Tissue-specific, high level expression of the rat whey acidic protein gene in transgenic mice.

The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

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

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.     

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

Rescue of an MMTV transgene by co-integration reveals novel locus control properties of the ovine β-lactoglobulin gene that confer locus commitment to heterogeneous tissues

In an attempt to enhance the frequency and level of expression of a poor-performing MMTV-driven transgene, we co-integrated this construct with the ovine -lactoglobulin (BLG) gene in transgenic mice. Seven lines of transgenic mice possessing co-integrated BLG and MMTV-RZ5 transgenes were compared with 12 lines of mice that possessed only the MMTV-RZ5 construct. Co-integration enhanced the frequency of expression in the mammary gland from two out of 12 lines for the MMTV-RZ5 transgene alone, to five out of seven when co-integrated with BLG. Surprisingly, co-integration also resulted in co-expression of the two transgenes in the salivary gland, lung and spleen in addition to the mammary gland. Furthermore, both transgenes were expressed in virgin animals, and throughout pregnancy and lactation, suggesting that the developmental regulation of the locus follows that of the MMTV-promoter. These findings represent a novel locus control property of the ovine BLG gene that confer s commitment of the locus to the mammary gland, but also to a range of heterogeneous tissues possibly defined by the second promoter at the locus     

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.     

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.     

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

EMMPRIN (basigin/CD147) expression is not correlated with MMP activity during adult mouse mammary gland development

Extracellular matrix metalloproteinase inducer (EMMPRIN/basigin/CD147) is a cell surface protein, which has been associated with the induction of matrix metalloproteinase (MMP) genes during cancer metastasis. EMMPRIN plays a role in a variety of physiological processes as is evident by the diverse deficiencies detectable in EMMPRIN knockout mice. We have analysed the role of EMMPRIN in the induction of MMP genes during mammary gland differentiation and involution. Co‐transfection studies showed that EMMPRIN has diverse effects on MMP promoter activity in different mammary and non‐mammary cell lines. Expression of EMMPRIN mRNA is enhanced markedly by insulin in a mammary gland cell line but appears to have no direct effect on MMP gene expression in these cells. Microarray analysis and quantitative PCR show that EMMPRIN is expressed throughout mammary gland differentiation in the mouse. Its expression decreases during early pregnancy and briefly after induction of mammary gland involution by litter removal. Immunohistochemical analysis shows that EMMPRIN expression is limited to the stromal compartment during pregnancy, whereas it is strongly expressed in the epithelium during lactation. In summary the data argue against a causal role for EMMPRIN for the induction of MMP gene expression during adult mammary gland development. These data therefore support a physiological role for EMMPRIN other than MMP induction in mammary gland biology. J. Cell. Biochem. 106: 52–62, 2009. © 2008 Wiley‐Liss, Inc.     

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.     

Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription

We investigated the consequences of augmented c-myc gene expression in the mammary gland of transgenic mice. For this purpose we directed the expression of a mouse c-myc transgene to the differentiating mammary epithelial cells by subjecting the protein coding region to the 5' regulatory sequences of the murine whey acidic protein gene (Wap). Analogous to the expression pattern of the endogenous Wap gene, the Wap-myc transgene is abundantly expressed in the mammary gland during lactation. The tissue-specific and hormone-dependent expression of the Wap-myc transgene results in an 80% incidence of mammary adenocarcinomas. As early as two months after the onset of Wap-myc expression, tumours occur in the mammary glands of the transgenic animals. The tumours express not only the Wap-myc transgene, but also the endogenous Wap and beta casein genes. The expression of the milk protein genes becomes independent of the lactogenic hormonal stimuli and persists even in transplanted nude mouse tumours.