The specific aims of this application are: (1) to clone and to express human Ep gene(s), (2) to purify and to characterize the cloned Ep gene product(s). Our long term objectives are: (1) to produce large amounts of pure Ep protein for the studies of its mode of action and for the developments of its therapeutic applications in treating various anemias, (2) to provide defined Ep gene sequence for the investigations of Ep gene structure, organization and expression during normal and abnormal red blood cell production. The following specific methods would be used to achieve our specific aims: (1) To identify Ep-producing human renal tissues by immunofluorescence and radioimmunoassay (RIA) with our monoclonal anti-Ep. (2) To prepare functional Ep mRNA and to synthesize double strand ds (Ep cDNA). (3) To insert Ep cDNA into plasmid pBR322 by homopolymeric G.C tailing and to clone the recombinant plasmid into E. coli C600 by transformation. (4) To select and identify clones containing recombinant Ep cDNA sequence by colony hybridization, RIA, and hybridization selection. (5) To characterize Ep cDNA clones by restriction mapping and sequencing overlapping and to construct full length Ep cDNA for subcloning and expression. (6) To purify and to characterize the cloned Ep gene product by immunoaffinity, cellular and molecular means.
| Lee-Huang, S; Lin, J J; Kung, H F et al. (1993) The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region. Gene 128:227-36 |
| Lee-Huang, S; Lin, J J; Kung, H F et al. (1993) The 3' flanking region of the human erythropoietin-encoding gene contains nitrogen-regulatory/oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements. Gene 137:203-10 |
