A method to identify cDNAs based on localization of green fluorescent protein fusion products

We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression clo ning method (Ft-REX; fluorescence localization-based retrovirus-mediated ex pression cloning) in which cDNAs can be isolated based on the subcellular l ocalization of their protein products. Complementary DNAs generated from mR NA using random hexamers were fused to the cDNA of green fluorescent protei n (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviru ses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP -fusion products, Using FL-REX we have identified 25 cDNAs, most of which s howed reasonable subcellular localization as GFP-fusion proteins, indicatin g that FL-REX is useful for identification of proteins that show specific i ntracellular localization.