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Transgenic Animals (transgenic + animals)
Selected AbstractsTransgenic Animals in Cardiovascular Disease ResearchEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Michael Bader Worldwide, the highest morbidity and mortality results from such cardiovascular diseases as hypertension, myocardial infarction, cardiac and renal failure, as well as stroke. Since the cardiovascular system and its regulation is quite complex, study of these disorders has been grossly limited to whole organism models. As a result, in recent years, transgenic technology has played a significant role in the discovery of specific gene products for cardiovascular regulation and disease aetiology. Genetic manipulation in rats and mice has altered the expression of numerous genes. In this review, some of the important new genetically modified animals (i.e. transgenic models) with alterations in hormone and second messenger systems involved in cardiovascular regulation are summarized. [source] Use of Transgenic Animals to Improve Human Health and Animal ProductionREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2005L-M Houdebine Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed. [source] Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2003M. Eren Summary., Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal ,2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-,1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-,1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal ,2.9 kb promoter. [source] Use of Transgenic Animals to Improve Human Health and Animal ProductionREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2005L-M Houdebine Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed. [source] Analysis of conserved residues in the ,pat-3 cytoplasmic tail reveals important functions of integrin in multiple tissuesDEVELOPMENTAL DYNAMICS, Issue 3 2010Xiaojian Xu Abstract Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans ,pat-3 cytoplasmic tail. The ,1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the ,1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a ,1C-like variant, which results in a frameshift, was constructed. The ,pat-3(,1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin. Developmental Dynamics 239:763,772, 2010. © 2010 Wiley-Liss, Inc. [source] Presynaptic secretion of mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environmentsDEVELOPMENTAL DYNAMICS, Issue 3 2009Emma Rushton Abstract Mind-the-Gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. Here, we generate MTG::GFP transgenic animals to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic-lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM-integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. We suggest that MTG pioneers a novel protein family involved in ECM-dependent synaptic differentiation. Developmental Dynamics 238:554,571, 2009. © 2009 Wiley-Liss, Inc. [source] Transient expression of thyroid hormone nuclear receptor TR,2 sets S opsin patterning during cone photoreceptor genesisDEVELOPMENTAL DYNAMICS, Issue 5 2007M.L. Applebury Abstract Cone photoreceptors in the murine retina are patterned by dorsal repression and ventral activation of S opsin. TR,2, the nuclear thyroid hormone receptor , isoform 2, regulates dorsal repression. To determine the molecular mechanism by which TR,2 acts, we compared the spatiotemporal expression of TR,2 and S opsin from embryonic day (E) 13 through adulthood in C57BL/6 retinae. TR,2 and S opsin are expressed in cone photoreceptors only. Both are transcribed by E13, and their levels increase with cone genesis. TR,2 is expressed uniformly, but transiently, across the retina. mRNA levels are maximal by E17 at completion of cone genesis and again minimal before P5. S opsin is also transcribed by E13, but only in ventral cones. Repression in dorsal cones is established by E17, consistent with the occurrence of patterning during cone cell genesis. The uniform expression of TR,2 suggests that repression of S opsin requires other dorsal-specific factors in addition to TR,2. The mechanism by which TR,2 functions was probed in transgenic animals with TR,2 ablated, TR,2 that is DNA binding defective, and TR,2 that is ligand binding defective. These studies show that TR,2 is necessary for dorsal repression, but not ventral activation of S opsin. TR,2 must bind DNA and the ligand T3 (thyroid hormone) to repress S opsin. Once repression is established, T3 no longer regulates dorsal S opsin repression in adult animals. The transient, embryonic action of TR,2 is consistent with a role (direct and/or indirect) in chromatin remodeling that leads to permanent gene silencing in terminally differentiated, dorsal cone photoreceptors. Developmental Dynamics 236:1203,1212, 2007. © 2007 Wiley-Liss, Inc. [source] Conditional ablation of neurones in transgenic miceDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001Anthony R. Isles Abstract Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 183,193, 2001 [source] ,-Amyloid immunization approaches for