Biomedical subjects

F J DeMayo

Publications and source records attributed to F J DeMayo.

At least 55 records · Page 3Linked to original sources

PubMed1997

Ligand-inducible and liver-specific target gene expression in transgenic mice.

Transgenic mice have been used as models for tissue-specific gene regulation and to examine the molecular and cellular effects of altered expression of specific gene in disease processes such as tumorigenesis. Because of the deleterious effects of constitutive expression of transgenes, which frequently result in prenatal or postnatal death, only a limited number of disease models have been established in transgenic mice. We report an inducible binary transactivation system that permits the control of transgene expression in a tissue-specific and inducible fashion in mice. In this system, transcription of the target transgene is kept silent until turned on by the administration of an exogenous compound. We also demonstrate that expression level of the target gene can be induced three to four orders of magnitude and can be controlled by the administrated compound in a dose-dependent manner.

Animals↗

PubMed1997

Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro.

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.

Animals↗

PubMed1997

Cyclin-dependent kinase inhibitor expression in pulmonary Clara cells transformed with SV40 large T antigen in transgenic mice.

Expression of cell cycle regulatory genes in mouse lung was investigated in transgenic models for Clara cell transformation. Clara cells were transformed by generating transgenic mice in which the SV40 large T antigen was expressed under the control of the mouse Clara cell M(r) 10,000 protein promoter. The resulting lung tumors express the large T antigen in normal Clara cells and in tumors, and these tumors express reduced levels of CC10 mRNA. The expression of cell cycle regulatory protein, p53, and the cyclin-dependent kinase inhibitors was analyzed by Northern blot analysis and in situ hybridization throughout the progression of Clara cell transformation in the lung. Increases in specific cyclin-dependent kinase inhibitor steady-state mRNA levels were detected in p15, p18, p27, and p57 during tumor progression. The expression of p15, p57, and p21 mRNAs were verified by in situ hybridization. Using this approach, regulatory genes have been identified that may be involved in the regulation of Clara cell differentiation.

Adenocarcinoma↗

PubMed1996

Metastatic prostate cancer in a transgenic mouse.

We have previously reported the development of a transgenic mouse model for prostate cancer derived from PB-Tag transgenic line 8247, henceforth designated the TRAMP (transgenic adenocarcinoma mouse prostate) model. We now describe the temporal and spatial consequences of transgene expression and report the identification and characterization of metastatic disease in the TRAMP model. TRAMP mice characteristically express the T antigen oncoprotein by 8 weeks of age and develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastases can be detected as early as 12 weeks of age. The common sites of metastases are the periaortic lymph nodes and lungs, with occasional metastases to the kidney, adrenal gland, and bone. By 28 weeks of age, 100% harbor metastatic prostate cancer in the lymph nodes or lungs. We have also demonstrated the loss of normal E-cadherin expression, as observed in human prostate cancer, as primary tumors become less differentiated and metastasize. The TRAMP model provides a consistent source of primary and metastatic tumors for histopathobiological and molecular analysis to further define the earliest molecular events involved in the genesis, progression, and metastasis of prostate cancer.

Adenocarcinoma↗

PubMed1996

Reproductive phenotpes of the progesterone receptor null mutant mouse.

Although progesterone has been traditionally associated with the establishment and maintenance of mammalian pregnancy, a number of studies have implicated physiological roles of this steroid hormone in other reproductive events. At present most of the downstream molecular and cellular mechanisms by which progesterone exerts its effects are unclear; however, the progesterone signal is known to be mediated initially by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most tissues studied, the PR is induced by ovarian estrogen via the estrogen receptor (ER), thereby implying that many of the observed reproductive physiological responses attributed to PR could conceivably be due to the combined effects of progesterone and estrogen. Therefore, to define clearly the distinct roles of progesterone and estrogen in vivo and to understand better progesterone function in a physiological context, we recently have generated a novel mouse strain in which both forms of the PR were ablated using gene targeting/embryonic stem cell techniques. Surprisingly, both male and female embryos, homozygous for the PR null mutation, developed to adulthood at the normal Mendelian frequency with no deviation in the sex ratio. Although developmental defects have yet to be detected in the adult male PR homozygote, extensive reproductive abnormalities were observed in the female. The reproductive phenotypes consisted of an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development and an impairment in the induction of a sexual behavioral response. Collectively, these results provide direct in vivo evidence for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure female fertility. Finally, we believe that this animal model will be an invaluable tool in exploring the effects of progesterone in physiological systems other than reproduction and may, in the future, help to redefine progesterone not just as a sex steroid hormone but also as a key regulator of diverse physiological processes.

