Glands Of Transgenic Mice Research Articles

Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary epithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of beta-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and beta-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine beta-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, beta-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of beta-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, beta-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy.

Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

A 6.1 kb 5′ upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5′ flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5′ flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5′ flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between −6.1 kb and −1.1 kb of 5′ flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.

Galanin gene expression is increased in the anterior pituitary gland of the human growth hormone-releasing hormone transgenic mouse.

The peptide galanin is synthesized within and secreted from specific cells of the anterior pituitary gland. Previous studies showed that GH-releasing hormone (GHRH) stimulates galanin release from pituitary cells in vitro. In the present study we used human (h) GHRH transgenic mice to examine the effects of high circulating levels of GHRH on pituitary galanin gene expression in vivo. Moreover, the hGHRH transgenic mice develop pituitary tumors and, thus, may be used as a model of estrogen-independent pituitary adenoma formation. We examined male hGHRH transgenic mice and nontransgenic siblings at 2, 4, 6, 8, and 10 months of age. Transgenic mice were identified using the polymerase chain reaction. Body weights and plasma mGH levels were higher in transgenic mice at all ages. Total protein contents in the anterior pituitary glands of transgenic mice were significantly greater at each age. Galanin peptide contents in the anterior pituitary gland of hGHRH mice were normalized for differences in total protein content and were significantly elevated at all ages examined. At 10 months of age, anterior pituitary galanin peptide concentrations were increased 7-fold. Hypothalamic concentrations of galanin peptide were also increased in hGHRH transgenic mice, but were not greater than those in nontransgenic siblings until 4 months of age. In contrast, no significant differences in galanin peptide concentrations of the neurointermediate lobes were evident. Galanin mRNA concentrations in the anterior pituitary of 6-month-old transgenic mice were increased 4-fold. In conclusion, 1) galanin peptide concentrations in the anterior pituitary gland and hypothalamus are increased in hGHRH transgenic mice compared to those in nontransgenic siblings, whereas galanin peptide concentrations in the neurointermediate lobe are not different; 2) pituitary galanin mRNA concentrations are increased 4-fold in 6-month-old transgenic mice; and 3) the development of pituitary hyperplasia is correlated to the increase in galanin mRNA and peptide concentrations.

Synergistic interactions between Pit-1 and other elements are required for effective somatotroph rat growth hormone gene expression in transgenic mice.

The role of the pituitary-specific POU-domain protein, Pit-1, in GH gene activation has been established by in vitro analyses and by the observation that mutations affecting the Pit-1 genomic locus result in genetically transmitted dwarfism. To define the quantitative contribution of the two Pit-1 response elements and the potential role of other factors in GH gene activation, we systematically assessed the ability of a series of GH promoter regions to activate transgenes in the mouse anterior pituitary gland. These studies revealed that the two GH Pit-1 binding sites are necessary, but not sufficient, for efficient transcriptional activation. Transgenes containing information including only these cis-active regions are expressed at extremely low levels in the pituitary glands of transgenic mice. The addition of 35 base pairs of 5'-flanking information, contributing other elements including a thyroid hormone/retinoic acid response element, results in much higher levels of transgene expression. Sequences located upstream of this segment contribute a further 5- to 10-fold activation. Thus, while Pit-1 is required for GH gene activation, it alone can only direct minimal expression in transgenic animals. Rather, synergistic interactions between other promoter elements and Pit-1 appear to be required for expression of the transgenes at approximately the 100-fold higher levels that are characteristic of somatotrophs, and are therefore likely to be critical components of somatotroph-specific expression of the GH gene.

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). The activities of acid phosphatase, alkaline phosphatase and beta-glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular delta 5-3 beta-HSD activity was higher in transgenic than in normal mice, while testicular 17 beta-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of beta-glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.

A mouse mammary tumor virus mammary gland enhancer confers tissue-specific but not lactation-dependent expression in transgenic mice.

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) is known to contain a number of transcriptional regulatory elements, including glucocorticoid response elements. In this study, we showed that a mammary gland/salivary gland enhancer found in the LTR of this virus directs expression of a heterologous promoter to both virgin and lactating mammary glands in transgenic mice. Using transgenic mice containing hybrid gene constructs with various deletions of the LTR sequences linked to marker genes, we also showed that the dramatic increase in MMTV expression that occurs during lactation is due to the glucocorticoid response elements. Thus, the MMTV LTR encodes two distinct elements, both of which are required for a high level of expression in lactating mammary glands.

A Heterologous Hormone Response Element Enhances Expression of Rat β-Casein Promoter-Driven Chloramphenicol Acetyltransferase Fusion Genes in the Mammary Gland of Transgenic Mice

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Restriction of neuroblastoma to the prostate gland in transgenic mice.

