Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones.

A bicistronic expression vector can be used to generate stable cell lines expressing green fluorescent protein (GFP)-tubulin and photoactivatable (PA)-GFP-tubulin. This vector, pIRES (originally available from BD Biosciences/Clontech), expresses the gene of interest (GFP-tubulin) and a selection marker from the same promoter. The vector uses an internal ribosome entry site (IRES) derived from the encephalomyocarditis virus and translates two open-reading frames from one mRNA. The gene of interest is inserted immediately downstream from the pCMV IE promoter, and the selection marker is downstream from the IRES site. Selection is more efficient using this vector system, presumably because of the linked expression of both genes. IRES vectors have been used with neomycin- and hygromycin-resistance markers. Cell lines can also be generated for proteins cloned into standard vectors (i.e., pCMV).