Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice.

Angiotensinogen is the precursor of the potent vasoactive peptide angiotensin II, and is therefore an important determinant of blood pressure and electrolyte homeostasis. In order to map the tissue-specific and inducible enhancer elements governing angiotensinogen gene expression in transgenic mice, we constructed minigenes containing either 0.75 kb or 4 kb or 5' flanking DNA from the BALB/c angiotensinogen gene. Sequences necessary and sufficient to mediate induction by glucocorticoids, oestrogen and bacterial endotoxin were contained on the minigene bearing 0.75 kb of DNA upstream of the capsite. This construct was also able to confer tissue specificity in the majority of organs producing angiotensinogen. In the testis and salivary gland, differences between the donor (BALB/c) and recipient (Swiss) strains were responsible for the apparently aberrant expression of the minigene constructs. The genetic lesion responsible for these expression polymorphisms has been characterized using recombinant inbred mice. An EcoRI restriction fragment length polymorphism which co-segregates with the angiotensinogen expression phenotypes into many inbred mouse strains is also described.     

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.     

Developmental regulation of human gamma-globin genes in transgenic mice.

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

Function and regulation of gastrin in transgenic mice: a review.

The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.     

Developmental regulation for collagen II gene expression in transgenic mice

In order to evaluate the involvement of the type II collagen regulatory sequences in development, we have injected a construct containing a toxin gene under the control of the rat type II collagen promoter and enhancer. The construct, pDAS10-DTA, contained the diphtheria toxin A chain gene under the control of type II collagen sequences which had been used previously to target cartilagenous tissues in transgenics. Inspection of developing fetuses at various stages of gestation revealed a high number of aborted implants as well as abnormally developing fetuses. These abnormal fetuses were of small size, had shortened and underdeveloped limbs, cleft palates, and generally resembled a phenotype similar to chondrodystrophic mice. Histological comparisons of normal and abnormal fetuses indicated a reduced amount of extracellular matrix surrounding chondrocytes, and a disorganized appearance of the tissue. These results suggest that the expression of the toxin has occurred in chondrocytes and altered the survival and development of the transgenic mice. These results also indicate that the promoter and enhancer sequences contained in the transgene controlled the developmental expression of the type II collagen gene expression.     

Characterization of transgenic mice with overexpression of spermidine synthase

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.     

Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring

A fusion plasmid, pMK, containing the mouse metallothionein-I promoter/regulatory region joined to the structural gene of herpesvirus thymidine kinase, was introduced into mice by microinjection into fertilized eggs followed by reinsertion of the eggs into foster mothers. Fifteen percent (10 of 69) of the mice developing from this procedure carried pMK sequences. Seven of these mice expressed high levels of viral thymidine kinase in the liver. This enzyme is inducible by heavy metals, as indicated by assay of thymidine kinase activity following sequential partial hepatectomies with or without cadmium treatment. However, glucocorticoid treatment has been ineffective in all transgenic mice tested. The pMK sequences are extensively methylated at a variety of restriction sites, indicating the existence of a de novo methylation enzyme. We have analyzed the inheritance of pMK sequences and their expression in several pedigrees. These fusion genes are inherited as though they were integrated into a single chromosome; however, their expression may be extinguished, diminished or enhanced in the offspring relative to that of the parent. In some animals there is a correlation between changes in DNA methylation and expression of these fusion genes.     

Differential regulation of the N-myc gene in transfected cells and transgenic mice.

The N-myc gene is expressed specifically in the early developmental stages of numerous cell lineages. To assay for sequences that could potentially regulate N-myc expression, we transfected constructs that contained murine N-myc genomic sequences linked to a reporter gene and genomic clones that contained the complete human or murine N-myc genes into cell lines that either express or do not express the endogenous N-myc gene. Following either transient or stable transfection, the introduced N-myc sequences were expressed regardless of the expression status of the endogenous gene. In contrast, when the clones containing the complete human N-myc gene were introduced into the germline of transgenic mice, expression in some transgenic lines paralleled the tissue- and stage-specific expression of the endogenous murine gene. These findings demonstrate differences in the regulation of N-myc genes in recipient cells following in vitro versus in vivo introduction, suggesting that early developmental events may play a role in the regulation of N-myc expression.     

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.