Day 0 Seed 293 cells and grow overnight.
Day 1 Transfect with retroviral vector containing gene of interest and an appropriate packaging vector.
Day 2 Replace medium.
Day 3 Harvest virus-containing supernatant. Virus may be stored at - 70C at this stage. Infect target cells,either for titer determination or for gene expression.
Day 4 Split infected target cells and grow for selecting stable virus producing cell lines. For transient expression experiments, you may harvest the cells at this stage.
Day 5 Start selection by replacing the medium with G418 containing medium.
Day 9 Change medium and continue selection.
Day 14 Count antibiotic resistant colonies and calculate titer.
Note: If you are using retroviral expression system for the first time, we strongly recommend using the LacZ control plasmid included in the kit. The B-galactosidase expression can be monitored using B-gal staining kit or any other standard protocol.
