Untitled

Abstract

A beta-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3' region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3' end, with BLG sequences of exon 6/intron 6/exon 7 and the 3' region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3' flanking sequences. The BLG 5' regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.

Luminal STAT5 mediates H2AX promoter activity in distinct population of basal mammary epithelial cells

“…Unless otherwise indicated, the effect of PRL and hydrocortisone supplementation was studied in a six-well culture plate (Corning, NY) for 5 days in DMEM:F12 containing 5% FCS, insulin (5 mg/ml), hydrocortisone (3 mg/ml, Sigma), and ovine PRL (3 mg/ml, provided by the NIDDK program). Stable transfections of the H2AX–GFP gene construct into non-transfected CID-9 cells, or BLG–CID9ca-transfected cells carrying a constitutively active STAT5 gene directed for expression in the mammary gland by BLG regulatory sequences (BLG–STAT5ca) [12, 25], were carried out with lipofectamine reagent (Gibco-BRL, Gaithersburg, MD) and pBABE plasmid [59]. Selection was performed with puromycin (2 μg/ml, Sigma).…”