Evaluation and comparison of the insulation efficiency of three enhancer-blocking insulators in plants

Enhancer-promoter interactions have the potential to compromise the precise engineering of gene function and agronomically important traits in crops; therefore, effective enhancer-blocking insulators are required in transgene constructs to block such interference. In this study, we evaluated and compared the insulation efficiency of three enhancer-blocking insulators using an enhancer activation assay in which a flower-specific AGAMOUS second intron-derived promoter (AGIP) that drives a beta-glucuronidase (GUS) reporter gene is activated in vegetative tissues by an adjacent CaMV35S enhancer. Of the insulators analyzed, both the EXOB fragment from the lambda genome and the TBS from petunia were found to effectively block enhancer activation, while the 16 bp NI29 fragment from Arabidopsis inserted either as a single copy or tandem repeat in the same assay vector failed to do the same as revealed by the detection of strong GUS expression in the leaves. PCR amplification and DNA sequencing analyses demonstrated that there were no deletions or mutation events within the transgenic NI29 sequence, indicating that the failure of NI29 to block 35S enhancer activation was not the result of gene rearrangements or sequence mutations. Quantitative analysis of GUS reporter expression at both the enzymatic and transcriptional levels exemplified that the EXOB is on average four times more efficient than the TBS in blocking enhancer-promoter communication. This highly effective insulator has an immediate application for remedying enhancer-promoter interactions within transformation vectors composed of multiple transcription units.