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HZE particle radiation induces tissue-specific and p53-dependent mutagenesis in transgenic animals.

Transgenic animals, with the integrated target gene, provide a unique approach for measuring and characterizing mutations in any tissue of the animal. We are using the plasmid-based lacZ transgenic mice with different p53 genetic background to examine radiation-induced genetic damage resulting from exposure to heavy particle radiation. We measured lacZ mutation frequencies (MF) in the brain and spleen tissues at various times after exposing animals to an acute dose of 1 Gy of 1GeV/amu iron particles. MF in the spleen of p53+/+ animals increased up to 2.6-fold above spontaneous levels at 8 weeks post irradiation. In contrast, brain MF from the same animals increased 1.7-fold above controls in the same period. In the p53-/- animals, brain MF increased to 2.2-fold above spontaneous levels at 1 week after treatment, but returned to control levels thereafter. Radiation also induced alterations in the spectrum of mutants in both tissues, accompanied by changes in the frequency of mutants with deletions extending past the transgene into mouse genomic DNA. Our results indicate that the accumulation of transgene MF after radiation exposure is dependant on the tissue examined as well as the p53 genetic background of the animals.

Animals↗

Neuropeptide gene expression in transgenic animals.

Transgenic animal techniques offer today's neuroscientist the ability to experimentally manipulate neurosecretory systems with a precision undreamt of by our predecessors. The range of techniques now available, building as it does on our growing knowledge of physiological systems at the inter- and intercellular level, allows us to critically define molecular lesions and ask about their consequences to the whole organism. Neuroscientist should grasp the opportunities afforded by these recent developments.

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Imaging transgenic animals.

Transgenic and eugenic animals as small as 30 g can be studied non-invasively by radionuclides with resolutions of 1-2 mm, by MRI with resolution of 100 microns and by light fluorescence and bioluminescence with high sensitivities. The technologies of radionuclide emission, magnetic resonance imaging, magnetic resonance spectroscopy, optical tomography, optical fluorescence and optical bioluminescence are currently being applied to small-animal studies. These technologies and examples of their applications are reviewed in this chapter.

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Piecing together the timetable for visual transduction with transgenic animals.

Transgenic mice bearing null or functional mutations are being used to define the roles of specific elements in phototransduction and also to time the molecular interactions. Genetic manipulation of the collision frequency between rhodopsin and transducin molecules identified this parameter as rate-limiting for the photoresponse onset. Genetic interference with rhodopsin phosphorylation and arrestin binding, transducin shut-off and calcium feedback has revealed their respective roles in shaping the response waveform. The timetable for all of these molecular events determines the amplitude, kinetics and reproducibility of the photoresponse.

Amino Acid Sequence↗

[The use of transgenic animals in biomedical research in Germany. Part 2: Ethical evaluation of the use of transgenic animals in biomedical research and perspectives for the changeover in research to research animal-free methods].

As a rule, transgenic animals are being used in in vivo experiments to examine gene functions, their regulation or the contribution of genetic alterations to the development of diseases. Many transgenic animals already are affected in their wellbeing due to the genetic modification alone regardless of the procedures performed with them. Moreover, it is to be questioned whether the experimental use of transgenic animals led to results that were of such outstanding scientific relevance that they legitimated the suffering of the animals. In order to point to possible approaches to avoiding the use of transgenic animals in the areas of research identified, subsequent investigations aimed at collecting information on non-animal test methods that might be applied in pursuing the aforesaid questions. In particular, these were non-animal test methods that make use of genetic techniques. Amongst these are in vitro cell culture methods with genetically modified cells, such as the so called Transfected Cell Array, as well as in vitro test methods, in which specifically targeted genes can be turned on or off selectively for example by the so-called RNA interference technique or by antisense oligonucleotide genes. Since such technologies can also be applied to cell cultures with human cells, investigations with these methods enable direct information on the function of human genes. Even though a one to one replacement of animal experiments with transgenic animals by non-animal test methods is considered unlikely, from the point of view of animal welfare the broad spectrum of already available non animal test methods with which to study the function of genes and genetically caused pathophysiological reactions proves that waiving of animal tests with transgenic animals is possible without impeding biomedical research. Even if it cannot be totally excluded that some very specific questions linked to the respective animal experiment might not be pursued for the time being, nevertheless research that would be restricted to modern and ethically acceptable in vitro test methods would certainly conceive its very own questions to pursue and solve the problems currently faced by biomedical research. It is against this background that it is to be welcomed that the German Federal Government currently actively promotes the further development of genetechnological non-animal test methods. In order to ensure that these funding measures will make an effective contribution to reducing animal experiments, as spelled out by the government itself, the conversion of genetechnological research, just like biomedical research as a whole, to non-animal testing methods should be supported by concrete political actions. From the point of view of the German Animal Welfare Federation the following issues are to be requested: (1)In order to enable a fast and comprehensive advancement of promising genetechnological non-animal test methods, it should be ensured that public funding is provided with an adequate budget and over a sufficiently long period of time. (2)The legislator should initiate broad discussions on the question if society would be willing to dispense with certain pieces of knowledge if they would necessarily have to be gained at the expense of a certain degree of animal suffering. As the case may be, in the German Animal Welfare Act it should be laid down that certain procedures should not be considered acceptable as such. (3)As long as animal experiments with transgenic animals continue to be performed, concrete legal measures should be laid down in the German Animal Welfare Act to ensure that the distress of the animals (taking into account all factors relevant for transgenic animals) and the expected benefit of the research project are determined objectively so that the outcome of the ethical evaluation process becomes comprehensible. (4)The legislator should provide the authorities responsible for the licensing of research projects with concrete instructions in order to ensure that all aspects relevant for the welfare of the animals are fully taken into account when evaluating the ethical acceptability and scientific indispensability of projects and that special attention is given to research projects with transgenic animals. (5)The German Decree on the Reporting of Laboratory Animals should be amended to ensure that all individual transgenic animals are included in the official statistical reports regardless of whether they end up being used in scientific procedures or not. From the point of view of animal welfare it is possible to redesign biomedical research to do without transgenic animals without impeding necessary scientific progress. The survey in hand sought to make a contribution to providing a scientifically sound background for initiating these discussions.

Animal Testing Alternatives↗

[Animal welfare problems concerning the use of transgenic animals]

Using transgenic animals as clinical models pose certain problems since they can suffer. Yet in single cases transgenic animals can reduce the suffering of (other) animals. The permission to generate transgenic animals is not yet clearly regulated in Switzerland. The term "dignity of creature", as formulated in the Swiss Constitution, has to be defined for the Swiss animal protection law. We present the recommendations of the commission for ethical questions concerning transgenic animals appointed by the Federal Council. Partly, these recommendations shall also be applied to the traditional breeding methods. We support the nomination of a national ethics committee for transgenic animals.

Journal Article↗

[An immunocytochemical analysis of the hormonal status of animals transgenic for growth hormone genes and for a mini-gene of human growth hormone-releasing factor].

An immunocytochemical analysis of hormonal status of transgenic rats containing human growth hormone gene has been done. The enhanced expression of the endogenous growth hormone gene was demonstrated with poly- and monoclonal antibodies inside somatatropes of pituitary. No activity of the heterologous growth hormone gene was revealed in kidney, pancreas or liver as it might be expected according to specificity of MT1 and TAT promotors. Transgenic animals of F0, F1 and F2 generation exhibited disturbance of functional morphology of glucagon and insulin producing cells. Lymphocyte infiltration was found in pancreatic islets. The transgenic rabbits and swine with the gene of releasing factor of human growth hormone did not reveal any severe disturbance. Although one swine demonstrated alterations in glucagon producing cells and one rabbit revealed a disturbed morphology of the stomach tissues. The data are discussed in relation to general problems of transgene activity and interaction with endogenous homolog.

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The methods to generate transgenic animals and to control transgene expression.

Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

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Activated and inactivated renin-angiotensin system in transgenic animals: from genes to blood pressure.

Much of our knowledge about blood pressure regulation has come from a huge number of biochemical, endocrinologic, pharmacologic, clinical, and physiologic studies. The renin-angiotensin system, composed of enzymatic and signal-transduction cascades, has a key role in regulating arterial blood pressure and in developing certain forms of experimentally induced hypertension and hypertension in human beings. Angiotensin peptides, the products of this system, exert a wide range of physiologically important effects on many tissues, including those of the cardiovascular system, through their actions on angiotensin receptors. Recently, several groups have succeeded in generating animals transgenic for the renin or angiotensinogen genes, or with a targeted disruption of the component genes of the renin-angiotensin system. This brief review describes advances of the in vivo analysis of blood pressure regulation by focusing on the renin-angiotensin system.

