Résumé
One way of targeting gene expression in vivo is to control transcription using a tissue-specific regulatory system. Tissue-specific promoters or enhancers are in use in transgenic animals and could be utilized in medicine for gene therapy. At present the usual method for selection of a tissue-specific promoter is to identify a gene, which is expressed at unusually high level in the target tissue, and then to use the promoter for this gene to drive expression of another therapeutic gene in the target tissue. This approach is logical but does not always lead to high levels of gene expression. A second approach is to investigate the scope for discovery of synthetic specific promoters using a target tissue. The objective of the work described in this paper was to use both approaches to design plasmid DNA expression vectors that would carry liver-specific promoter/enhancer linked to a reporter gene [i.e. luciferase]. Then transfect these vectors to both liver-derived and non-liver cell lines. This is followed by evaluation of the liver-specificity of each construct by measuring the basal level expression of the reporter gene [i.e. luciferase activity] in both cell lines. Hepatocyte nuclear factor-4 [HNF-4] is liver-enriched transcription factor used to design new synthetic enhancers by inserting a tandem array of 1', 3' or 5' repeats of the HNF-4 binding site upstream of the SV40 promoter linked to the luciferase reporter gene within an Epstein-Barr virus [EBV]-based vector, p706. The results of transfection revealed that unexpectedly the HNF-4 binding sites in these constructs act as a repressor rather than enhancer of the liver-specific expression of the luciferase gene