Male transgenic mice that carry a construct containing 5'-flanking sequences of the gp91-phox gene linked to the early region of the simian virus 40 (SV40) genome reproducibly develop tumors arising from the prostate gland. As gp91-phox is expressed exclusively in terminally differentiating hematopoietic cells of the myelomonocytic lineage, the induction of tumors arising from the prostate gland was unexpected. These lesions appear to be due to a novel transcription signal that was generated during the construction of the transgene. Surprisingly, the histopathological and biochemical properties of the tumor are diagnostic of neuroblastoma rather than of adenocarcinoma of the prostate gland. Tumors produce SV40 T antigen and isoforms of neural cell adhesion molecule characteristic of neuronal cells, and they occur in a testosterone-independent manner. Microscopic examination of prostate glands from young transgenic mice reveals the presence of small lesions arising outside of the prostate gland epithelium, which is consistent with the diagnosis of neuroblastoma and further distinguishes this tumor from prostatic adenocarcinoma. Prostate gland tumors occur in all male animals of susceptible lines carrying the gp91-phox promoter/SV40 early-region transgene. However, variability in the time at which gross tumors appear and the presence of cells expressing T antigen prior to tumorigenesis suggest that somatic events in addition to T-antigen production are required for the development of a malignancy. The extraordinary restriction of the site of tumorigenesis in these animals indicates the presence in the prostate gland of a novel, tissue-specific neuroectodermal cell of origin. These transgenic animals provide a model system for the study of neuroectodermal malignancies.

Restriction of neuroblastoma to the prostate gland in transgenic mice.

Male transgenic mice that carry a construct containing 5'-flanking sequences of the gp91-phox gene linked to the early region of the simian virus 40 (SV40) genome reproducibly develop tumors arising from the prostate gland. As gp91-phox is expressed exclusively in terminally differentiating hematopoietic cells of the myelomonocytic lineage, the induction of tumors arising from the prostate gland was unexpected. These lesions appear to be due to a novel transcription signal that was generated during the construction of the transgene. Surprisingly, the histopathological and biochemical properties of the tumor are diagnostic of neuroblastoma rather than of adenocarcinoma of the prostate gland. Tumors produce SV40 T antigen and isoforms of neural cell adhesion molecule characteristic of neuronal cells, and they occur in a testosterone-independent manner. Microscopic examination of prostate glands from young transgenic mice reveals the presence of small lesions arising outside of the prostate gland epithelium, which is consistent with the diagnosis of neuroblastoma and further distinguishes this tumor from prostatic adenocarcinoma. Prostate gland tumors occur in all male animals of susceptible lines carrying the gp91-phox promoter/SV40 early-region transgene. However, variability in the time at which gross tumors appear and the presence of cells expressing T antigen prior to tumorigenesis suggest that somatic events in addition to T-antigen production are required for the development of a malignancy. The extraordinary restriction of the site of tumorigenesis in these animals indicates the presence in the prostate gland of a novel, tissue-specific neuroectodermal cell of origin. These transgenic animals provide a model system for the study of neuroectodermal malignancies.

Studies on the thyroid in transgenic mice expressing the genes for human and bovine growth hormone

The thyroid glands of transgenic mice (TM) expressing the genes for human (h) and bovine (b) growth hormone (GH) were studied. The percentages of larger follicles in hGH TM of either sex were significantly greater than in the corresponding normal littermates, and follicles ranging up to 350 microns in diameter were present in male hGH TM. In contrast, thyroid follicles were only slightly enlarged in male bGH TM, and were unchanged in female bGH TM. The serum concentrations of T4 were significantly decreased in male bGH TM and not altered in the other groups. Serum concentrations of T3 were slightly, but significantly increased in female hGH TM and female bGH TM, but were unaffected in male TM of either type. Since the principal difference between these foreign GHs in rodents is the additional lactogenic activity of human GH, these results may indicate that the effects of prolactin can influence the development of the thyroid.

A salivary amylase transgene is efficiently expressed in liver but not in parotid gland of transgenic mice

Two distinct mouse amylase cDNAs, corresponding to the genes Amy-1.1 and Amy-1.2, have been isolated from a YBR/Ki parotid cDNA library. A cosmid clone containing the intact Amy-1.1 gene from strain YBR/Ki, including both the parotid and liver promoters, was transferred to the germ line of C57BL/6J mice. Two independent transgenic lines were produced. The transferred genes are organized as a 4-copy autosomal locus in line Tg518 and an 8-copy Y-linked locus in line Tg2736. Serum of both transgenic lines contained high levels of the AMY1B isozyme encoded by the transferred gene. Transcripts were detected in liver and, at a lower level, in several other tissues including white and brown fat. The anticipated expression in parotid was not observed. Constructs containing 270 or 540 bp of the 5' flanking region of the parotid promoter, cloned upstream of the chloramphenicol acetyltransferase (CAT) structural gene, were also not expressed in transgenic mice. The results suggest that sequences located more than 5 kb upstream of the Amy-1 parotid promoter and/or more than 10 kb downstream from the structural gene are required for parotid-specific expression. The results also demonstrate that non-parotid sources can produce a normal level of AMY1 in serum. Liver is the probable source of AMY1 in serum of these transgenic mice.

A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.

Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.