Applications of Tissue-specific Transcription Factor in Animal Gene Therapy

在pSEAP载体中产生报告质粒构建体，其中报告基因在源自加塔-1基因5'上游序列的转录激活元件之后，包括红系特异性启动子和外显子1，以及内含子1。当转染到K562细胞而不是COS 7或CHO细胞中时，观察到报告基因表达的激活。制备了含有加塔-1基因5 '上游和内含子1序列与正常牛带3 cDNA融合的逆转录病毒载体（pLNCX 2bebWT），并通过G418筛选分离了几个K562细胞克隆（K562 bebWT）。免疫荧光染色显示带3蛋白定位于质膜，而突变体构建体（R664 X）在细胞表面没有表现出显著的信号。K562 bebWT具有DIDS敏感性的氯离子转运功能，证明该蛋白具有正常的功能。将带3基因导入受影响动物的骨髓细胞并使其表达的研究正在进行中。

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.