Amino Acid Sequence↗

[Application of genetically engineered animals in pharmacology: the use of green fluorescent protein for selective production of transgenic animals].

Transgenic animals are a very important tool not only for basic science but also for the pharmaceutical industry. The use of genetically engineered farm animals are suitable as a disease model. Production of a therapeutic protein such as human clotting factors by transgenic animals will reduce the risk of infection with human immunodeficiency virus and hepatitis virus. The efficiency of transgenic animal production, however, has been low and thus limited its application. We improved the efficiency using a green fluorescent protein (GFP) as a marker of gene integration. Using this method, we obtained 12 fetuses, and Southern blot analysis showed eight of them were transgenic, indicating transgenic embryos were successfully selected at the preimplantation stage. We also observed similar GFP expression in rat and bovine blastocysts. Application of this GFP selection method should improve the efficiency of transgenic livestock production.

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Production of bioproducts through the use of transgenic animal models.

Transgenic livestock that produce recombinant proteins in their milk can provide an economic and safe system for production of valuable proteins, such as pharmaceutical proteins for treatment or prevention of human disease or biomaterials for medical use. This method of production is frequently referred to as biopharming. The promise of biopharming, that is the actual commercial production of pharmaceuticals and other bioproducts, is nearing fulfillment. Improvements in molecular and reproductive techniques and strong economic incentives have continued to drive the implementation of transgenic technology to domestic animals. Nuclear transfer using transgenic donor cells is rapidly becoming the predominant technique used in the production of transgenic livestock, replacing the direct injection of DNA into the zygotic pronuclei. Production of transgenic founder animals by nuclear transfer in combination with traditional reproductive technologies can result in the propagation of transgenic herds of sufficient size to meet market demands for commercially important proteins. While some of the companies that have established transgenic programs have run into setbacks owing to a combination of economic, scientific and regulatory difficulties, other companies are continuing to make significant advances. While further improvements are needed to increase efficiencies of production, economically viable production of recombinant proteins using livestock species is not only possible but should be a commercial reality in the very near future.

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Use of transgenic animals to improve human health and animal production.

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.

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Exploring pathogenetic mechanisms using transgenic animals.

Molecular technologies for the permanent germ-line transformation of animals are now well established and routine. These new strains of animals, called transgenic, offer an unprecedented opportunity to gain a basic understanding of human genetic disorders. In this brief review we discuss the role of transgenic animals in the creation of new models of human disease and their experimental use in biomedical research. Models are now available for the study of the genetic processes involved in the pathogenesis of neoplasia, diabetes, atherosclerosis, and developmental abnormalities. Many others are available and new ones are being produced at a great rate. Principles of gene replacement therapy are amenable to analysis with transgenic animals and the information gained will be important for the development of rational therapy.

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[Transgenic animals in medicine].

Transgenic animals are an experimental model of human diseases and enable us to study pathophysiological mechanisms, the interaction between genetic environmental factors and new therapeutic approaches incl. Their long-term effect. By using various methods it introduces into the genome of the experimental organism alien genes which become expressed. Mutated genes are introduced as well as the regulation sequence of expression of various genes, the sequence disrupting a certain gene or gene construction. Investigations of transgenic animals, whose production is rapidly increasing, are used in all spheres of medicine and biology. The author presents some examples from the sphere of oncology, cardiovascular, pulmonary, inflammatory and immunological diseases, human reproduction and early ontogenetic development, neuropsychiatric diseases and toxicology.

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Transgenic animals and nutrition research.

Transgenic animals are useful tools for the study of biological functions of proteins and secondary gene products synthesized by the action of protein catalysts. Research in nutrition and allied fields is benefiting from their use as models to contrast normal and altered metabolism. Although food, nutritional products, and ingredients from transgenic animals have not yet reached consumers, the technologies for their production are maturing and yielding exciting results in experimental and farm animals. Regulatory governmental bodies are already issuing guidelines and legislation in anticipation of the advent of these products and ingredients. This review summarizes available technology for the production of transgenic animals, discusses their scientific and commercial potential, and examines ancillary issues relevant to the field of nutrition.

Journal Article↗