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PubMed2003

Precision-cut tissue slices from transgenic mice as an in vitro toxicology system.

In these experiments precision-cut tissue slices from two existing transgenic mouse strains, with transgenes that couple promoting or binding elements to a reporter protein, were used for determination of reporter induction. This approach combines the power of transgenic animals with the practicality of in vitro systems to investigate the biological impact of xenobiotics. Additionally, the normal cellular architecture and heterogeneity is retained in precision-cut tissue slices. Two transgenic mouse strains, one of which couples the promoting region of CYP 1A1 to beta-galactosidase, and another which couples two forward and two backward 12-O-tetradecanoyl phorbol-13-acetate (TPA) repeat elements (TRE) to luciferase (termed AP-1/luciferase), were used to determine the feasibility of this approach. Precision-cut kidney and liver slices from both transgenic strains remain viable as determined by slice K(+) ion content and LDH enzyme release. Liver slices harvested from the CYP 1A1/beta-galactosidase transgenic mice exhibit a 14-fold increase in beta-galactosidase activity when incubated with beta-napthoflavone for 24 h. Kidney and liver slices obtained from the AP-1/luciferase transgenic mice demonstrate induction of luciferase (up to 2.5-fold) when incubated with phorbol myristate acetate (PMA or TPA) up to 4 h. These data indicate that precision-cut tissue slices from transgenic mice offer a novel in vitro method for toxicity evaluation while maintaining normal cell heterogeneity.

Animals↗
PubMed2001

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice.

The transforming growth factor alpha (TGFalpha) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGFalpha and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGFalpha is evident in the transgenic mice overexpressing TGFalpha in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGFalpha transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGFalpha double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGFalpha, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.

Animals↗
PubMedDate not supplied

Mechanism of portal venous tolerant long-term MHC Class I L(d)-specific skin graft survival in transgenic 2CF1 mice.

BACKGROUND: Administration of alloantigen via the portal vein (PV) in non-transgenic animals has been shown to promote immunologic tolerance and enhance transplant allograft survival. The underlying mechanisms remain unclear. In 2C x dm2 F1 (2CF1) transgenic mice, the monoclonal antibody, 1B2, identifies specific 2C TCR transgenic CD8+ T cells that are cytotoxic against Class I MHC L(d). In these mice, the specific response by these cells to L(d+) skin grafts after PV administration of L(d+) antigen was determined. MATERIALS AND METHODS: Saline (control) or allogeneic C57BL/6 x BALB/c F1 (CB6F1) spleen cells (25 x 10(6)), which differ from 2CF1 only at L(d), were injected PV into 2CF1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these 2CF1 mice. Skin graft rejection was defined as >50% loss of the graft. Parallel experiments were performed in non-transgenic littermates [B6F1 (C57BL/6 x dm2)]. FACS analysis of 2CF1 peripheral blood for 1B2+, CD4+, and CD8+ T cells was performed 2 days before PV injection (9 days prior to skin grafting), 5 days after PV injection (2 days prior to skin grafting), and 7, 14, 21, 28, and 60 days after skin grafting. FACS analysis of nai;ve, saline control, and CB6F1 PV-treated 2CF1 thymocytes was also performed. Responsiveness of saline (control)-treated and PV-treated 2CF1 splenocytes was measured by in vitro cytotoxic T lymphocyte (CTL). RESULTS: All CB6F1 skin grafts were rejected in <14 days by PV saline controls. However, a single PV injection of donor L(d+) CB6F1 cells was sufficient to induce indefinite CB6F1 (L(d+)) skin allograft survival in 100% of non-transgenic B6F1 and transgenic 2CF1 (anti-L(d)) TCR transgenic recipients. FACS analysis of 1B2+ T cells demonstrated that PV injection of donor antigen followed by a CB6F1 skin graft led to a 70% decrease in peripheral donor-reactive 1B2+ CD8+ T cells by day 7, while central thymocytes were unchanged. CTL of 2CF1 splenocytes following PV CB6F1 demonstrated that they were hyporesponsive to L(d) compared to saline-treated 2CF1 splenocytes. Despite recovery of peripheral CD8+ T cells to near normal levels by 60 days post-transplantation, skin graft survival persisted indefinitely. CONCLUSIONS: Administration of specific PV antigen results in exquisite long-term L(d+) skin allograft acceptance. This tolerance induction is related to a significant peripheral deletion of donor-reactive 1B2+ CD8+ transgenic T cells and anergy of the residual T cells.

