Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

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 extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells 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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.905-914.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 905-914

Author(s):

T C Dale ◽

M J Krnacik ◽

C Schmidhauser ◽

C L Yang ◽

M J Bissell ◽

...

Keyword(s):

Extracellular Matrix ◽

Transgenic Mice ◽

Untranslated Region ◽

Whey Acidic Protein ◽

Acidic Protein ◽

Developmental Expression ◽

Control Element ◽

High Level Expression ◽

Promoter Sequences ◽

High Level

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 extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells 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.

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A distal region, hypersensitive to DNase I, plays a key role in regulating rabbit whey acidic protein gene expression

Biochemical Journal ◽

10.1042/bj3590557 ◽

2001 ◽

Vol 359 (3) ◽

pp. 557-565 ◽

Cited By ~ 5

Author(s):

Benjamin MILLOT ◽

Marie-Louise FONTAINE ◽

Dominique THEPOT ◽

Eve DEVINOY

Keyword(s):

Mammary Gland ◽

Sequence Similarity ◽

Distal Region ◽

Dnase I ◽

Whey Acidic Protein ◽

Acidic Protein ◽

Upstream Region ◽

Lactating Mammary Gland

The aim of the present study was to identify the functional domains of the upstream region of the rabbit whey acidic protein (WAP) gene, which has been used with considerable efficacy to target the expression of several foreign genes to the mammary gland. We have shown that this region exhibits three sites hypersensitive to DNase I digestion in the lactating mammary gland, and that all three sites harbour elements which can bind to Stat5 in vitro in bandshift assays. However, not all hypersensitive regions are detected at all stages from pregnancy to weaning, and the level of activated Stat5 detected in the rabbit mammary gland is low except during lactation. We have studied the role of the distal site, which is only detected during lactation, in further detail. It is located within a 849bp region that is required to induce a strong expression of the chloramphenicol acetyltransferase reporter gene in transfected mammary cells. Taken together, these results suggest that this region, centred around a Stat5-binding site and surrounded by a variable chromatin structure during the pregnancy–lactation cycle, may play a key role in regulating the expression of this gene in vivo. Furthermore, this distal region exhibits sequence similarity with a region located around 3kb upstream of the mouse WAP gene. The existence of such a distal region in the mouse WAP gene may explain the differences in expression between 4.1 and 2.1kb mouse WAP constructs.

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Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2063 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2063-2070 ◽

Cited By ~ 108

Author(s):

S Li ◽

J M Rosen

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Transgenic Mice ◽

Binding Sites ◽

Transgene Expression ◽

Protein Gene ◽

Whey Acidic Protein ◽

Nuclear Factor ◽

Factor I ◽

Nuclear Factor I

The rat whey acidic protein (WAP) gene contains a mammary gland-specific and hormonally regulated DNase I-hypersensitive site 830 to 720 bp 5' to the site of transcription initiation. We have reported previously that nuclear factor I (NFI) binding at a palindromic site and binding at a half-site are the major DNA-protein interactions detected within this tissue-specific nuclease-hypersensitive region. We now show that point mutations introduced into these NFI-binding sites dramatically affect WAP gene expression in transgenic mice. Transgene expression was totally abrogated when the palindromic NFI site or both binding sites were mutated, suggesting that NFI is a key regulator of WAP gene expression. In addition, a recognition site for mammary gland factor (STAT5), which mediates prolactin induction of milk protein gene expression, was also identified immediately proximal to the NFI-binding sites. Mutation of this site reduced transgene expression by approximately 90% per gene copy, but did not alter tissue specificity. These results suggest that regulation of WAP gene expression is determined by the cooperative interactions among several enhancers that constitute a composite response element.

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