Comparative analysis of the effect of genomic isolators flanking transgenes to avoid positional effects in Arabidopsis

Abstract

HighlightWe have studied the effect of different insulator sequences over transgene expression levels and variability, and over transgene integration, using NGS. Our results compare the benefits obtained by their use.\n\nAbstractFor more than 20 years, plant biologists have tried to achieve complete control of transgene expression, but until gene targeting techniques become routine, flanking transgenes with genetic insulators can help avoid positional effects. Insulators are DNA sequences with barrier activity that protect transgenes from interferences with the host genome. We have, for the first time, compared the effect of three insulator sequences previously described in the literature and of a matrix attachment region from Arabidopsis never tested before. Our results indicate that the use of all sequences increases transgene expression, but only the last one reduces variability between lines and between individuals to a minimum. We have analyzed the integration of insulator-flanked T-DNAs using whole genome re-sequencing (to our knowledge, also the first time) and found chiMAR lines with insertions located within heterochromatic regions of the genome, characterized by DNA methylation that did not spread into the T-DNA, suggesting that chiMAR can shelter transgene insertions from neighboring repressive epigenetic states. Finally, we could also observe a loss of accuracy of the RB insertion in the lines harboring insulators, evidenced by a high frequency of truncation of T-DNAs and of insertion of vector backbone that, however, did not affect transgene expression.