Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation
Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation
复制标题
DOI：
10.1023/b:mole.0000031363.35839.46
复制
发表时间：
2004-06-01
影响因子：
2.8
通讯作者：
Lipps, HJ
中科院分区：
文献类型：
--
作者：
Jenke, ACW;Scinteie, MF;Lipps, HJ
目前用于哺乳动物细胞的可用载体受到许多限制，这使得它们仅部分可用于真核细胞和生物体的遗传修饰以及基因治疗。虽然载体的整合可导致与宿主基因组的不可预测的相互作用和整合的转基因的沉默，但大多数非整合载体仅介导转基因的瞬时表达。所有可用的载体类型都可以导致受体细胞的转化，其中许多载体可以在生物体中引起严重的免疫副作用。理想的载体必须是免费的这些副作用，并应允许长期表达的转基因的情况下selection.In这份报告中，我们描述了一种新的非病毒附加型表达系统，满足这些标准。将编码CMV启动子控制下的截短的大鼠NGF受体基因的基因插入含有支架/基质附着区（S/MAR）的载体构建体中。然后将该载体转染到CHO细胞和人HaCat细胞中。我们表明，该载体复制附加在这些细胞中，是有丝分裂稳定的选择超过100代。此外，我们提供了第一个实验数据，CMV-启动子在附加体是不受胞嘧啶甲基化沉默，从而允许长期表达的转基因在没有选择。
Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection.In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.