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

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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