Direct Selection of Functional Insulators in Arabidopsis thaliana

Abstract Background: Insulators are DNA sequences found in all eucaryotes. By forming DNA-protein complexes in vivo they regulate enhancer-promoter interactions and define heterochromatin-euchromatin boundaries. Studies to date have focused mainly on well-studied elements from Saccharomyces cerevisiae, Homo sapiens and Drosophila melanogaster. More recently there has been increased interest in insulators in plants such Petunia hybrida and Arabidopsis thaliana, in part due to the increased production of transgenic plants and there use in agriculture. The production of transgenic plants requires the correct tissue-specific and developmentally regulated gene expression of the transgene; however, their construction identified several problems such as nonspecific enhancer–promoter interactions and transgene silencing or “position effects”. Insulators have the potential to mitigate these unwanted effects. Through the study of insulator mutations, we have identified several non-plant insulators that function in the model plant A. thaliana. For the potential of insulators to be maximized, more, different insulators need to be identified. In this paper we describe a novel protocol to isolate de novo insulators, enhancer-blocking DNAs that function as regulatory elements in A. thaliana.Results: The selection of insulators depends upon three vectors we have constructed. pC1 is a negative selection vector. The presence of an insulator blocks the expression of the gene cytosine deaminase (codA) than depends upon the CaMV35S enhancer. Cytosine deaminase converts the non-toxic, cytosine analogue 5-Fluorocytosine (5-FC) into a highly toxic metabolite 5-Fluorouracil (5-FU). Plants that survive selection are potential insulators. These sequences can to cloned into pB31 that is similar to pC1 except GUS expression is monitored to confirm lack of expression in most tissues. Finally cloning into pL1 is used to verify GUS expression from the tissue-specific expression from the napin, seed-coat specific promoter is unaffected by the CaMV35S enhancer. As a proof of concept, we have recovered five, relatively-short functional insulators sequences from a library of random DNA sequences generated in vitro. Conclusions: The protocol we describe can be easily scaled-up to generate more insulators. Furthermore, the source of the DNA could be any species, and modifications specific for a defined species in other genera could lead to the identification of a wide range of species-specific insulators. The elimination of unwanted enhancer-promoter interactions will impact the design of transformation systems and the recovery of transgenic plants. Now you can create your own!