Alzheimer's diseaseDRUG DEVELOPMENT RESEARCH, Issue 2 2002Bruno P. Imbimbo Abstract Alzheimer's disease (AD) represents the third leading cause of death in the U.S. and the leading cause of dementia in the elderly population. Until recently, there was little hope of efficiently combating this devastating disease. The deposition of ,-amyloid (A,) is the major pathological hallmark of AD brains. Genetic, biochemical, and pharmacological evidence support the hypothesis that A, plays a key role in the development of the disease. Thus, in the last 5 years a number of pharmacological strategies have been developed to interfere with the A, cascade. The most revolutionary of these approaches was proposed in 1999 by scientists at Elan Pharmaceuticals, which immunized against A, transgenic mice with spontaneously developing A, pathology. The immunization was achieved by subcutaneous injections of a preaggregated form of the synthetic human 42-amino acid A, emulsified with Freund's adjuvant, an immune stimulant. The vaccination caused a near complete inhibition of A, plaque formation in younger animals and a marked reduction of the A, burden in older animals. The effects on A, plaques were accompanied by a reduction of A,-associated astrogliosis and neuritic dystrophy. These results were later confirmed by other groups with similar vaccination protocols, which also demonstrated that the A, immunization of transgenic animals normalize or reduce the cognitive impairment associated with A, pathology. Interestingly, effective removal of brain A, plaques was also obtained by peripherally administering A, antibodies. The mechanism with which the vaccine increases A, clearance is not fully understood. Centrally, the vaccine appears to activate A, phagocytosis by microglial monocytes. Peripherally, serum A, antibodies bind and sequester A,, thus altering its equilibrium between CNS and plasma. The dramatic results obtained in animal models of AD raised unprecedented hopes for both a preventive and a curative intervention for this devastating disorder. A vaccine preparation for human use (AN-1792) composed of preaggregated human A,42 peptide and a highly purified saponin derivative (QS-21) was developed by Elan Pharmaceuticals and Wyeth Ayerst and tested in AD patients. Unfortunately, a Phase IIa study aimed at evaluating the safety and immunological activity of AN-1792 in 360 AD patients was discontinued because 15 subjects receiving the vaccine developed serious signs of CNS inflammation. Both central activation of cytotoxic T cells and autoimmune reactions were proposed as potential mechanisms of toxicity. Other therapeutic A, vaccination strategies are being pursued, including immuno-conjugates and monoclonal antibodies. The future of these and other A, immunization approaches depend on a clear understanding of the mechanism of A, clearance and additional insight into the role of inflammation in the AD brain. Drug Dev. Res. 56:150,162, 2002. © 2002 Wiley-Liss, Inc. [source] Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transferEQUINE VETERINARY JOURNAL, Issue 1 2008B. A. BALL Summary Reasons for performing study: Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objectives: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647 -pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. Results: Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647 -pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. Conclusions: The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7,10 embryos, there was no evidence of expression of EGFP in these embryos. Potential relevance: Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids. [source] Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2007Jared Clarke Abstract Cerebral ischaemia leads to a transient accumulation of ,-amyloid precursor protein (APP) and ,-amyloid (A,) peptides adjacent to the ischaemic lesion. There is conflicting evidence that APP/A, fragments may either enhance neuronal plasticity or be neurotoxic. The aim of the current study was to assess the effect of overexpression of human APP in rats on functional recovery following cerebral ischaemia. Adult APP-overexpressing (hAPP695 Tg) rats subjected to transient middle cerebral artery occlusion (MCAO) had significantly smaller infarct volumes than non-transgenic littermates, yet did not perform better on a series of sensorimotor or learning tests during a 6-month follow-up period. In fact, transgenic animals were found to be significantly more impaired in both the beam-walking and Morris water maze tests following MCAO. Immunohistochemistry showed human A,-positive staining in the cortex and hippocampus of APP transgenic rats. The present data suggest that while overexpression of APP in rats may provide some histological neuroprotection in the event of cerebral ischaemia, this does not translate into significant functional recovery. [source] Synaptic plasticity in the basolateral amygdala in transgenic mice expressing dominant-negative cAMP response element-binding protein (CREB) in forebrainEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2000G. Rammes