Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Androgens, progestins, and glucocorticoids induce follicle-stimulating hormone beta-subunit gene expression at the level of the gonadotrope

FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LbetaT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHbeta promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHbeta promoter where six putative hormone response elements exist. The -381 site is critical for FSHbeta induction by all three steroid hormones, whereas the -197 and -139 sites contribute to maximal induction. Interestingly, the -273 and -230 sites are also necessary for androgen and progestin induction of FSHbeta, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHbeta promoter, in vivo, and specifically bind the -381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHbeta by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotrope.     

Introduction and expression of the human Bs-globin gene in transgenic mice.

Owing to the episodic and unpredictable nature of the sickling crisis, many aspects of the disease sickle cell anemia have resisted in vivo analysis. The lack of an animal model has hindered the pathophysiological investigation of this disease, as well as deterred the development of pharmacological therapies. The transgenic mouse system offers a new means for creating animals that make a specified mutant gene product, and we have used this system to create a series of mice that contain the human beta s-globin gene. These animals express this gene in the appropriate tissues and at the same point in development as the adult mouse globin genes are expressed. We have crossed the human beta s-containing transgenic mice with a beta-thalassemic mouse line and examined the hemoglobins produced by these mice. Their red cells contain 10% mouse alpha/human beta s hybrid hemoglobin, which partially corrects the thalassemic phenotype of the homozygous beta-thalassemic animals. Though the red cells do not sickle, other properties of the human beta s gene in these mice indicate the potential for the eventual development of a transgenic animal model for sickle cell anemia.     

Chemical synthesis of peptide fragments of the hormone-specific beta-subunit of human follicle-stimulating hormone

In order to determine the specific antigenic determinants of human follicle-stimulating hormone (hFSH), hFSH-beta peptides with amino acid residues 33-49 (V2), 95-118 (V3), 76-118 (V3 + 1/2 C2), 1-33 (V1 + C1), 22-33 (1/2C1), and 95-107 (V3 + 1/4C2) according to the nomenclature of Stewart and Stewart [Stewart, M., & Stewart, F. (1977) J. Mol. Biol. 116, 175] as well as additional peptides with the residues 93-107, 91-107, 89-107, 87-107, and 85-107 were chemically synthesized. The peptides were examined in radioimmunoassay systems of FSH, luteinizing hormone (LH), or human chorionic gonadotropin (hCG). V3 + 1/2C2 and V1 + C1 showed immunological activity, whereas the other peptides did not. Antibodies were raised in rabbits against these peptides and examined for specific binding with hFSH, LH, thyroid-stimulating hormone (TSH), and hCG. V3 + 1/2C2 as well as V1 + C1 produced antisera, which specifically bound hFSH, hLH, and hTSH, indicating that the amino acid sequences contained in hFSH-beta peptides V3 + 1/2C2 and V1 + C1 share common antigenic sites with hLH and hTSH. Antisera were produced in rabbits against hFSH-beta, against reduced and S-aminoethylated hFSH-beta (AE-FSH-beta), and against AE-FSH-beta coupled to hemocyanin. Reduced and S-aminoethylated beta-subunit of FSH-beta coupled with hemocyanin produced antisera in rabbits that specifically bound only hFSH and not hLH, hTSH, or hCG.     

Tissue-specific expression of the human renin gene in transgenic mice

Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5'-flanking sequence and 1.2 kb of 3'-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.     

New AccI polymorphism in the follicle-stimulating hormone beta-subunit gene and its prevalence in three Southeast Asian populations.

A thymine-cytosine substitution was identified in exon 3 (codon 76, TAT to TAC) of the human follicle-stimulating hormone (FSH) beta-subunit gene. The nucleotide change led to creation of an AccI digestion site. The frequencies of the A allele (with AccI site) in Chinese (n = 201), Malays (n = 168) and Indians (n = 132) were 0.358, 0.333 and 0.402, respectively. The new FSH beta-subunit marker may be useful in gene tracking and association studies.     

UV activation of human immunodeficiency virus gene expression in transgenic mice.

Human immunodeficiency virus (HIV) infection is associated with a clinical latency of as long as 10 years before the development of disease. One explanation for this delay is the requirement of cofactors such as other DNA or RNA viruses, cytokines critical for immune modulation, or environmental UV light. At least in tissue culture studies, these agents are capable of inducing HIV gene expression in cell lines which either harbor the entire viral genome or contain a reporter gene under the control of the viral long terminal repeat regulatory region. The role of these cofactors in terminating clinical latency and inducing disease has been difficult to ascertain because of the lack of an appropriate animal model. We now report that UV light can markedly induce HIV gene expression in transgenic mice carrying both the cis-acting (long terminal repeat) and trans-acting (the tat gene) elements which are essential for viral transactivation and replication in infected cells. Our finding may explain the clinical observations that cutaneous lesions in HIV-infected individuals are often seen in the sunlight exposed areas of the skin, including the face and neck.     

Regulation of the human apolipoprotein AIV gene expression in transgenic mice

The apolipoprotein (Apo) AI-CIII-AIV gene cluster has a complex pattern of gene expression that is modulated by both gene- and cluster-specific cis-acting elements. In particular the regulation of Apo AIV expression has been previously studied in vivo and in vitro including several transgenic mouse lines but a complete, consistent picture of the tissue-specific controls is still missing. We have analysed the role of the Apo AIV 3' flanking sequences in the regulation of gene expression using both in vitro and in vivo systems including three lines of transgenic mice. The transgene consisted of a human fragment containing 7 kb of the 5' flanking region, the Apo AIV gene itself and 6 kb of the 3' flanking region (-7+6 Apo AIV). Accurate analysis of the Apo AIV mRNA levels using quantitative PCR and Northern blots showed that the 7+6 kb Apo AIV fragment confers liver-specific regulation in that the human Apo AIV transgene is expressed at approximately the same level as the endogenous mouse Apo AIV gene. In contrast, the intestinal regulation of the transgene did not follow, the pattern observed with the endogenous gene although it produced a much higher intestinal expression following the accepted human pattern. Therefore, this animal model provides an excellent substrate to design therapeutic protocols for those metabolic derangements that may benefit from variations in Apo AIV levels and its anti-atherogenic effect.     

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.     

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.