Vectors used

Most of the full-length-enriched cDNA libraries by oligo-capping method were constructed using the expression vector pME18SFL3 (Fig. 3-4 & Fig. 3-5), which is a eukaryotic expression vector (for part of the libraries, pUC19FL3 (Fig. 3-6 & Fig. 3-7) was used). In pME18SFL3, the SR alpha-promoter is incorporated in the region upstream from the cloning site, and an SV40 poly A-ligated signal sequence is inserted in the downstream region. The cloning site in pME18SFL3 is the asymmetric DraIII sites, and SfiI sites complementary to those DraIII sites are added to the termini of the cDNA fragment. Therefore, cloned cDNA fragments can be inserted directionally in the region downstream of the SR alpha-promoter. Thus, the gene product encoded by the full-length cDNA can be transiently expressed by directly introducing the plasmid containing the full-length cDNA into COS cells. Thus, the gene product, a protein, or its biological activity can be experimentally analyzed with great ease.