The rat beta-casein gene is a member of a small gene family, encoding the principal milk proteins. In order to understand the mechanisms by which its stage- and tissue-specific expression are regulated, initially, a 14 kb genomic clone containing the entire 7.5 kb rat beta-casein gene with 3.5 kb of 5' and 3.0 kb of 3' flanking DNA was microinjected into the germline of mice. Eight F0 transgenic mice were generated with copy numbers ranging from 1-10; five transmitted the transgene to their offspring in a Mendelian pattern. A specific RNase protection assay was developed to quantitate the level of expression of the rat beta-casein transgene as compared to the endogenous mouse beta-casein gene. Using this assay expression was demonstrated predominantly in the lactating mammary gland of transgenic mice at a level of 0.01-1% of the endogenous mouse beta-casein gene. The transgene employed the authentic transcription initiation site observed previously in the analogous rat beta-casein gene. In one line, a reduced level of expression of the transgene was also observed in the brain. The site of integration, therefore, plays an important role in influencing the level of expression of the transgene, but not its general pattern of tissue-specificity. The transgene appears to be developmentally-regulated in accordance with the endogenous mouse beta-casein gene. These lines of mice generated carrying the rat beta-casein transgene should provide useful models for studying the developmental and hormonal regulation of milk protein gene expression.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat j-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. In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat I8-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 0-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 ,l-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.Development of the fetal mammary gland is characteristically sexually dimorphic (35). The ultimate function of the female mammary epithelium is to synthesize and secrete milk. Milk protein gene expression is regulated by a variety of factors, including peptide and steroid hormones and cell-cell and cell-substratum interactions (7,15,36). Caseins are the predominant milk proteins encoded by a small gene family, whose expression exhibits both tissue and stage specificity. The rat 1-casein gene encodes the principal murine casein and displays a several-hundred-fold increase in expression during adult mammary development from virgin to midlactation (12). Thus, it should provide an excellent model to elucidate the molecular mechanisms by which milk protein gene expression is regulated.The ability to introduce cloned or manipulated genes into mice via microinjection into fertilized eggs provides a novel approach to studying the regulation of gene expression (24), especially for those genes whose regulation requires cell-cell and cell-substratum interactions. An increasing number of cellular genes have been introduced into mice, and most of them resembling their mouse counterparts have been expressed and regulated correctly (25).We have previously generated several lines of transgenic mice, bearing the entire rat p-casein gene along with 3....

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In order to identify DNA sequences responsible for the regulation beta-casein gene expression, lines of transgenic mice bearing the entire rat beta-casein gene and two rat beta-casein promoter chloramphenicol acetyltransferase (CAT) fusion genes have been established. All three transgenes have been shown previously to be regulated in a tissue- and stage specific manner. To investigate the relative contribution of promoter and intragenic sequences in the hormonal regulation of the beta-casein gene, mammary explant cultures derived from these lines of mice have now been performed, and the effects of PRL and glucocorticoids on transgene as compared with endogenous beta-casein gene expression have been quantified. After the addition of PRL to cultures performed in the presence of insulin and glucocorticoids, a 25- to 40-fold induction of endogenous mouse beta-casein mRNA was observed after 48 hr. A comparable greater than 25-fold induction of transgene expression after PRL addition was observed in explant cultures derived from a line of mice expressing the entire rat beta-casein gene. In contrast, PRL addition elicited only a 1- to 4.5-fold increase in CAT activity in cultures derived from two lines of mice bearing casein-CAT fusion genes with either 524 or 2300 base pairs of 5'-flanking DNA. In the presence of insulin, glucocorticoid or PRL addition alone increased endogenous beta-casein gene expression 2- to 2.5-fold and 5- to 10-fold, respectively, but only a 1.2- to 2.5-fold induction of CAT activity was observed for each hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

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