Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.
Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.
复制标题
DOI：
10.1016/0042-6822(89)90600-4
复制
发表时间：
1989-08
期刊：
影响因子：
3.7
通讯作者：
Li Xu;J. Yee;J. Wolff;T. Friedmann
中科院分区：
文献类型：
--
作者：
Li Xu;J. Yee;J. Wolff;T. Friedmann
我们已经检测了逆转录病毒载体在受感染的小鼠细胞中的长期功能和结构稳定性。我们以表达人hprt、萤火虫荧光素酶(Luc)和大肠杆菌β-半乳糖苷酶(LacZ)的Moloney鼠白血病病毒为报告基因，以人hprt和转座子Tn5新霉素抗性(Neo)基因为选择标记。所有载体，无论是单基因还是双基因，都能产生稳定和不稳定的克隆。前病毒的稳定性取决于许多因素，包括感染细胞的性质、报告基因、前病毒的整合位置、组成基因在多基因载体中的相对位置以及选择压力的存在或不存在。选择压力有助于维持前胡萝卜体结构和功能的完整性，但并非普遍有效。来自内部启动子的报告基因的表达很可能不稳定，无论有没有选择上游的LTR驱动的新基因。在一些克隆中，前病毒基因表达的丧失伴随着缺失，而另一些不活跃的克隆则保留了明显完整的前病毒。在后一个克隆中，5-氮胞苷处理未能激活报告基因，但辅助病毒的重叠感染导致可传递载体的重新出现，表明前病毒关闭是一种可逆的表观遗传机制。有效的逆转录病毒载体的设计及其在活体中的可能使用需要进一步表征这些前病毒稳定性的决定因素。
We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors epxressing human HPRT, firefly luciferase (luc), andEscherichia coliβ-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-drivenneogene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible usein vivowill require further characterization of these determinants of provirus stability.