Abstract Electrophysiological and behavioural experiments were performed in transgenic mice expressing a dominant-negative form of cAMP response element-binding protein (CREBA133) in the limbic system. In control littermate in vitro slice preparation, tetanizing the lateral amygdala,basolateral amygdala (BLA) pathway with a single train (100 Hz for 1 s) produced short-term potentiation (STP) in the BLA. Five trains (10-s interstimulus interval) induced long-term potentiation (LTP), which was completely blocked by the N-methyl- d -aspartate (NMDA) receptor antagonist d(,)-2-amino-5-phosphonopentanoic acid (AP5; 50 ,m). When GABAergic (,-aminobutyric acid) inhibition was blocked by picrotoxin (10 ,m), LTP became more pronounced. Low-frequency stimulation (1 Hz for 15 min) induced either long-term depression (LTD) or depotentiation. LTD remained unaffected by AP5 (50 ,m) or by the L- and T-type Ca2+ -channel blockers nifedipine (20 ,m) and Ni2+ (50 ,m), but was prevented by picrotoxin (10 ,m), indicating a GABAergic link in the expression of LTD in the BLA. When conditioned fear was tested, a mild impairment was seen in one of three transgenic lines only. Although high levels of mRNA encoding CREBA133 lead to downregulation of endogenous CREB, expression of LTP and depotentiation were unaltered in BLA of these transgenic animals. These results could suggest that residual CREB activity was still present or that CREB per se is dispensable. Alternatively, other CREB-like proteins were able to compensate for impaired CREB function. [source] Overexpression of spermidine/spermine N1 -acetyltransferase in transgenic mice protects the animals from kainate-induced toxicityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2000Kyllikki Kaasinen Abstract 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. [source] Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesisEXPERIMENTAL DERMATOLOGY, Issue 9 2007Markus Walz Abstract:, The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases. [source] Constitutive androstane receptor (CAR) ligand, TCPOBOP, attenuates Fas-induced murine liver injury by altering Bcl-2 proteins,HEPATOLOGY, Issue 1 2006Edwina S. Baskin-Bey The constitutive androstane receptor (CAR) modulates xeno- and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild-type (CAR+/+) and CAR knockout (CAR,/,) mice were treated with the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP-treated CAR+/+ but not in CAR,/, mice. Likewise, acute and chronic ConA-mediated liver injury and fibrosis were also reduced in wild-type versus CAR,/, TCPOBOP-treated mice. The proapoptotic proteins Bak (Bcl-2 antagonistic killer) and Bax (Bcl-2-associated X protein) were depleted in livers from TCPOBOP-treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor-1 (Mcl-1) was increased fourfold. Mcl-1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl-1 transcription. Indeed, site-directed mutagenesis of a putative CAR consensus binding sequence on the Mcl-1 promoter decreased Mcl-1 promoter activity. Mcl-1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl-1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas-mediated hepatocyte injury via a mechanism involving upregulation of Mcl-1 and, likely, downregulation of Bax and Bak. (HEPATOLOGY 2006;44:252,262.) [source] Comparison of commissural sprouting in the mouse and rat fascia dentata after entorhinal cortex lesionHIPPOCAMPUS, Issue 6 2003Domenico Del Turco Abstract Reactive axonal sprouting occurs in the fascia dentata after entorhinal cortex lesion. This sprouting process has been described extensively in the rat, and plasticity-associated molecules have been identified that might be involved in its regulation. To demonstrate causal relationships between these candidate molecules and the axonal reorganization process, it is reasonable to analyze knockout and transgenic animals after entorhinal cortex lesion, and because gene knockouts are primarily generated in mice, it is necessary to characterize the sprouting response after entorhinal cortex lesion in this species. In the present study, Phaseolus vulgaris -leucoagglutinin (PHAL) tracing was used to analyze the commissural projection to the inner molecular layer in mice with longstanding entorhinal lesions. Because the commissural projection to the fascia dentata is neurochemically heterogeneous, PHAL tracing was combined with immunocytochemistry for calretinin, a marker for commissural/associational mossy cell axons. Using both techniques singly as well as in combination (double-immunofluorescence) at the light or electron microscopic level, it could be shown that in response to entorhinal lesion mossy cell axons leave the main commissural fiber plexus, invade the denervated middle molecular layer, and form asymmetric synapses within the denervated zone. Thus, the commissural sprouting response in mice has a considerable translaminar component. This is in contrast to the layer-specific commissural sprouting observed in rats, in which the overwhelming majority of mossy cell axons remain within their home