Abstract
For more than 20 years, plant biologists have tried to achieve complete control of transgene expression. Until the techniques to target transgenes to safe harbor sites in the genome become routine, flanking transgenes with genetic insulators, DNA sequences that create independent domains of gene expression, can help avoid positional effects and stabilize their expression. We have, for the first time, compared the effect of three insulator sequences previously described in the literature and one never tested before. Our results indicate that their use increases transgene expression, but only the last one reduces variability between lines and between individuals. We have analyzed the integration of insulator-flanked T-DNAs using whole genome re-sequencing (to our knowledge, also for the first time) and found data suggesting that chiMARs 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. Our data supports that the effect of each genetic insulator is different and their use in transgenic constructs should depend on the needs of each specific experiment.
Highlights
Due to the random nature of transgene insertion in the majority of higher eukaryotes, transgenic DNA may integrate into regions of the genome that are transcriptionally repressed, which can result in many cases in transgene silencing
The same group later found that a significant reduction in variation of gene expression was conferred upon the GUS gene driven by the double cauliflower mosaic virus 35S promoter, but not to the NPTII gene, Centre for Plant Biotechnology and Genomics Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Campus Montegancedo UPM Pozuelo de Alarcón (Madrid), Madrid, Spain
Taking advantage of the capacities of modular cloning systems, we generated five identical constructs harboring the firefly luciferase transgene driven by the constitutive mannopine synthase Agrobacterium gene promoter www.nature.com/scientificreports and followed by the Basta resistance selection marker cassette
Summary
Due to the random nature of transgene insertion in the majority of higher eukaryotes, transgenic DNA may integrate into regions of the genome that are transcriptionally repressed (heterochromatin), which can result in many cases in transgene silencing. Boundary elements, are DNA sequences with the capacity to define a chromatin domain because of two key activities, the first is the ability to interfere with enhancer-promoter communication when placed between the two (enhancer blocking activity) and the second one is the ability to protect a flanked transgene from position-dependent silencing (barrier activity)[2]. These boundary elements have been characterized extensively in animals. Later studies applying different transformation methods reported no boost effect on transgene expression of Arabidopsis wild type plants[10], but an increase in silencing mutant backgrounds[3]
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