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Design and Application of a shRNA 2193.4. Infection of CellsThe infectious retrovirus is generated by transfecting the plasmid DNA intoa packaging cell line, for example, Phoenix cells (http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html). Transfection quality DNA of theretrovirus plasmid is made using a Qiafilter plasmid Maxi kit, according to themanufacturer’s instructions. After transfection, the Phoenix cells reverse transcribethe plasmid DNA into an RNA that is packaged into infectious virus andexpelled from the cells. Then, the tissue culture supernatant containing the virusis applied to the cells of interest, to deliver the shRNA and the shRNA-resistantcDNA to the cells in a single virus. The infected cells can be selected inpuromycin to enrich for the infected cells. To assess the efficiency of killing ofuninfected cells by puromycin, perform a mock virus infection. To assess theefficiency of infection, infect one plate with a virus known to generate goodtiter (e.g., vector pQCXIP, Clontech).1. The day before transfection, plate 5 × 10 6 Phoenix cells in 10 mL of medium in a10-cm dish. Culture in a 37°C, 5% (v/v) CO 2incubator overnight.2. Remove the medium from the 10-cm dish 4 h before transfection, and replace with9 mL of prewarmed fresh medium.3. Dilute the required amount of 2.5 M CaCl 2to 250 mM and aliquot 0.5 mL pertransfection to separate sterile 15-mL polystyrene tubes.4. Add supercoiled plasmid DNA of the intended virus, to a total of 30 µg per tube.5. Add 0.5 mL of 2X BES Buffered Saline (BBS), by dripping slowly from a 1-mL pipet,vertically down the center of the tube (1–2 drops per second). Do not mix. Wait15 min. At this time, the precipitate should be barely visible to the naked eye.6. Use a 1-mL pipet to blow air bubbles through the solution to mix the precipitate.Distribute the mixture drop-wise into the medium, evenly over the plate ofPhoenix cells.7. Rock the plates back and forth very gently to mix the calcium phosphate precipitateand then place in humidified 37°C incubator with 5% (v/v) CO 2overnight.8. Remove the medium and replace with 6 mL of fresh medium 24 h after transfectionand return to the humidified 37°C incubator with 5% (v/v) CO 2.9. On the same day, split the target WI38 primary fibroblast cells in 10 mL of mediumin a 10-cm plate. The cell density the next day should be about 50% confluent.10. Harvest the virus containing supernatant 24 h after step 8, and then filter througha 0.45-µm filter.11. Remove 10-mL medium from the target WI38 cell plate, and replace with 5 mLof fresh WI38 cell medium. Add 5 mL of virus containing supernatant fromPhoenix cells dropwise into the target WI38 cell plate.12. Add polybrene to each plate of WI38 cells to a final concentration of 8 µg/mL.Mix polybrene into the medium by gently shaking the plate. Put the cell plate backinto a 5% (v/v) CO 2-containing incubator.