construction problem, the overexpression of the target gene at the mRNA level was verified by q-PCR, and an easily expressed cell such as 293T was used as a control at the same time.
(3) If the mRNA level can be overexpressed, the protein level overexpression may be insignificant due to factors such as post-translational modification, codon composition, translation efficiency, and rapid protein degradation. At the same time, when using WB for protein level analysis, a positive control needs to be set to verify the experimental conditions, especially the antibodies.
3 , how to improve the efficiency of viral infection
In addition to improving the MOI, it is possible to try to improve the efficiency of infection from other angles.
(1) Appropriately reduce the volume of the incubation medium (the amount of virus added), which can increase the virus density and improve the infection efficiency. The effect on cell status and fluid change time should be considered when reducing the volume of incubation. For example, 96-well plate (50 ul), 24-well plate (250 ul), 6-well plate (1 ml), incubate for 4 h in a small volume, then replenish to normal culture volume, continue to culture for 24 h and then change.
(2) Prolong the incubation time appropriately. If you change to 24 h and then change the fluid, pay attention to the need to take into account the state of the cells, mainly for the short incubation time (such as 4-6 h). Because the lentivirus has a half-life of about 8 hours at 37 ° C, the effect of prolonging the incubation time is not obvious.
(3) Polybrene can improve the infection efficiency for many cells, but it also has cytotoxicity. It is more toxic to some types of cells (such as neurons and DC cells) and should not be used. When the MOI is lower than 20, there is no need to add Polybrene. The concentration of Polybrene used in specific cells can be reported in the literature and determined by preliminary experiments. Usually in the range of 4-10 ug/ml. Protamine sulfate is less toxic to some cells as a replacement for Polybrene.
(4) If there is a flat-angle centrifugal head that can be centrifuged, it can be infected at 2200 rpm for 2 h (37 ° C) while centrifugation, and then continue to incubate in an incubator and culture according to standard conditions. This method is particularly beneficial for infection of suspended cells.
(5) If the cell state permits, repeated infections can be performed. After the incubation, the virus-free complete medium was replaced. After 16-24 h of culture, the infection process was repeated 1-2 times to improve the infection efficiency.
Fourth, "virus packaging + proteome" exclusive program
Option 1: Find downstream action factors based on interest factors
After the virus is transfected into the cell, the gene to be studied is overexpressed or knocked down. Through SILAC and iTRAQ technology, we can find the differentially expressed protein in the relevant pathway, and then verify the specific protein of the relevant pathway, and easily find the factor of your research. Key pathway effect factors for related pathways.
Option 2: Find specific action factors based on specific conditions
Through SILAC and iTRAQ technology, we can screen changes in cell (sample) protein levels under specific conditions (drugs, stimulating factors, etc.) and find relevant proteins for different pathways based on the direction of the research topic. Further, the specific gene is interfered or overexpressed at the cellular level or at the body level, and the cell function or in vivo verification is performed.
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