LV ()

The production of lentivirus was mainly based on the co-transfection of three plasmids (core plasmid pLV-GOI/shRNA, helper plasmids psPAX2 and pMD2G) into HEK293T cells. The core plasmid contains the basic components of the virus, 5 'LTR and 3' LTR, as well as foreign target genes. The psPAX2 plasmid mainly carries gag, pol and rev genes, which encode the main structural proteins of the virus, specific enzymes and regulatory factors regulating the expression of gag and pol genes respectively. pMD2G vector contains vsvg gene, which provides the envelope protein needed for virus packaging.

Figure 1 Lentivirus packaging flowchart

1 LV plasmid construction

1) Select different rLV carriers according to the experimental purpose and the tested cells. The target gene was cloned into LV expression vector and the expression function of the vector was verified by simple enzyme-ligation-transformation-sequencing.

2) In order to obtain plasmid with high quality, high concentration and low endotoxin content, plasmid DNA was purified by column centrifugation and endotoxin detoxification reagents.

Figure 2 Flowchart of lentivirus core plasmid construction

2 Virus Packaging

1 Cell preparation
The frozen cells were removed from liquid nitrogen irrigation and resuscitated in a water bath at 37℃. After centrifugation, fresh medium was added to the cells and cultured at 37℃ and 5%CO2. Cell passage was performed every 2-3 days. After the cells grew normally, they were transferred to a 10cm petri dish for adherent culture.

2 plasmid transfection
Transfection could be performed when the cell density reached a confluence rate of about 80-90%. After the transfection triplasmid system was configured in a certain proportion, the plasmid was transfected into the prepared 293T cells using transfection reagent (PEI max), and transfection and culture were performed according to the system of 1000μL per dish. After transfection, fresh medium was replaced after 6h. (Transfection conditions refer to PEI max transfection reagent use method)

3-cell culture
One day after culture, the cell transfection was observed by microscope.

4 Virus collection and decontamination
After 48 hours of culture, the virus supernatant was collected and continued to be cultured with fresh media. After 72 hours, the virus supernatant was collected and filtered by 0.45μm filter membrane.

5 Virus Purification
The filtered virus supernatant was centrifuged at 25000rpm at 4 ° C for 2.5h. After centrifugation, the supernatant was discarded, the virus precipitation was re-suspended with the virus preservation solution, dissolved at 4 ° C and placed overnight.

6 Virus Collection
Collect and package the venom.

Lentivirus (LV) quality detection

• 1 Titer detection

  The titer unit of lentivirus is TU/mL, which refers to the number of biologically active virus particles per milliliter, that is, the number of viral genomes that infect and enter the target cell. According to the expression of the target gene after infection, the content of lentivirus in unit volume can be converted, that is, the infection titer of lentivirus.

  1) Lentivirus titer detection with fluorescent tags
  a. Cell preparation (day 1)The 293T cells were inoculated into 96-well plates with about 3~5x104 target cells/pores, and cultured overnight at 37℃ and 5%CO2. Generally, the cell fusion degree was about 70% during viral infection.
  b. Virus dilution and infection (day 2) The lentivirus stock solution to be detected was diluted by 10 times gradient using cell culture medium, and 6-8 groups of viruses with dilutive degree were generally set. According to the actual situation of the experiment, the dilution ratio can be increased or decreased, and the number of virus dilution groups can be adjusted. The diluted virus was added to the prepared 293T cells and incubated overnight, and 3 Wells were repeated for each dilution.
  c. Changing the culture medium (day 3) Change the fresh culture medium within 24 hours of virus infection and continue culture.
  d. Fluorescence counting and titer calculation (day 5) The number of cells with fluorescence in the pores was observed with a microscope, and each dilution sample was repeated 3 times to calculate the average number of cells with fluorescence in the 3 complex pores. The lentivirus titer is calculated using the following formula. Assume that A is the mean value of fluorescent cells in the penultimate observable fluorescence hole, and B is the mean value of fluorescent cells in the penultimate observable fluorescence hole. Titer (TU/ml) = (A+10xB) x1000/2/ Pore virus amount (uL) Detection standard: titer ≥1.0E+8 TU/mL

  2) Lentivirus titer detection without fluorescent labels a Cell Preparation (day 1) 293T cells were inoculated into 24-well plates with about 3~5x104 target cells/pores, and cultured overnight at 37℃ and 5%CO2. Generally, the cell fusion degree was about 70% during viral infection. b virus dilution and infection (day 2) The lentivirus stock solution to be tested and the lentivirus stock solution with fluorescent label known TU (experimental control group) were diluted by 10 times gradient using cell culture medium, and 3 groups of viruses with dilution were generally set. The diluted virus was added to the prepared 293T cells and incubated overnight, and 3 Wells were repeated for each dilution. c Change of culture medium (day 3) Change the fresh culture medium within 24 hours of virus infection and continue culture. d Genomic DNA extraction and titer calculation (day 5) 72h after viral infection, cells were collected and genomic DNA was extracted (refer to the Takara genomic DNA extraction kit instructions). Specific primers were used for fluorescence quantitative PCR to detect the gene copy number of the virus samples to be tested, and the gene copy number and TU value of the control group were used to convert the lentivirus titer (TU/mL) value without fluorescent labels. Each sample was repeated 3 times, and the average copy number of the 3 multipore genes was taken. The lentivirus titer is calculated using the following formula. Titer (TU/mL) = (gene copy number of sample to be tested/gene copy number of control sample) * Titer of control sample Detection standard: titer ≥1.0E+8 TU/mL

2.Sterility test

Detection methods:Virus samples were added to 293T cells in 96-well plates, cultured at 37℃ for 72h, and the growth of bacteria and fungi was detected by microscope.

Test criteria:The medium should be clear and transparent, with no obvious particles in the intercellular space and no bacterial or fungal contamination.

3. Detection of mycoplasma

Detection methods:Primers were designed using the conserved region of mycoplasma 16S rRNA gene, and the contamination of mycoplasma in viral samples was quantitatively detected by gel electrophoresis and fluorescence.

Detection criteria:PCR glue map without strip.