Example 3: Overexpression of circ-NFATC3 lentivirus and its stable cell line construction
 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.
 2. Lentivirus packaging
 (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.
 (2) Change the cell culture medium to serum-free medium before transfection.
 (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.
 (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.
 (5) Mix the diluted DNA with the diluted Lipofectamine 2000, gently invert and mix without shaking.
 (6) After mixing, incubate at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution.
 (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.
 (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.
 3. Harvesting and concentration of viruses
 (1) Collect the 293T cell supernatant at 48 hours and 72 hours after transfection (starting from 0 hours after transfection).
 (2) Centrifuge at 4000g for 10 min at 4°C to remove cell debris.
 (3) Filter the supernatant with a 0.45μm filter in a 50ml centrifuge tube.
 (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.
 (5) After the combination is completed, balance it and place it on the rotor.
 (6) Centrifuge at 5000×g to the required virus concentration volume. It usually takes 10-15 minutes.
 (7) After centrifugation, the filter cup is the virus concentrate.
 (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.
 4. Cells infected by lentivirus:
 (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.
 (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.
 (3) Aspirate the mixed solution from the tube and add it to the petri dish or hole.
 (4) Add a sufficient amount of fresh culture medium.
 (5) Change the liquid after 12 hours.
 (6) After 48 hours, 2ug/ml puromycin was added to screen for stable cell lines.
 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.