Method of screening tumor metastasis suppressor gene using RNA interference library
A technique for RNA interference and tumor metastasis, which is applied in the field of screening tumor metastasis suppressor genes using RNA interference libraries, which can solve the problems of inability to form tumor metastasis, unfavorable screening of tumor metastasis suppressor genes, and elevated
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[0056] 1. Using RNA interference library DNA to transfect Phoenix retrovirus packaging cells
[0057] Day 1: Preparing Phoenix Retroviral Packaging Cells
[0058] 1. 18-24 hours before transfection, press 3×10 6 / 10 cm plate inoculation of Phoenix cells onto the plate;
[0059] 2. Let the cells adhere to the wall for 10-24 hours;
[0060] 3. When the cell density reaches 2 / 3, a 10 cm plate has about 5×10 6 cell. At this time packaging cells are most easily transfected and produce the highest titers of virus at 37°C.
[0061] *Medium: DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine.
[0062] Day 2: Transfection
[0063] 1. Prepare the DNA for transfection and dissolve it in HEPES solution;
[0064] 2. About 5 minutes before transfection, add 25 μM chloroquine to the plate (the storage concentration of chloroquine is 50 mmol);
[0065] * Chloroquine inhibits DNase in lysosomes by neutralizing the pH inside intracellular transport microvesicles;
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