Animals↗

PubMed1996

Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX).

This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.

Animals↗

PubMed1996

Immunohistochemical localization of mouse Clara cell 10-KD protein using antibodies raised against the recombinant protein.

To investigate the developmental regulation of the mouse Clara cell 10-KD protein (mCC10), we raised an antibody against the recombinant mCC10 protein. The coding region for the mature mCC10 protein was placed in frame with the glutathione-S-transferase gene in the pGEX2-T bacterial expression vector. The GST-mCC10 fusion protein was expressed in E. coli DH5 alpha cells. The fusion protein was purified and eluted using glutathione-Sepharose beads. The GST-mCC10 fusion protein was injected into rabbits to raise antibodies. The rabbit anti-mCC10 antibody was tested by immunoblot analysis using both purified protein as well as extracts of lung, liver, and uterus. The antibodies produced were used in immunohistochemistry and immunoelectron microscopy to detect the cellular localization of this protein in the above organs. This anti-mCC10 antibody will be useful for future investigation of the developmental biology of the lung.

Animals↗

PubMed1996

Dopamine requires the unoccupied progesterone receptor to induce sexual behavior in mice.

Using the recently generated mutant mice strain (PRKO) carrying a null mutation for the progesterone receptor (PR) gene by gene targeting, we examined the critical role of PR as a coordinator of key regulatory events involved in the steroid hormone and dopamine-facilitated sexual behavior in female mice. In vitro one-point binding analyses of estradiol benzoate (EB)-induced cellular PRs and immunohistochemistry of PR in the mediobasal hypothalamus demonstrated a reduction in binding in the homozygous females, equivalent to background levels seen in EB-unresponsive tissue. The biochemical findings correlated well with the behavioral observations, with the wild type females exhibiting high levels of lordosis, while the homozygous females showed minimal lordosis in response to mating by male mice. As a critical validation of our earlier studies on ligand-independent activation of PRs by dopamine, we examined the facilitation of sexual behavior by a dopamine agonist in the null mutants. Wild type females having the full complement of PRs exhibited high levels of lordosis, while the homozygous females showed minimal lordosis in response to dopamine. To determine whether this reduced response was due to a general lack of ability to express lordosis, mice were treated with another neurotransmitter, serotonin. No significant difference in the serotonin-facilitated lordosis response was observed between the wild type and the homozygous females. We conclude that multiple signal transduction pathways coexist in the neuroendocrine system for reproductive behavior, with PR acting as a transcriptional mediator for dopamine, as well as progesterone, to achieve integration of neural communication in the central nervous system.

Animals↗

PubMed1995

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival.

Animals↗

PubMed1995

cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis.

Transient transfection and murine germ line gene transfer analysis was used to determine the regions of DNA necessary to confer the appropriate level and cell specificity of the expression of the gene coding for the murine Clara cell 10-kDa protein, mCC10. To identify the cis-acting elements involved in the regulation of mCC10 gene, different lengths of the 5'-flanking sequence were ligated to the bacterial chloramphenicol acetyltransferase gene for transient transfection to H441 cells (human lung adenocarcinoma cell line). The corresponding sequences were also fused to the human growth hormone gene and transferred to the murine genome for an in vivo analysis of mCC10 promoter activity. The results of the transient transfection analysis identified the region from -166 to -124 of the 5'-flanking region of the mCC10 gene as necessary for the expression of this gene in H441 cells. The transgenic mouse analysis confirmed that the 166 base pairs of 5'-flanking DNA was sufficient to confer cell-specific expression. However, the transgenic mouse analysis also showed that, to achieve the full quantitative level of transgene (human growth hormone) expression, regions between -803 and -166 base pairs of the 5'-flanking sequences are required for maximum expression of mCC10 gene promoter activity.

