【摘要】 转基因沉默是转基因基础研究和临床基因治疗应用研究中的两大障碍之一,本文以转导单拷贝逆转录病毒载体MGPN2的HT1080细胞克隆为研究模型,利用一系列分子生物学技术探索转基因沉默的机制,为构建高效表达转基因的载体提供帮助。结果显示:在无GFP蛋白生成的115号细胞克隆中,载体的表观遗传修饰与其它克隆比没有明显变化,载体启动子能有效启动报告基因GFP的转录,但是转录本的大小和序列发生了显著改变,导致转录本阅读框改变。因此,除染色体位置效应引起转基因载体表观遗传修饰改变而导致转基因的沉默以外,逆转录病毒载体转录本阅读框的改变也可以引起转基因的完全沉默,这可能与载体自身重组有关。更多还原

【Abstract】 Transgene silencing is one of two major obstacles in both basic biomedical research for transgene and clinical practice of gene therapy.Based on the model of HT1080 cell clones,which transduced single copy of retroviral vector MGPN2,the mechanism of transgene silencing was explored in this investigation by a serial molecular techniques.In the HT1080 cell clone that absence of GFP protein synthesized,no significant aberration of epigenetic modification was detected,but the transcript size and the sequence changed that resulted in the reading frame shift.In addition to chromosomal position effect leading to transgene silencing,the transcript reading frame shift associated with retroviral vector rearrangements could induce complete silencing of transgene.更多还原