Integration site-specific transcriptional reporter gene analysis using Flp recombinase targeted cell lines.
While high-throughput genome-wide approaches are useful to identify important regulatory regions, traditional reporter gene methodologies still represent the ultimate steps in fine structure analysis of transcriptional control elements. However, there are still several inherent limitations in the currently available transient and stable transfection systems often leading to aberrant function of specific cis elements. In this study we overcome these problems and have developed a novel and widely applicable system that permits the comparison of transcriptional reporter gene activities following site-specific genomic integration. By using Flp recombinase-mediated integration, the system allows the integration and expression of a series of reporter gene constructs at exactly the same genomic location and orientation in all cells of any one culture. The resulting reporter gene lines carry a single reporter gene, which is incorporated within a measurably active chromatinized setting, thus more closely reflecting the endogenous gene environment.
Karimi
Mahdad
M
University of Western Australia, Crawley, WA, Australia.
Goldie
Lauren C
LC
Ulgiati
Daniela
D
Abraham
Lawrence J
LJ
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D017931:DNA Primers; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D010957:Plasmids; D014158:Transcription, Genetic
D017931:DNA Primers; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase