Lentiviruses are used widely to generate stable expression mammalian cell lines. They are used for gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of the gene of interest). This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. Unlike the short term protein expression observed using transient transfection methods, generating cell lines using lentiviral vectors enables long-term protein expression studies. In addition, repeating experiments in a stable cell line as opposed to transiently-transfected cells increases reproducibility, since it eliminates the variation associated with repeated transient transfection.