territory. These data demonstrate an important species difference in the commissural/associational sprouting response between rats and mice that needs to be taken into account in future studies. © 2003 Wiley-Liss, Inc. [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosisJOURNAL OF NEUROCHEMISTRY, Issue 3 2006Céline Vermeiren Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1G93A) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate,aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease. [source] Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemiaJOURNAL OF NEUROCHEMISTRY, Issue 2 2005Purnima Narasimhan Abstract Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen,glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia. [source] Insulin-like signalling in neurons controls lifespan in C. elegansJOURNAL OF NEUROCHEMISTRY, Issue 2002C. A. Wolkow Insulin-like signalling controls C. elegans lifespan, development and metabolism. Mutations that weaken this insulin-like signalling pathway extend lifespan. Severe mutations abolishing insulin-like signalling cause animals to arrest development as dauer larvae, a larval form specialized for stress resistance and long-term survival. A number of the genes acting in this pathway have been cloned, including daf-2, which encodes a homolog of vertebrate insulin/IGF-I receptors, and age-1, encoding the C. elegans homolog of the PI(3)K p110 catalytic subunit. In order to identify cells from which insulin-like signalling controls lifespan and development, transgenic animals were constructed which possessed insulin-like signalling only in specific cell types. To achieve this, cell-type specific promoters were used to drive expression of daf-2 or age-1 cDNAs in daf-2(,/,) or age-1(,/,) backgrounds, respectively. By utilizing this strategy, we could restore wild-type daf-2 or age-1 activity only in cells that are capable of expressing each transgene. Restoring insulin-like signalling to the nervous system of daf-2 or age-1 mutants could rescue long lifespan. This result was specific for transgenes restoring insulin-like signalling to the nervous system. Expressing daf-2 or age-1 cDNAs from muscle- or intestinally-restricted promoters was insufficient to rescue lifespan. In contrast, age-1 and daf-2 expression in either neuronal or non-neuronal cell types rescued dauer larval arrest in the mutants. These findings demonstrate that insulin-like signalling pathways in the nervous system control C. elegans lifespan. [source] In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin GenesJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2003D. Murphy Abstract Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one-cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis -acting sequences mediating the regulation of the vasopressin-oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell-specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High-throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication-deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long-lasting expression of biologically active proteins in vasopressin and oxytocin MCNs. [source] Proactive consumer consultation: the effect of information provision on response to transgenic animalsJOURNAL OF PUBLIC AFFAIRS, Issue 3-4 2005David Castle A national study is reported which proactively engaged 1365 Canadian consumers and solicited their opinions concerning new transgenic salmon and pork products which have not yet entered the marketplace. Respondents were methodically requested to provide initial free-association responses, and then scaled responses to product concepts about which progressively more information was revealed. This combined qualitative and quantitative method was pursued in order to determine initial knowledge levels and subsequent responses with a minimal amount of cueing via question probes. The results indicate that disclosure concerning benefits and risks of these new technologies did not harm judgements about them or estimates of purchase intent. A significant determinant of opinions was the gender of the respondent. Females were more negatively predisposed overall to the concepts and more sensitive to specific information regarding product benefits and risks. The research offers a methodological template for public consultation and communication pre-testing for new biotechnological products. Implications for regulatory policy and information dissemination for new food biotechnology products are discussed. Copyright © 2005 John Wiley & Sons, Ltd. [source] The promise and challenges of bioengineered recombinant clotting factorsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2005S. W. PIPE Summary., The past 10 years of clinical experience have demonstrated the safety and efficacy of recombinant clotting factors. With the adoption of prophylactic strategies, there has been considerable progress in avoiding the complications of hemophilia. Now, insights from our understanding of clotting factor structure and function, mechanisms of hemophilia and inhibitors, gene therapy advances