Animals↗

PubMed1994

Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) genes encode transcription factors belonging to the orphan subfamily of the steroid/thyroid hormone receptor superfamily. Two COUP-TF counterparts have been cloned from mouse. In an attempt to study the function of these genes in the developing central nervous system (CNS), the spatiotemporal expression patterns of the two mouse genes have been examined by in situ hybridization. Both genes are widely expressed in the developing CNS, with patterns that are overlapping yet distinct from each other. The differential expression of murine COUP-TFI and -II in the diencephalon is striking in that high levels of expression from each gene are confined to specific segmental compartments--the neuromeres. Our results suggest that murine COUP-TFs may play important roles in the development and differentiation of the CNS, including the specification of diencephalic neuromeres.

Amino Acid Sequence↗

PubMed1994

The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice.

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Androgen-Binding Protein↗

PubMed1993

Cloning and characterization of the mouse Clara cell specific 10 kDa protein gene: comparison of the 5'-flanking region with the human rat and rabbit gene.

The mouse Clara Cell 10 kiloDalton (kDa) protein (mCC10) cDNA was used to isolate a recombinant phage containing the mCC10 gene sequence in a 14 kilobase (kb) insert from a mouse genomic library. A total of 7.7 kb of this clone was sequenced. The sequenced region included: 3.3 kb of 5'-flanking region, 4.2 kb intragenic sequence and 0.2 kb of DNA flanking the 3' end of the gene. Computer assisted sequence analysis identified potential cis acting response elements for the glucocorticoid receptor, hepatocyte nuclear factor (HNF3) and octamer (Oct1) binding protein. The presence of B1 murine repetitive sequence also has been identified in a similar position reported in rat CC10 5'-flanking sequence. As with the rat CC10, the mCC10 5'-flanking region also contains deletions of a 2.1 kb and a 0.3 kb sequence present in the rabbit uteroglobin gene, these regions are reported to contain a cluster of glucocorticoid/progesterone receptor binding sites and estrogen receptor binding sites, respectively.

Animals↗

PubMed1993

Cloning and tissue-specific expression of the cDNA for the mouse Clara cell 10 kD protein: comparison of endogenous expression to rabbit uteroglobin promoter-driven transgene expression.

Uteroglobin (UG) is a hormonally regulated secretory protein produced in the lung and urogenital system of rabbits. It is homologous to rat and human Clara cell 10 kD protein (CC10); however, there are significant differences in the tissue-specific expression between these species. Mouse CC10 (mCC10) protein has been less well characterized. In this study, we cloned and sequenced the cDNA encoding the mCC10 protein. The mouse cDNA showed 90, 52, and 51% amino acid homology to rat and human CC10 and rabbit UG cDNA, respectively. The cellular and tissue-specific expression of mCC10 was examined in adult and developing mice. Endogenous mCC10 expression was compared with transgenic mice expressing a fusion gene of the rabbit 3.3 kb UG promoter linked to human growth hormone (hGH) as an easily detectable marker. Northern blot analysis detected mCC10 mRNA only in the lung. hGH mRNA was detected in the lung in levels similar to the endogenous mCC10 transcripts. However, it was also present in trace quantities in the uterus and ovary of normal adult female mice and in the epididymus of adult male mice. hGH and mCC10 proteins were identified in the trachea and lung, where they were localized to Clara cells. Ultrastructurally, hGH was present in secretory granules in the Clara cell cytoplasm and appeared to be secreted into the airways. hGH was initially detectable in 16 day gestation developing mice; however, CC10 was not detectable until the eighteenth day of gestation. We have created an attractive model for comparing the cis-acting DNA elements governing the interspecies variation in tissue-specific expression of CC10.

Amino Acid Sequence↗

PubMed1992

Identification of a new brain-specific transcription factor, NURR1.

