Expression from the transferrin gene promoter in transgenic mice.

Abstract

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.