Targeted expression of enhanced green fluorescent protein to steroidogenic cells expressing steroidogenic acute regulatory protein by bacterial artificial chromosome transgenesis in vivo

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In order to explore structure-function aspects of steroidogenic acute regulatory protein (StAR) within steroidogenic cells, the present research defined the regulatory elements sufficient for expression of mouse Star gene in steroidogenic cells in vivo. First, we identified a bacterial artificial chromosome (BAC) clone that included 47 kb upstream of the transcription initiation codon, the entire mouse Star structural gene, and 62 kb downstream of the termination codon of the gene. To examine the ability of these genomic sequences to target gene expression correctly, we inserted a cassette encoding enhanced green fluorescent protein (eGFP) and a polyadenylation signal from bovine growth hormone into the BAC clone at the normal initiator methionine, Following BAC Modification by homologous recombination in E. cohi, independent transgenic lines with supercoiled BAC DNA were generated. The Star/eGFP transgene was expressed at high levels throughout the adrenal cortex, in Leydig cells in the testes, and in theca and interstitial cells in the ovary. Immunohistochemical analysis with either polyclonal antibodies against mouse StAR protein or monoclonal antibodies against eGFP protein confirmed the expression of eGFP in steroidogenic cells of the adrenal glands and gonads. We did not observe fluorescence or immunological activity of the eGFP in other tissues, including brain, thymus, heart, and kidney. These results indicate that the cis-element including 47 kb upstream of the transcription initiation codon, the entire mouse Star structural gene, and 62 kb downstream of the termination codon of the gene is sufficient for the expression of the Star gene in adrenal glands and gonads and that the Star/eGFP BAC transgenic mice provide an eGFP lineage marker for the endogenous expression of Star gene.