and a worldwide demand for clotting factor concentrates leave us on the brink of embracing targeted bioengineering strategies to further improve hemophilia therapeutics. The ability to bioengineer recombinant clotting factors with improved function holds promise to overcome some of the limitations in current treatment, the high costs of therapy and increase availability to a broader world hemophilia population. Most research has been directed at overcoming the inherent limitations of rFVIII expression and the inhibitor response. This includes techniques to improve rFVIII biosynthesis and secretion, functional activity, half-life and antigenicity/immunogenicity. Some of these proteins have already reached commercialization and have been utilized in gene therapy strategies, while others are being evaluated in pre-clinical studies. These novel proteins partnered with advances in gene transfer vector design and delivery may ultimately achieve persistent expression of FVIII leading to an effective long-term treatment strategy for hemophilia A. In addition, these novel FVIII proteins could be partnered with new advances in alternative recombinant protein production in transgenic animals yielding an affordable, more abundant supply of rFVIII. Novel rFIX proteins are being considered for gene therapy strategies whereas novel rVIIa proteins are being evaluated to improve the potency and extend their plasma half-life. This review will summarize the status of current recombinant clotting factors and the development and challenges of recombinant clotting factors bioengineered for improved function. [source] The mechanisms of tumor suppressor effect of glucocorticoid receptor in skinMOLECULAR CARCINOGENESIS, Issue 8 2007Dmitry Chebotaev Abstract Glucocorticoid hormones exert a tumor suppressor effect in different experimental models, including mouse skin carcinogenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well-known transcription factor that regulates genes by DNA-binding dependent transactivation, and DNA-binding independent transrepression through negative interaction with other transcription factors. In this perspective, we analyze known mechanisms that underlie the anticancer effect of GR signaling, including effects on cell growth, differentiation, apoptosis, and angiogenesis. We also discuss a novel mechanism for the tumor suppressor effect of the GR in skin: through the regulation of the number and status of follicular epithelial stem cells (SC), which are a target cell population for skin carcinogenesis. Our studies on keratin5.GR transgenic animals that are resistant to skin carcinogenesis, demonstrated that the GR diminishes the number of follicular epithelial SCs, reduces their proliferative and survival potential and affects the expression of follicular SC "signature" genes. The analysis of global effect of the GR on gene expression in follicular epithelial SCs, basal keratinocytes, and mouse skin tumors provided an unexpected evidence that gene transrepression by GR plays an important role in the maintenance of SC and in inhibition of skin carcinogenesis by this steroid hormone receptor. It is known that antiinflammatory effect of glucocorticoids is chiefly mediated by GR transrepression. Thus, our findings suggest the similarity between the mechanisms of antiinflammatory and anticancer effects of the GR signaling. We discuss the potential clinical applications of our findings in light of drug discovery programs focused on the development of selective GR modulators that preferentially induce GR transrepression. © 2007 Wiley-Liss, Inc. [source] Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertilityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2007Khailun Tseden Abstract During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3,untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3,UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3,UTR of Tnp2 gene was replaced by 3, 3,UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3,UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Orthodontically stressed periodontium of transgenic mouse as a model for studying mechanical response in bone: The effect on the number of osteoblastsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 2 2000Dubravko Pavlin A better understanding of cellular and molecular mechanisms involved in response to mechanical stress is a prerequisite for future improvements in orthodontic treatment. To expand the application of molecular biology techniques in this area of research, we developed and characterized a mouse tooth movement model. The aim of this study was to biomechanically characterize this model and to evaluate the effect of orthodontic stress on the proliferation of periodontal osteoblasts. We used an orthodontic coil spring appliance with a low force/deflection rate, which produced an average force of 10,12 g. This design provided a predictable tipping movement of the molar with the center of rotation at the level of root apices. Histological observations of paradental tissues revealed a response favoring a fast onset