High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region.

Abstract

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extraceUlular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in ceUs cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.

Nuclear Factor I and Mammary Gland Factor (STAT5) Play a Critical Role in Regulating Rat Whey Acidic Protein Gene Expression in Transgenic Mice

“…The two lines without detectable expression contained either one or two copies of the transgene. This result was similar to those obtained previously for the ϩ2020 wildtype transgene (8,24), i.e., when the copy number of the ϩ2020 transgene was very low, the transgene was inactive. On the basis of the results for the three lines expressing ϩ2020m4, mice with higher copy numbers appeared to express the transgene at higher levels, indicating that copy number-dependent expression may not depend upon the presence of an intact MGF-binding site (Fig.…”

“…Three conserved bases in each of the NFI half-binding sites, which have been shown by contact point analysis to be essential for binding of NFI to the rat WAP promoter (28), were changed as indicated. All of these mutated constructs were based on the rat WAP genomic transgene ϩ2020, which displays high-level, tissuespecific, and position-independent expression in transgenic mice (8). This permitted the functional definition of individual regulatory elements in mutated transgenes.…”

“…Transgenic mice were generated, and mouse tail DNA was isolated as described previously (26). PCR was employed to screen for positive transgenic mice (8).…”

“…Our laboratory has employed the rat whey acidic protein (WAP) gene, a major whey protein gene expressed in rodents, as a model system to study milk protein regulation. Previous studies have demonstrated that a 3.0-kb rat WAP genomic transgene (designated ϩ2020) with 949 bp of 5Ј-and 70 bp of 3Ј-flanking DNA is efficiently expressed specifically in the mammary glands of transgenic mice in a copy-number-dependent manner (8). Two mammary gland-specific DNase I-hypersensitive sites (HSS) have been identified in the 5Ј-flanking region of the endogenous rat WAP gene and the ϩ2020 transgene during lactation, i.e., HSS I at Ϫ800 to Ϫ700 and HSS II at Ϫ150 to Ϫ50 bp relative to the transcriptional initiation site.…”

Production and secretion of recombinant human fibrinogen by the transgenic murine mammary gland

“…It was postulated that the clone contained some but not all regulatory elements necessary for correct expression. Due to conservation of 3' UTRs between milk protein genes (Dale et al, 1992) and the potentially high expression of the full clone, several constructs were developed using cDNAs inserted into the first exon of the clone (Tomasetto et al, 1989;Velander et al, 1992a;Jhappan et al, 1993). Expression of protein from these constructs was in the 5 to 30 :g/ml milk range.…”