Novel expression cloning methods based on retrovirus mediated gene transfer.

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We previously established a high-efficiency retrovirus-mediated expression cloning method. Using this system, we have now developed a novel expression cloning method in which cDNAs can be isolated based on the subcellular localization of their products. We make cDNAs using random hexamer and fuse them to 5' endo of the cDNA of green fluorescent protein(GFP)in the pMX retrovirus vector to construct a cDNA-GFP fusion retrovirus library. The derived retroviruses are then infected to NIH3T3 cells to identify a cDNA of interest based on the subcellular localization of its protein product such as nucleus, nucleoli, Golgi apparatus, and cell surface. Using this strategy, we have identified a novel zinc finger protein from fetal mouse liver cells that contain hematopoietic progenitor cells.We have also established a novel signal sequence trap method which can identify cDNA fragments containing signal sequence that encode secreted and cell-surface. Signal sequence trap was originally developed in Kyoto University, and was improved by a group in Genentech, isolate cDNAs with signal sequences that encode such proteins have been established. In our method termed, cDNA fragments fused to an extracellular deletion mutant of the constitutively active receptor for thrombopoietin(MPL)were introduced into IL-3-dependent cells via retrovirus infection followed by the selection of factor-independent clones. Our method is much quicker and more accurate than the previously published methods. We have identified three novel receptors(a type I cytokine receptor, a TNF receptor-like molecule, and a novel low-density lipoprotein receptor-related protein)and several novel secreted molecules by SST-REX, and are now characterizing these molecules using various strategies including knockout mice.