The chloramphenicolresistance gene was subsequently removed of using Flp recombinase to leave a single FRT site in place of the conserved region. It was not always possible to design homology arms of the targeting cassette to precisely delete all of the conserved sequences due to the presence of repetitive elements or lack of unique sequences immediately surrounding some conserved regions. In such cases, some additional flanking sequence was also removed. The integrity of the required constructs was verified by assessing chloramphenicol-sensitivity and by PCR and restriction digest analyses. In order to investigate the effect of the deletion of each conserved non-coding region on FXN gene expression, each constructPR-957 960374-59-8was analyzed in transient transfection assays in a mammalian cell line. We have previously shown that the BHK-21 cell line is readily transfectable with large DNA constructs and suitable for the in vivo characterization of FXN gene expression. The EGFP/DsRed-Express ratio produced by the BAC containing a deletion of conserved region 1 was significantly lower than the unmodified control RP11-265B8::Ex5a-EKDsAmp construct. The deletion of conserved region 6 also resulted in significantly lower FXN gene expression. The deletion of conserved region 7 caused a reduction in expression, but not to the same extent as deletion of conserved regions 1 and 6. In contrast, the removal of conserved regions 4 and 5 resulted in higher FXN expression levels. EGFP and DsRed-Express expression from each of the BAC dual-reporter deletion constructs was also analyzed by fluorescence microscopy. Supporting the flow cytometry data, a clear reduction in EGFP expression was observed in cells transfected with the BAC clones containing Paclitaxel 33069-62-4a deletion of either conserved region 1 or 6. Deletion of conserved region 7 also demonstrated lower EGFP expression although not to the same extent as in the constructs containing deletions of conserved regions 1 and 6. To complement the data obtained using the genomic reporter assay, the examination of FXN gene regulatory mechanisms was also assessed using small plasmid luciferase reporter constructs. A set of progressively longer, DNA fragments containing the endogenous human FXN promoter and one or multiple conserved non-coding regions was amplified by PCR and cloned into the pGL3-Basic vector. The set of pGL3-Basic derivative constructs containing the FXN promoter and one or multiple conserved non-coding regions were separately transfected into two mammalian cell lines, HeLa and BE -M17. In both cell lines, the construct containing the shortest insert elicited much higher levels of luciferase expression than the unmodified pGL3-Basic vector without an insert.