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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。
Transgenic silencing is one of the two major obstacles in genetically modified basic research and clinical gene therapy. In this study, HT1080 cell cloning transduced with single copy retroviral vector MGPN2 was used as a model to explore the molecular mechanisms of gene silencing Mechanism for the construction of highly efficient expression of the carrier of genetically modified to help. The results showed that there was no significant change in the epigenetic modification of the vector and the other clones in the 115 cell clone without GFP protein. The promoter of the vector could effectively activate the transcription of the reporter gene GFP, but the size and sequence of the transcript Significant changes that lead to changes in the reading frame of the transcript. Therefore, in addition to the chromosomal location effect causes the epigenetic modification of the transgene carrier to cause the silencing of the transgene, the alteration of the reading frame of the retroviral vector transcript can also cause complete silencing of the transgene, which may be related to the self-recombination of the vector.