How can I create a stable cell line with a functional shRNA expression vector?
Stable cell lines can be generated by two different methods. First, target cells can be transfected with the shRNA plasmid. One day after transfection, the transfected cells can be selected with 0.5 – 1.0 ug/ml puromycin for 1-2 weeks with passages as needed. Alternatively, retroviral packaging cell lines can be used to generate retroviruses for stable cell line generation. For example, the plasmid can be transiently transfected into the Phoenix helper-free amphotropic packaging cells line. Two days after the transfection, the cell culture supernatant is collected, filtered and used to infect the target cell lines. Stable cell lines can be established following drug selection.
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- How can I create a stable cell line with a functional shRNA expression vector?
