Study on the higher order structure of DNA that enhances the integration of transgene into the host genome

从烟草细胞的转基因位点克隆的507-bp序列（命名为TJ 1）被发现是核基质/支架附着区（MAR/SAR），该序列以四个不同的方向侧接于绿色荧光蛋白（GFP）（一种非选择性标记）基因的表达盒的5'和3'侧。在这些表达盒的每一个中，GFP基因在花椰菜花叶病毒35 S启动子和胭脂碱合酶多腺苷酸化信号的控制下。将这些构建体分别引入烟草细胞中，以研究MAR对转基因整合到宿主基因组中的频率的影响。与缺乏MAR序列的对照构建体相比，盒中MAR序列的存在似乎使PCR产率增加2倍以上。由于在本研究中没有获得这些转化体的选择压力，因此可以想象，基于MAR的基因表达增强不涉及本研究的拟南芥产量结果。因此，观察到的转基因产量应该纯粹反映转基因整合到宿主基因组中的频率。因此得出结论，TJ 1 MAR使积分频率比对照增加2倍以上。没有观察到MAR序列的取向对结晶产率的显著影响。我们的研究结果首次为从转基因位点克隆的MAR刺激转基因整合到真核基因组中提供了直接证据

A 507-bp sequence (designated TJ1) that has been clone from a transgene locus of tobacco cell, and found to be a nuclear matrix / scaffold attachment region (MAR/SAR) was flanked to the 5' and 3' side of the expression cassette, in four different orientations, of the green fluorescent protein (GFP), a non-selective marker, gene. In each of these expression cassette, the GFP gene was under the control of cauliflower mosaic virus 35 S promoter and nopaline synthase polyadenlyation signal. These constructs were introduced separately into tobacco cells in order to investigate the effect of the MAR on the integration frequency of the transgene into the host genome. The presence of the MAR sequences in the cassette appeared to increase the transformant yield more than 2 times as compared with the control construct lacking the MAR sequences. Since there is no selection pressure to obtain those transformants in the present study, it is conceivable that MAR-based enhancement of the gene expression is not involved in the present result of the transformant yield. Therefore, the observed transformant yield should purely reflect the frequency of the integration of the transgene into the host genome. It is thus concluded that TJ1 MAR increases the integration frequency more than 2 times over the control. No significant effect of the orientations of the MAR sequence in the transformant yield was observed. Our findings provide direct evidence for the first time that the MAR cloned from the transgene locus stimulates the integration of transgene into the eukaryotic genome