2Western Australian Institute for Medical Research, Perth, WA, Australia
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The mechanisms controlling gene transcription are complex, and understanding these processes requires the identification and characterization of gene regulatory regions. While recent advances in high-throughput genome-wide approaches to identifying gene regulatory elements are informative (1,2), reporter gene methodologies still represent the end game of any gene-specific analysis of transcrip-tional regulation (3). As such, reporter gene analysis has provided remarkable insights into the mechanisms governing gene expression. However, there are still several inherent limitations in the currently available systems whose ultimate objective is mimicking the expression pattern of the endogenous gene and testing the effects of mutations or changes in cis elements in the promoter of an introduced reporter gene.
The most common functional assay used to map cis-acting regulatory regions is the transient transfection assay in which a reporter is expressed under the control of a regulatory region of the gene to be analyzed. In this assay, hundreds or even thousands of copies of the reporter gene construct can enter the cell (3). Since most transcription factors are present at low concentration, only a fraction of the constructs entering the cell receive the full complement of proteins needed for the proper function of the control region (3), often leading to aberrant function of specific control elements (4,5).
Many gene regulatory regions have been shown to function inappropriately in transient transfection assays. In addition to the concentration effects outlined above, aberrant activity may also be due to the lack of an appropriate chromatin conformation (5). More specifically, many long-range enhancer and silencer elements do not function unless integrated within the genome using either stable transfection assays or transgenic mouse models (6,7,8). As chromatin context is likely to effect how transcription factors interact with their cognate cis elements (9,10), the transient transfection assay, while a reasonable indicator of important regulatory regions, is not a completely appropriate model to assess the full influence of regulatory regions on the expression of a particular gene.
The use of stable transfection assays, where a reporter gene expression cassette is integrated into the genome of a cell lines, has extended our knowledge of the function of context-critical cis-acting elements (3). However, stable transfections have additional problems such as integration site concerns as well as an inability to directly compare between wild-type and mutant reporters due to the random nature of the integration.
In this study, we overcome these problems and have developed a novel and widely applicable system, which permits the comparison of transcriptional reporter gene activities following site-specific genomic integration. By using site-specific Flp recombinase-mediated integration (11), 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, which are isogenic and of single-gene copy, are incorporated within a measurably active chromatinized setting, thus more closely reflecting the endogenous gene environment.
Materials and Methods Cell LinesCell lines Flp-In™ -Jurkat T (Invitrogen, Carlsbad, CA, USA) and K562 (ATTC, Manassas, VA, USA) and its Flp recombinase target (FRT) derivatives, were cultured in RPMI-1640 (Trace Biosciences, Castle Hill, NSW, Australia) supplemented with 100 µg/mL each of penicillin and streptomycin (Trace Biosciences) and 10% fetal bovine serum (FBS; Thermo Trace Ltd, Noble Park, VIC, Australia) at 37°C in 5% CO2. FRT-K562 cells were generated by transfection with the FRT vector, pFRTLacZeo, exactly as described by the manufacturer (Invitrogen). Single site integration of the FRT sequence was checked by Southern hybridization using a probe obtained by use of the PCR primers SV40-F and LacZeo-R (see below) and pFRTLacZeo plasmid DNA (Figure 1).
Construction of Promoter-Green Fluorescent Protein Reporter Plasmids
A 1110-bp KpnI and blunted HindIII fragment containing -993 to +110 of the tumor necrosis factor (TNF) promoter (12) was cloned into the KpnI and blunted BamHI multiple cloning sites (MCS) of the pd2EGFP-N1 (Clontech Laboratories, Mountain View, CA, USA) expression vector. The TNF promoter-green fluorescent protein (GFP) expression cassette was then excised using BglII, which occurred just upstream of the KpnI site in the MCS of pd2EGFP-N1, and NotI, which occurred downstream of the GFP sequence of pd2EGFP-N1, and subcloned into Kpn1 and Not1 sites in the MCS of the pcDNA5/FRT expression vector (Invitrogen), which had 967 bp of the cytomegalovirus (CMV) promoter and regulatory sequences removed.