Adjuvants, Immunologic↗
PubMed1997

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C.

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100-1800 micrograms ml-1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100-400 micrograms ml-1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein.

Animals↗
PubMed2003

Increased efficiency of transgenic livestock production.

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.

Animals↗
PubMed2004

Macrophage-specific overexpression of human matrix metalloproteinase-12 in transgenic rabbits.

Increased matrix metalloproteinase-12 (MMP-12) has been implicated in atherosclerosis and many other inflammatory processes. To define MMP-12 functions in vivo, we generated transgenic rabbits that expressed human (h) MMP-12 gene under the control of a macrophage-specific promoter, the human scavenger receptor promoter. Two transgenic founder rabbits were found to have hMMP-12 transgene integration by Southern blot analysis. hMMP-12 mRNA was expressed in peritoneal and alveolar macrophages, and in tissues enriched in macrophages in transgenic rabbits. High levels of hMMP-12 protein were detected in the conditioned media of cultured peritoneal and alveolar macrophages from transgenic rabbits. Zymography showed that hMMP-12 secreted from macrophages possessed enzymatic activity toward beta-casein. To evaluate the expression of hMMP-12 in inflammatory sites, we used carrageenan-induced granulomas as an in vivo model for tissue macrophages and foam cells. Granuloma size in transgenic rabbits was significantly increased compared to that in control rabbits, and histological examination revealed that granulomas of transgenic rabbits were enriched in macrophages associated with increased hMMP-12 expression. We believe that this transgenic rabbit model with increased expression of hMMP-12 may become a useful model for further mechanistic studies of MMP-12 in inflammatory diseases and cancer invasion; it is also an ideal model for testing the in vivo action of MMP-12 inhibitors.

Animals↗
PubMed2002

The amplicon-plus system for high-level expression of transgenes in plants.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

Cysteine Endopeptidases↗
PubMed2000

Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells.

Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, alpha(v)beta5- and alpha(v)beta3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was 'trans-complementation' of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.

Adenoviridae↗
PubMed1997

Overexpression of insulin-like growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in transgenic mice.

Transgenic animals were developed to assess the role of insulin-like growth factor 1 (IGF-1) in skin growth, differentiation and organization, as well as its importance in tumor formation. Expression of a human IGF-1 cDNA was targeted to the interfollicular epidermis of transgenic mice using a human keratin 1 promoter construct (HK1). Transgenic animals (HK1.IGF-1 mice) could be identified at birth by early ear unfolding and excessive ear and skin growth compared to non-transgenic littermates. Further examination of the skin from these mice showed epidermal hyperplasia and hyperkeratosis, marked thickening of the dermis and hypodermis, and early hair follicle generation in newborns. The severity of this phenotype correlated with transgene expression both of which subsided with age. Adult HK1.IGF-1 mice developed spontaneous tumors following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and exhibited an exaggerated epidermal proliferative response following treatment with the tumor promoter compared to non transgenic littermates. Additionally, HK1.IGF-1 transgenic mice developed papillomas faster and in markedly greater numbers compared to non-transgenic littermates in standard initiation-promotion experiments. The data presented suggest an important role for IGF-1 in the process of multistage carcinogenesis in mouse skin.

Animals↗
PubMed2000

Hepatocellular carcinoma in WHV/N-myc2 transgenic mice: oncogenic mutations of beta-catenin and synergistic effect of p53 null alleles.

