The eight CPI-613 regions are evolutionarily conserved from human to mouse and some to even more divergent species. Each of the identified regions is within the size range of 40�C750 bp and harbor between 40% to greater than 70% homology between species. The linear order of the identified conserved regions is maintained across the species analyzed. These findings make the eight regions strong candidates to contain key regulatory elements for FXN gene expression. The eight identified conserved sequences were analyzed via their deletion in the context of the whole FXN locus within a dualreporter LEE011 in vivo BAC-based assay. BACs are very useful research tools as they can contain the entire intact locus of a gene, including surrounding regulatory elements, thus allowing the recapitulation of normal gene expression patterns. In addition, the positional information of most regulatory elements required for normal gene expression will be naturally preserved. In this approach, a reporter gene is fused to the coding sequence of the gene of interest in the context of its intact genomic locus and the whole construct is then used to study gene expression in transient transfection studies. An advantage of transient assays over the integration of BAC constructs into the host genome is the avoidance of variation produced by different integration sites, the influences of flanking genomic sequences, and in differences in transgene copy number at the site of integration. The RP11-265B8 BAC clone utilized in this study contains the FXN gene and significant amounts of flanking sequences, thus making it likely to contain the intact FXN locus. This BAC construct has also been shown to rescue the embryonic lethality of the homozygous knockout of the mouse Fxn gene indicating that most, if not all, regulatory elements required for normal FXN gene expression are present within this clone. This BAC clone was also used to develop the first genomic reporter for FXN expression, based on the targeted insertion of an EGFP cassette following the final codon of exon 5a. The DsRed-Express reporter was introduced into the backbone of BAC clone to serve as an internal control for transfection efficiency and vector copy number. The presence of the two fluorescent markers enabled the monitoring of the effects of modifications of conserved regions and permitted correction for differing transfection efficiencies. Modifications of each of the conserved non-coding regions identified in silico were carried out on the dual-reporter BAC construct. The deletion of conserved region 4 and the deletion of conserved region 5 resulted in an increase in FXN gene activity by 30% and 20%, respectively. It is therefore hypothesized that these regions contain silencer elements or binding sites for repressors, which normally serve to dampen FXN gene expression.