of tooth movement and deposition of new osteoid starting after 3 days of treatment. The effect of treatment on the histomorpometric parameter of the number of osteoclasts per unit bone perimeter was determined after 1, 2, 3, 4, 6, and 12 days of treatment. Starting with day 2, the osteoblast number showed a modest but consistent increase in treated periodontal sites at all time-points, ranging from 14 to 39% and becoming significant only at day 6. Only a moderate increase in the number of osteoblasts in the areas of otherwise intense bone matrix synthesis suggests that, during bone formation, proliferation of cells has a smaller role compared to a marked increase in differentiation of individual cells. The mouse model, which allows for a controlled, reproducible, orthodontic mechanical loading, can be applied to both wild-type and transgenic animals and should enhance the research of the transduction of mechanical orthodontic signal into a biological response. [source] Use of Transgenic Animals to Improve Human Health and Animal ProductionREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2005L-M Houdebine Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed. [source] Biglycan Overexpression on Tooth Enamel Formation in Transgenic MiceTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 10 2008Xin Wen Abstract Previously, it was shown that the volume of forming enamel of molar teeth in biglycan-null mice was greater than that in genetically matched wild-type mice. This phenotypic change appeared to result from an increase in amelogenin expression, implying that biglycan directly influences amelogenin synthesis. To determine whether biglycan overexpression resulted in decreased amelogenin expression, we engineered transgenic mice to overexpress biglycan in the enamel organ epithelium. Biglycan overexpression did not significantly affect the amelogenin expression in incisor and molar teeth in 3-day postnatal transgenic mice. In the transgenic animals, we observed that the immature and mature enamel appeared normal. These results suggested that increasing the biglycan expression, in the cells that synthesize the precursor protein matrix for enamel, has a negligible influence on amelogenesis. Anat Rec, 2008. © 2008 Wiley-Liss, Inc. [source] Characteristics of Rabbit Transgenic Mammary Gland Expressing Recombinant Human Factor VIIIANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009P. Chrenek Summary The objective of this research was to compare (i) the content of milk protein and recombinant human factor VIII (rhFVIII) in the milk of transgenic and non-transgenic rabbit females at three lactations and (ii) histological structure, ultrastructural morphology and occurrence of apoptosis in rabbit transgenic and non-transgenic mammary gland during third lactation and involution. Significant differences (t0.05) in milk protein content were found between transgenic and non-transgenic at all three lactations. The percentage of apoptotic cells was significantly higher (t0.01) in non-transgenic ones compared with transgenic mammary gland tissues (6.5% versus 2.4%) taken at the involution stage. Morphometrical analysis of histological preparations at the involution stage detected a significantly higher (t0.05) relative volume of lumen in transgenic animals compared with non-transgenic ones (60.00 versus 46.51%). Ultrastructural morphology of the transgenic mammary gland epithelium at the involution stage revealed an increased relative volume of protein globules (t0.05); at the lactation stage, a significantly higher volume of mitochondria (13.8%) compared with the non-transgenic (9.8%) ones was observed. These results, although revealing differences in some parameters of ultrastructure and histology, indicate no harmful effect of the mouse whey acid protein-hFVIII transgene expression on the state of mammary gland of transgenic rabbit females. [source] Ultrastructural Morphometry of Mammary Gland in Transgenic and Non-transgenic RabbitsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2006S. Dragin Summary The mammary gland of transgenic animals has been used for the production of recombinant proteins of therapeutic and nutraceutical use. The objective of this study was to compare the ultrastructure of transgenic and non-transgenic rabbit mammary gland tissue. New Zealand White transgenic rabbits were obtained by breeding non-transgenic rabbits with transgenic founder rabbits containing a whey acidic protein-human factor VIII (WAP-hFVIII) transgene integrated into their genome. Samples of mammary gland tissue from lactating rabbit females were isolated by surgical procedures. These samples were examined by optical and electron microscopy and photographs were taken. Measurements of ultrastructural organelles were made from digital images of the mammary cells. No differences were found in the cellular structure of mammary tissue, but significant differences t(0.001) in the relative volume of mitochondria and vacuoles between transgenic and non-transgenic mammary gland epithelium were observed. [source] | |||||||||||||||||||||||||