The intronless N-myc2 gene was originally identified as the major target of hepatitis virus insertion in woodchuck liver tumors. Here we report that transgenic mice carrying the N-myc2 gene controlled by woodchuck hepatitis virus (WHV) regulatory sequences are highly predisposed to liver cancer. In a WHV/N-myc2 transgenic line, hepatocellular carcinomas or adenomas arose in over 70% of mice, despite barely detectable expression of the methylated transgene in liver cells. Furthermore, a transgenic founder carrying unmethylated transgene sequences succumbed to a large liver tumor by the age of two months, demonstrating the high oncogenicity of the woodchuck N-myc2 retroposon. Stabilizing mutations or deletions of beta-catenin were found in 25% of liver tumors and correlated with reduced tumor latency (P<0.05), confirming the important role of beta-catenin activation in Myc-induced tumorigenesis. The ability of the tumor suppressor gene p53 to cooperate with N-myc2 in liver cell transformation was tested by introducing a p53-null allele into WHV/N-myc2 transgenic mice. The loss of one p53 allele in transgenic animals markedly accelerated the onset of liver cancer (P=0.0001), and most tumors of WHV/N-myc2 p53+/Delta mice harbored either a deletion of the wt p53 allele or a beta-catenin mutation. These findings provide direct evidence that activation of N-myc2 and reduction of p53 levels act synergistically during multistage carcinogenesis in vivo and suggest that different genetic pathways may underlie liver carcinogenesis initiated by a myc transgene. Oncogene (2000).

Animals↗
PubMed2002

Lung-specific expression of human mutant p53-273H is associated with a high frequency of lung adenocarcinoma in transgenic mice.

To investigate the tumorigenic potential of mutant p53 when selectively expressed in lung tissue, a transgenic mouse model was developed in which a mutant form of p53 (p53-273H) was placed under the transcriptional control of the lung-specific human surfactant protein C (SP-C) promoter. Two founder mice were identified, and a line of SP-C/p53-273H transgenic mice was established from one of the founders. Human p53-273H protein was detected specifically in lung tissue from transgenic mice. Malignant tumors, which were histologically characterized as adenocarcinomas, were observed in transgenic mice, with the earliest onset documented at 4 months of age. To further evaluate incidence and onset of tumor formation, transgenic mice (n=113) were sacrificed at age intervals ranging from 4-15 months. At 13-15 months of age, transgenic mice were significantly more likely to have lung tumors at necropsy than age-matched non-transgenic littermates (9 out of 39 (23%) versus 2 out of 35 (5.7%), chi(2) test, P=0.036). The SP-C/p53-273H transgenic mice described here thus represent a genetically defined model with which to study the role of p53 mutations in lung tumorigenesis, as well as the potential complementary contributions of other genetic alterations or environmental carcinogens to lung tumor development.

Adenocarcinoma↗
PubMed2005

Biological, cellular, and molecular characteristics of an inducible transgenic skin tumor model: a review.

The genetically initiated Tg.AC transgenic mouse carries a transgene consisting of an oncogenic v-Ha-ras coding region flanked 5' by a mouse zeta-globin promoter and 3' by an SV-40 polyadenylation sequence. Located on chromosome 11, the transgene is transcriptionally silent until activated by chemical carcinogens, UV light, or full-thickness wounding. Expression of the transgene is an early event that drives cellular proliferation resulting in clonal expansion and tumor formation, the unique characteristics now associated with the Tg.AC mouse. This ras-dependent phenotype has resulted in the widespread interest and use of the Tg.AC mouse in experimental skin carcinogenesis and as an alternative carcinogenesis assay. This review examines the general biology of the tumorigenic responses observed in Tg.AC mice, the genetic interactions of the ras transgene, and explores the cellular and molecular regulation of zeta-globin promoted transgene expression. As a prototype alternative model to the current long-term rodent bioassays, the Tg.AC has generated a healthy discussion on the future of transgenic bioassays, and opened the doors for subsequent models for toxicity testing. The further exploration and elucidation of the molecular controls of transgene expression will enhance the usefulness of this mouse and enable a better understanding of the Tg.AC's discriminate response to chemical carcinogens.

Animals↗
PubMed2006

Endothelial dysfunction in growth hormone transgenic mice.

