Lentiviral vector for suspension cells and application of lentiviral vector
A technology of lentiviral vector and recombinant lentivirus, applied in the field of lentiviral vector, can solve the problems of difficult lentivirus, infection, etc.
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Embodiment 1
[0028] Example 1 Construction of circRNA overexpression into a circular vector
[0029] 1. Design circRNA overexpression into a circular vector
[0030] Design a promoter sequence suitable for suspension cells; according to the sequence information of the commercialized vector pLVX-EGFP-IRES-Puro, design a complete Ubi promoter-Cbh promoter nucleotide sequence (SEQ ID NO .1), and use the primer design software Primer Premier 5.0 to design primers, the primers are as shown in Table 1, and the primers are handed over to the nucleic acid synthesis company for primer synthesis.
[0031] Table 1 Primer design
[0032]
[0033] 2. PCR amplification
[0034] The PCR system is shown in Table 2, and two DNA sequences were obtained by PCR amplification.
[0035] Table 2 PCR reaction system
[0036]
[0037] PCR reaction conditions:
[0038] 98°C for 5min; 98°C for 10s, 62°C for 5s, 72°C for 30s, 30cycles; 72°C for 10min, 4°C for 4min.
[0039] 3. DNA fragments were recovered...
Embodiment 2
[0060] Example 2 Purpose Gene-pLVX-Ubi-MCS-Cbh-EGFP-IRES-Puro Vector Construction
[0061] 1. The target gene sequence is amplified by PCR, and the PCR reaction system is shown in Table 2.
[0062] PCR reaction conditions:
[0063] 98°C for 5min; 98°C for 10s, 60°C for 5s, 72°C for 15s, 30cycles; 72°C for 10min, 4°C for 4min.
[0064] 2. DNA fragments were recovered by agarose gel electrophoresis.
[0065] 10 μL of the reaction product was subjected to 1% agarose gel electrophoresis to recover the target gene fragment. The gel recovery kit was purchased from Qingke Biotech.
[0066] 3. Restriction digestion of pLVX-Ubi-MCS-Cbh-EGFP-IRES-Puro vector
[0067] Plasmid pLVX-Ubi-MCS-Cbh-EGFP-IRES-Puro was double digested with AgeI-EcoRI, and the restriction system is shown in Table 5.
[0068] Table 5 enzyme digestion system
[0069]
[0070] After reacting at 7°C for 5 hours, large fragments were recovered on 1% agarose gel.
[0071] 4. Ligation of target gene fragment and ...
Embodiment 3
[0083] Example 3 pLVX-Ubi-MCS-Cbh-EGFP-IRES-Puro lentiviral packaging
[0084] 1. Cell preparation
[0085] 1) One day before transfection, inoculate 3-5*106 cells / dish of 293T cells in a 10mm cell culture dish, add DMEM medium containing 10% fetal bovine serum, and place at 37°C, 5% CO 2 Cultured in an incubator.
[0086] 2) On the day of transfection, transfection was performed when the cell density reached 80%. According to psPAX2: pMD2.G: target plasmid = 2: 1: 1, add it to the serum-free DMEM culture medium, mix gently, and let it stand for 5 minutes.
[0087] 3) Gently mix the transfection reagent with serum-free DMEM medium and let stand for 5mim
[0088] Mix the liquids in steps 2) and 3) and let stand for 20 minutes.
[0089] 4) Evenly drop the mixed solution into a cell culture dish, and incubate for 6 hours in a 37° C., 5% CO2 cell culture incubator.
[0090] 5) Collect the cell supernatant after continuing to culture for 48h
[0091] 10 μL of the reaction pro...
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