Applications of Tissue-specific Transcription Factor in Animal Gene Therapy

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Reporter plasmid constructs were created in pSEAP vectors in which the reporter followed the transcription activating element derived from 5' upstream sequence of GATA-1 gene, including erythroid-specific promoter and exon 1, and intron 1. When transfected into K562 cells but not COS7 or CHO cells, activation of the reporter gene expression was observed. Thus, the region involving 5' upstream and intron 1 sequences was demonstrated to possess activity in enhancing gene transcription.Retroviral vector containing the 5' upstream and intron 1 sequence from GATA-1 gene fused to normal bovine band 3 cDNA (pLNCX2bebWT) was prepared and several clones of K562 cells (K562bebWT) were isolated by G418 selection. Immunofluorescence staining showed that band 3 proteins localized to the plasma membrane, whereas the mutant construct (R664X) exhibited no significant signals at the cell surface. K562bebWT showed DIDS-sensitive chloride transport, demonstrating the protein have normal function. Introduction and expression of band 3 gene into bone marrow cells from affected animals are under investigation.