Analysis of Potential DNA Insulators in Arabidopsis thaliana

Abstract

The control of transgene expression is the key to producing transgenic organisms with beneficial traits; however, the prevention of transgene misexpression in transformed organisms has been a recurring challenge and may lead to the expression of undesirable traits. Insulators are DNA sequences that regulate gene expression through their interaction with protein factors. They function as cis-acting DNA sequences with the ability to act as enhancer-blocking elements or barrier elements, or both depending on the molecular mechanism. The functions of insulators have been tested using a model system based upon transgenic Arabidopsis thaliana plants to determine the expression of transgenes and to decrease non-specific interactions resulting in misexpression. To date, various insulators have been identified in invertebrates and vertebrates but few have been discovered and analyzed in plants. This study aims to identify insulators that can function to block enhancer-promoter interactions in transgenic plants preventing transgene misexpression. An oligonucleotide library was produced allowing for subsequent screening of putative insulators. Screening of recombinant clones was initiated to identify strong insulator candidates using vectors with different promoter/reporter systems in Arabidopsis. To date, three insulators (CLOI-3, CLOII-10, and CLOII-12) have been identified as candidate insulators that block enhancer-promoter interaction. The CLOI-3 insulator has been further analyzed through the introduction of deletions to identify crucial regions. Two replicates for both CLOII-10 and CLOII-12 indicate possible insulator activity. By characterizing new insulator sequences we will be able to further understand the mechanisms involved in gene expression and apply them to transgenic research involving other biological systems, ultimately addressing important issues such as crop development.