The Isolation and Transfection of the Entire Rat <i>β</i>‐Casein Gene

David-Inouye, Yvonne; Couch, C. H.; Rosen, Jeffrey M.

doi:10.1111/j.1749-6632.1986.tb15539.x

Cited by 6 publications

(5 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although DNA-mediated gene transfer into cell culture has been a powerful approach for identifying cis-acting DNA sequences for several genes responsible for tissue-specific expression and hormonal regulation (35), this approach has so far been refractory when applied to defining the cis-acting DNA sequences involved in tissue-specific and hormonal regulation of rat 8-casein gene expression. For example, a genomic rat 6casein gene sequence cloned into a bovine papilloma virus (BPV) vector failed to be regulated correctly when transfected into a human mammary derived cell line, T47-D, in which endogenous human casein genes are not expressed, but functional prolactin receptors exist (3).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, several rat B-casein-CAT (chloramphenicol acetyl transferase) fusion genes have not shown cell-specific expression and hormonal responsiveness when transfected into COMMA-D and NIH/3T3 cells (36). The failure of these gene transfer experiments in cell culture may be due to: (1) the lack of important cis-acting DNA sequences or the presence of vector sequences, e.g., BPV, in those constructions preventing the normal expression of the transferred genes; (2) the lack of appropriate recipient cells for gene transfer; or perhaps (3) the transferred genes need to be processed through germ line "imprinting" in order to acquire the necessary developmentally-regulated chromatin structure and DNA modifications required for normal expression. Transgenic mice, therefore, provided an excellent alternative approach for addressing the question of regulation of rat 8-casein gene expression because the "manipulated" genes free of vector sequences could be introduced into the germ line to mimic the in vivo environment and subsequently to examine the expression of the transgenes in various tissues throughout development.…”

Section: Discussionmentioning

confidence: 99%

“…To further define the cis-acting DNA sequences within or proximal to the gene, and eventually trans-acting factors, involved in the regulation of rat 8casein gene expression, gene transfer experiments in cell culture have been carried out in our laboratory. However, at present this approach has failed to generate either cell-type specific or hormonally-regulated expression, and instead constitutive expression has been observed (3).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Tissue-specific expression of the rat β-casein gene in transgenic mice

et al. 1988

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Tissue-specific expression of the rat β-casein gene in transgenic mice

et al. 1988

View full text Add to dashboard Cite

show abstract

“…One direct test of the function of these putative regulatory sequences would be to transfect the cloned a-lactalbumin gene into cells in culture. Although this approach has worked for other hormonally responsive genes (Kelly & Darlington, 1985), we (Laird et al, 1988) and others (David-Inouye, 1986;Lee et al, 1988) have been unable to obtain correct hormonal regulation of milk protein gene expression in tissue culture. An alternative approach to this problem is to generate transgenic mice (Palmiter & Brinster, 1986) carrying foreign milk protein genes.…”

Section: Introductionmentioning

confidence: 99%

Transgenic mice carrying the guinea-pig α-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation

Maschio

Brickell

Kioussis³

et al. 1991

Biochemical Journal

View full text Add to dashboard Cite

We have generated transgenic mice carrying the entire guinea-pig alpha-lactalbumin gene. Lactating transgenic mice expressed high levels of correctly initiated and processed guinea-pig alpha-lactalbumin mRNA in the secretory epithelium of their mammary glands, and secreted guinea-pig alpha-lactalbumin in their milk. Transcripts were detectable after 7 days of pregnancy, indicating that the transgene was under correct hormonal control. Whereas no or negligible transcription was detectable in all other tissues tested, high levels of transcripts were found in the skin of lactating transgenic mice. Guinea-pig alpha-lactalbumin protein was undetectable in the skin, however. In situ hybridization analysis showed that expression was localized to the undifferentiated cells in the basal layer of the sebaceous glands. Further studies revealed high levels of endogenous beta-casein mRNA in normal lactating mouse skin, demonstrating that the transcription of milk protein genes in lactating mouse skin is a normal event, and is not peculiar to the transgene. This surprising finding highlights the developmental relationship of the mammary gland to other specialized structures of the skin, supports a role for epithelial-extracellular matrix interactions in the regulation of milk protein gene expression in vivo, and identifies the skin as a particularly accessible model system in which to study the regulation of milk protein gene expression. In addition, the guinea-pig alpha-lactalbumin gene will be a source of regulatory sequences with which to direct heterologous gene expression to the sebaceous glands of transgenic mice.

show abstract

“…Recently, a 2.8-kb 5'-flanking region of the WAP gene has been demonstrated to direct Ha-ras, c-myc, and tissue plasminogen activator (TPA) expression to the mammary gland of lactating female mice (1,28,30) and the secretion of TPA in transgenic mouse milk (8). Despite the previous inability to use cell culture models to demonstrate mammary-specific expression of the rat 13-casein-cat fusion gene (2,6), CAT activity was detected predominantly in the lactating mammary gland of transgenic mice carrying either construct. These results demonstrate that 0.5 kb of flanking DNA sequences along with noncoding exon I and 0.5 kb of intron A are sufficient to target a heterologous protein to be synthesized in the mammary gland.…”

mentioning

confidence: 99%

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

Lee¹,

Atiee²,

Rosen³

1989

Mol. Cell. Biol.

View full text Add to dashboard Cite

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....

show abstract

The Isolation and Transfection of the Entire Rat β‐Casein Gene

Cited by 6 publications

References 5 publications

Tissue-specific expression of the rat β-casein gene in transgenic mice

Tissue-specific expression of the rat β-casein gene in transgenic mice

Transgenic mice carrying the guinea-pig α-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation

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

Contact Info

Product

Resources

About