Acromegaly [overproduction of GH (growth hormone)] is associated with cardiovascular disease. Transgenic mice overexpressing bGH (bovine GH) develop hypertension and hypercholesterolaemia and could be a model for cardiovascular disease in acromegaly. The aims of the present study were to investigate the effects of excess GH on vascular function and to test whether oxidative stress affects endothelial function in bGH transgenic mice. We studied the ACh (acetylcholine)-induced relaxation response in aortic and carotid rings of young (9-11 weeks) and aged (22-24 weeks) female bGH transgenic mice and littermate control mice, without and with the addition of a free radical scavenger {MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride]}. We also measured mRNA levels of eNOS (endothelial nitric oxide synthase) and EC-SOD (extracellular superoxide dismutase). Intracellular superoxide anion production in the vascular wall was estimated using a dihydroethidium probe. Carotid arteries from bGH transgenic mice had an impaired ACh-induced relaxation response (young, 46 +/- 7% compared with 69 +/- 8%; aged, 52 +/- 5% compared with 80 +/- 3%; P < 0.05), whereas endothelial function in aorta was intact in young but impaired in aged bGH transgenic mice. Endothelial dysfunction was corrected by addition of MnTBAP in carotid arteries from young mice and in aortas from aged mice; however, MnTBAP did not correct endothelial dysfunction in carotid arteries from aged bGH transgenic mice. There was no difference in intracellular superoxide anion production between bGH transgenic mice and control mice, whereas mRNA expression of EC-SOD and eNOS was increased in aortas from young bGH transgenic mice compared with control mice (P < 0.05). We interpret these data to suggest that bGH overexpression is associated with a time- and vessel-specific deterioration in endothelial function, initially caused by increased oxidative stress and later by other alterations in vascular function.

Acetylcholine↗
PubMed1997

Growth hormone-releasing hormone (GHRH)-GH-somatic growth and luteinizing hormone (LH)RH-LH-ovarian axes in adult female transgenic mice expressing human GH gene.

We have examined alterations in the hypothalamo-pituitary GH-somatic growth axis and the hypothalamo-pituitary LH-ovarian axis in a line of transgenic ICR mice expressing human GH (hGH) under the influence of the whey acid protein promoter. Transgenic female mice weighed twice as much as control females and were infertile. The size of the anterior pituitary (AP) was 1/3 that of the controls. In transgenic mice, acinar cells in the mammary and mandibular glands displayed hGH-immunoreactivity, and plasma hGH was detected by radioimmunoassay. In the medial basal hypothalamus (MBH) of transgenic females, the immunoreactive-GHRH level was decreased (P<0.01). There was a corresponding reduction in the number of GHRH-immunoreactive neurons in the arcuate nucleus (ARC) and in the immunostaining of GHRH nerve terminals in the median eminence. The level of somatostatin (SRIH) in the MBH was increased (P<0.05), and SRIH-immunoreactive neurons in the periventricular nucleus (PeV) were increased in size and number in transgenic mice. The MBH level of LHRH in transgenic animals was greater (P<0.01) than in controls, although there was no apparent difference in the number of LHRH-immunoreactive neurons or in LHRH level in the preoptic area. There are fewer SRIH- and LHRH-immunoreactive neurons in the ARC in transgenic mice. Cells in the AP for GH, PRL, and LH were fewer in transgenic mice. The ovary suffered disturbance of follicular development and of corpora lutea formation. These results demonstrate that chronic overproduction of hGH may profoundly affect the organization of the GHRH/SRIH-GH-somatic growth axis and the LHRH-LH-ovarian axis due to reduction of GHRH-, SRIH- and LHRH-neurons in the ARC and increase of SRIH-neurons in the PeV.

Animals↗
PubMed2002

Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration.

To obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results confirm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identified in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strand-annealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.

Base Sequence↗
PubMed1998

The 3'-untranslated region of the urokinase gene enhances the expression of chimeric genes in cultured cells and correlates with specific brain expression in transgenic mice.

Transgenic mice were previously described, carrying the cDNA of the human or murine protease urokinase-type plasminogen activator (uPA) while linked to the cell-specific promoter of the alphaA-crystallin gene. Surprisingly, these mice produced transgenic uPA in an ectopic manner specifically in the brain. Here we tested the possibility that this ectopic expression could have been contributed primarily by the uPA transgenic moiety. Several experimental approaches have been used. (a) Constructs consisting of uPA cDNA linked to the cell-specific promoters of the alphaA-crystallin or insulin genes yielded active uPA after transfection into cells where these promoters are thought to be inactive. (b) When reporter genes were inserted into these constructs between the promoter and the cDNA, the cDNA enhanced the chimeric reporter expression 5-50-fold. This effect was obtained upon stable or transient construct transfection into four different cell types. (c) Reporter enhancement also took place in the presence of the homologous uPA gene promoter. (d) Mapping of the cDNA through deletion-substitution analysis has detected fragments mediating positive or negative effects on reporter expression, all fragments residing in the 3'-untranslated region (3'UTR) of the uPA gene that was included in the cDNA. Some fragments exhibited cell-specific effects. One fragment (2002/2187) behaved like a classical transcriptional enhancer, enhancing reporter expression from different positions and orientations. (e) Transgenic mice have now been generated that carry a transgene consisting of the alphaA-crystallin promoter, the luciferase reporter gene and mouse uPA cDNA. Among four transgenic lines producing luciferase activity in the eye lens, three lines exhibited ectopic luciferase activity exclusively in the brain, where luciferase mRNA was localized through in situ hybridization. From these results we conclude that the 3'UTR of the uPA gene contains sequences capable of exerting variable effects on gene expression, including transcriptional enhancement. In addition, uPA cDNA correlates with transgenic brain expression. Therefore, we suggest that the 3'UTR of the uPA gene is involved in brain expression of the transgenes containing uPA cDNA as well as of the normal uPA gene.

