A gene fusion method to screen for regulatory effects on gene expression: application to the LDL receptor

We have evaluated the feasibility of using fusion genes to link transcriptional promoters of biomedical interest to reporter genes to screen for pharmacological or genetic regulatory effects. Using gene targeting, we generated two lines of embryonic stem (ES) cells expressing human alpha-fetoprotein (hAFP) under control of the endogenous promoter for the LDL receptor (LDLR). In one line, hAFP was introduced into the first intron after the translational start codon; in the other, hAFP was positioned in the 3'-untranslated region leaving the potential for expression of LDLR intact. In both lines, an internal ribosome entry site (IRES) was included to facilitate translation. Readily measurable levels of hAFP were found in the medium with both targeted ES cell lines, compared with undetectable levels with the starting cell line. The expectation that the level of hAFP would reflect the steady-state level of mRNA for the fusion transcript and secondarily transcriptional control of LDLR was confirmed by correlating hAFP levels with the abundance of LDLR and fusion transcripts. We also generated mice carrying the LDLR-hAFP fusion in the 3'-untranslated region and these mice produced detectable levels of hAFP in serum. Levels of hAFP in culture medium and in serum were increased by simvastatin, a drug known to up-regulate LDLR. These ES cell clones and mice are suitable for pharmacological and genetic screening to detect effects on expression of LDLR. The data demonstrate the feasibility of using gene fusions to screen for drugs and genetic factors that affect expression of a wide variety of genes of interest.