We have identified and cloned a novel member of the nuclear receptor superfamily. The cDNA was isolated from a mouse brain cDNA library and encodes a protein 598 amino acids in length with a predicted mol wt of 66 kilodaltons. The amino acid sequence of the protein is closely related to an additional family member and immediate early gene product, Nur77, and the novel factor is referred to as Nurr1 (Nur-related factor 1). The relationship between Nurr1 and Nur77 suggests that these proteins constitute an additional subfamily within the nuclear receptor superfamily. Like Nur77, the expression of Nurr1 is induced by membrane depolarization of PC12 cells. However, while Nur77 shows an early transcriptional response to nerve growth factor stimulation, the failure of Nurr1 to respond to this agent suggests a differential selectivity of the two proteins in terms of their transcriptional responses to specific stimuli. Finally, both proteins are differentially expressed during development and in tissues of the adult mouse. Unlike Nur77, Nurr1 appears to be predominantly located in brain tissue, suggesting a primary role for this putative transcription factor in regulation of gene expression in the central nervous system.

Action Potentials↗

PubMed1991

Production of transgenic mice from cryopreserved fertilized ova.

Cryopreserved fertilized mouse ova were used to generate transgenic mice via micromanipulation. Five-DNA constructions were injected into a total of 1,052 cryopreserved ova, of which 683 (65%) survived the injection and were transferred into recipients. Of 35 recipients, 66% became pregnant and littered a total of 88 pups. As controls, these DNA constructions were also injected into 1,123 fresh ova, of which 744 (66%) survived and were transferred. Of 42 recipients, 79% became pregnant and littered a total of 167 pups. That is, 22% of fresh ova that were transferred developed into live pups, whereas only 13% of cryopreserved ova did so. Of the pups born, 42 of the 167 (25%) produced from fresh ova were transgenic, and 28 of the 88 (32%) produced from cryopreserved ova were transgenic. In terms of the injected ova that had been transferred, 5.6% of the 744 fresh and 4.1% of the 683 frozen ova developed into transgenic mice. These data indicate that the efficiency of production of transgenic mice from cryopreserved ova is close to that from fresh ova. That observation and the fact that cryopreserved ova allow more efficient utilization of animals suggest that cryopreserved ova can be used instead of fresh ova to produce transgenic mice.

Animals↗

PubMed1991

Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

Animals↗

PubMed1991

Expression and regulation of the rabbit uteroglobin gene in transgenic mice.

The rabbit uteroglobin (UG) gene, with varying lengths of 5' flanking sequence, was introduced into the mouse genome to investigate the DNA sequences required for tissue-specific expression and regulation by steroid hormones. The pattern of expression and steroid hormone regulation of the transgene was compared to the expression and regulation of the endogenous mouse UG-like gene. In the rabbit, UG is induced in the uterus by progesterone and is expressed constitutively in the lungs, where it is weakly regulated by glucocorticoids. Genomic DNA fragments containing the complete UG-coding sequence with 4.0 (UG4.0), 3.0 (UG3.0), 2.3 (UG2.3), or 0.6 (UG0.6) kilobases of 5' flanking sequence were used to establish lines of transgenic mice. Expression of UG mRNA was observed in the lungs of UG4.0 (2/4 lines), UG3.0 (4/4 lines), UG2.3 (1/2 lines), and UG0.6 (4/4 lines) mice. Uterine expression was observed in UG3.0 (3/4 lines), UG2.3 (1/2 lines), and UG0.6 (2/4 lines). In the lungs of UG3.0 and UG2.3 mice, RNA expression was stimulated by treatment with dexamethasone. In the one line of UG3.0 mice examined, UG was regulated by ovarian steroids in the uterus. The endogenous mouse UG-like gene showed the major site of expression to be in the lung. Unlike the transgene, the endogenous gene was more strongly stimulated by glucocorticoids. Thus, we conclude that the cis elements needed for pulmonary expression of UG are contained within the UG2.3 fragment used to generate transgenic mice, but that other elements are required for full glucocorticoid regulation. Also, the transgene did not show the full uterine expression observed in the rabbit, but regulation by the ovarian steroids was observed.

Animals↗