Mir-124 and HER2-shRNA double-gene expression cassette virus vector, construction method,virus and application
A mir-124, 1.mir-124 technology, applied in the field of molecular biology, can solve the problems of breast cancer patients with recurrence and metastasis, the effect of inhibiting proliferation cannot meet the actual needs, and the overall survival rate is less than 25%.
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Embodiment 1
[0052] To construct the mir-124 and HER2-shRNA double gene expression cassette lentiviral vector, the specific steps are as follows:
[0053] 1) Artificial synthesis of the mir-124 sequence shown in SEQ ID NO.1 and the HER2-shRNA sequence shown in SEQ ID NO.2 and SEQ ID NO.3;
[0054] 2) Using KOD high-fidelity enzyme, using step 1) artificially synthesized mir-124 sequence and HER2-shRNA sequence as templates, respectively, PCR amplification was performed with the primers shown in Table 1:
[0055] Table 1
[0056] Primer name
[0057] The specific reagent dosage for PCR amplification is shown in Table 2 below:
[0058] Table 2
[0059]
[0060]
[0061] The program conditions for PCR amplification are shown in Table 3 below:
[0062] table 3
[0063]
[0064] 3) Agarose gel electrophoresis to check whether the size of the PCR amplified product is consistent with expectations, cut the target fragment band in the agarose gel electrophoresis gel, recover and purify it with a DNA recovery...
Embodiment 2
[0084] To construct a mir-124 and HER2-shRNA double gene expression cassette lentivirus, the specific steps are as follows:
[0085] 1. Digest the HEK 293 cells in the logarithmic growth phase with membrane protease, with a cell density of 5×106, and re-inoculate them in a cell culture dish containing 25 mL of antibiotic-free DMEM medium containing 10% (v / v) FBS with a diameter of 15 cm , 37℃, 5% CO2 incubator;
[0086] 2. Prepare four plasmid DNA solutions in the lentivirus packaging system, and the granulation map is as follows Figure 1~4 Shown:
[0087] Table 9
[0088] Plasmid name
quality
pRSV-Rev
1Oμg
pMDLg-pRRE
15μg
pMD2G
7.5μg
pCDH-CMV-mir124-HER2shRNA-EF1-copGFP-T2A-Puro
20μg
[0089] Add the above plasmid to 1800μl of sterile water, then add 200μl of CaCl2 (2.5mol / L) solution and mix well, add 2000μl of 2×PBS buffered salt solution, and leave it at room temperature for 20-30min;
[0090] 3. Transfection is performed when the cell density in step 1 reaches ab...
Embodiment 3
[0098] Cell transfection and screening of the mir124 and HER2-shRNA double gene expression cassette lentivirus constructed in Example 2: The specific steps are:
[0099] Ⅰ: Culture MCF-7 cells in a 6-well cell culture plate, and cell transfection can be carried out after the cells are fully adherent and confluent at 30%-40%: Before transfection, cells in each well are replaced with fresh DMEM for complete culture Add 1μL of polybrene to each well of the 6-well plate and place it in the incubator to incubate for 1h;
[0100] Ⅱ: Add 0.1 μL of the mir124 and HER2-shRNA double gene expression cassette lentiviral solution obtained in Example 2 to the 5 wells of the above 6-well cell culture plate, shake well and put it in 37°C, 5% ( v / v) Incubate in an incubator with CO2 and 95% relative humidity for 1 to 5 days, add 20μL / well of CCK8 reagent every day, and continue to incubate for 3 hours, then measure the absorbance OD value of each well at 450nm;
[0101] Ⅲ: Follow the same method as ...
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