[0064] Example 3: Overexpression of circ-NFATC3 lentivirus and its stable cell line construction
[0065] 1. Overexpression circ-NFATC3 lentiviral vector construction: Synthesize the circ-NFATC3 linear full sequence in Shanghai Jierui Company, the sequence is annealed into a double-stranded DNA fragment, inserted into the LVminiCirc vector through multiple cloning sites, and the recombinant plasmid is identified by sequencing , Control negative control is an empty LVminiCirc vector with no sequence inserted.
[0066] 2. Lentivirus packaging
[0067] (1) 24h before transfection, digest 293T cells in logarithmic growth phase with trypsin and pass them to a 10cm cell culture dish at 37℃, 5% CO 2 Cultivate in an incubator. It can be used for transfection when the cell density reaches 70% to 80% in 24 hours. Cell state is very important for virus packaging, so it is necessary to ensure good cell state and fewer passages.
[0068] (2) Change the cell culture medium to serum-free medium before transfection.
[0069] (3) Add each prepared DNA solution (LVminiCirc-Circ-NFATC3/LVminiCirc vector 10μg, pGag/Pol vector 5μg, pRev vector 5μg, pVSV-G vector 5μg) into a sterile centrifuge tube, and add the corresponding volume of Opti -MEM is mixed evenly and the total volume is adjusted to 1.5ml.
[0070] (4) Shake Lipofectamine 2000 reagent gently, take 60 μl Lipofectamine 2000 reagent and mix with 1.5 ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes.
[0071] (5) Mix the diluted DNA with the diluted Lipofectamine 2000, gently invert and mix without shaking.
[0072] (6) After mixing, incubate at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution.
[0073] (7) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well, and incubate at 37°C, 5% CO 2 Culture in a cell incubator.
[0074] (8) After 6 hours of incubation, aspirate the medium containing the transfection mixture, add 10ml of cell culture medium containing 10% serum to each bottle of cells, and hold at 37°C, 5% CO 2 Continue to incubate for 48 hours in the incubator.
[0075] 3. Harvesting and concentration of viruses
[0076] (1) Collect the 293T cell supernatant at 48 hours and 72 hours after transfection (starting from 0 hours after transfection).
[0077] (2) Centrifuge at 4000g for 10 min at 4°C to remove cell debris.
[0078] (3) Filter the supernatant with a 0.45μm filter in a 50ml centrifuge tube.
[0079] (4) Add the crude virus extract sample to the filter cup (maximum 19ml), and close the lid. Insert the filter cup into the filtrate collection tube.
[0080] (5) After the combination is completed, balance it and place it on the rotor.
[0081] (6) Centrifuge at 5000×g to the required virus concentration volume. It usually takes 10-15 minutes.
[0082] (7) After centrifugation, the filter cup is the virus concentrate.
[0083] (8) Remove the virus concentrate and store it in virus tubes after aliquots. It can be stored at 4°C for a week or -80°C for long-term storage. Take one of them for virus biological titer determination.
[0084] 4. Cells infected by lentivirus:
[0085] (1) Collect the cells by centrifugation in a 1.5ml tube according to the amount of cells, and then dilute the cell pellet with 100-200ul of serum-free culture medium, subject to the cells being completely immersed in the culture medium.
[0086] (2) Aspirate the over-expressed circ-NFATC3 virus solution and add it to the cells, and place the 1.5ml tube in a 37°C incubator for 30 minutes. Another LVminiCirc empty vector control virus infection was used as a control cell line.
[0087] (3) Aspirate the mixed solution from the tube and add it to the petri dish or hole.
[0088] (4) Add a sufficient amount of fresh culture medium.
[0089] (5) Change the liquid after 12 hours.
[0090] (6) After 48 hours, 2ug/ml puromycin was added to screen for stable cell lines.
[0091] 5. Identification of stable cell lines: The constructed stable cell lines are photographed and observed under a fluorescence microscope, the positive rate of GFP> 95%, and some cells were collected at the same time for QPCR testing, confirming the overexpression efficiency of circ-NFATC3> 2 times. See the result Figure 4 , The circ-NFATC3 gene-specific sequence was constructed on the LVminiCirc lentiviral vector, and the Huh7-circ-NFATC3 stable cell line overexpressing circ-NFATC3 was constructed by lentivirus. The result showed that the GFP positive rate was> 95%, QPCR test confirmed that circ-NFATC3 was successfully overexpressed, and the overexpression effect reached 2 times.