Animals↗
PubMed2000

Overexpression of spermidine/spermine N-acetyltransferase in transgenic mice protects the animals from kainate-induced toxicity.

We recently generated a transgenic mouse line with activated polyamine catabolism through overexpression of spermidine/spermine N1-acetyltransferase (SSAT). A detailed analysis of brain polyamine concentrations indicated that all brain regions of these animals showed distinct signs of activated polyamine catabolism, e.g. overaccumulation of putrescine (three- to 17-fold), appearance of N1-acetylspermidine and decreases in spermidine concentrations. In situ hybridization analyses revealed a marked overexpression of SSAT-specific mRNA all over the brain tissue of the transgenic animals. The transgenic animals appeared to tolerate subcutaneous injections of high-dose kainate substantially better as their overall mortality was less than 50% of that of their syngenic littermates. We used the expression of glial fibrillary acidic protein (GFAP) as a marker of brain injury in response to kainate. In situ hybridization analysis with GFAP oligonucleotide up to 7 days after the administration of sublethal kainate doses showed reduced GFAP expression in transgenic animals in comparison with their non-transgenic littermates. This difference was especially striking in the cerebral cortex of the transgenic mice where the exposure to kainate hardly induced GFAP expression. The treatment with kainate likewise resulted in loss of the hippocampal (CA3) neurons in non-transgenic but not transgenic animals. These results support our earlier findings indicating that elevated concentrations of brain putrescine, irrespective whether derived from an overexpression of ornithine decarboxylase, or as shown here, from an overexpression of SSAT, play in all likelihood a neuroprotective role in brain injury.

Acetylation↗
PubMed2003

Cux-1 transgenic mice develop glomerulosclerosis and interstitial fibrosis.

BACKGROUND: Cux-1 is a murine homeobox gene that is highly expressed in the nephrogenic zone of the developing kidney. Transgenic mice ectopically expressing Cux-1 develop renal hyperplasia associated with down-regulation of the cyclin kinase inhibitor p27. Because the reduction of p27 has been associated with mesangial cell proliferation and glomerular disease, we evaluated glomerular changes in Cux-1 transgenic mice. METHODS: Adult kidneys from Cux-1 transgenic mice were analyzed morphologically for changes in glomerular cell number and for changes in mesangial and interstitial extracellular matrix deposition. Mesangial matrix expansion was identified by light microscopy. Glomerular cell number was performed following immunohistochemistry. Type IV collagen deposition was analyzed by immunofluoresence and Western blotting. Renal function was evaluated by serum protein, blood urea nitrogen (BUN), creatinine, and electrolyte analysis, and by urine protein and creatinine analysis. RESULTS: In adult transgenic glomeruli, Cux-1 was ectopically expressed in mesangial cells, and this was associated with an increase in mesangial cell number, resulting from an increase in proliferation. There was a marked increase in mesangial matrix area in transgenic mice compared to non-transgenic littermates, related to an increase in type IV collagen. Podocyte foot process effacement was observed in transgenic mice, and this was related to an increase in urinary albumin. Interstitial fibrosis was also observed in transgenic kidneys. CONCLUSION: These observations indicate that increased expression of Cux-1 in mesangial cells results in cell proliferation and mesangial expansion. In addition, these changes are potentially related to disruption of podocyte architecture leading to loss of filtration. These results suggest that expression of Cux-1 is sufficient to induce the early events of mesangioproliferative glomerulonephritis.

Animals↗