Use Of Transgenic Animals Research Articles

How does angiotensin AT2 receptor activation help neuronal differentiation and improve neuronal pathological situations?

The angiotensin type 2 (AT2) receptor of angiotensin II has long been thought to be limited to few tissues, with the primary effect of counteracting the angiotensin type 1 (AT1) receptor. Functional studies in neuronal cells have demonstrated AT2 receptor capability to modulate neuronal excitability, neurite elongation, and neuronal migration, suggesting that it may be an important regulator of brain functions. The observation that the AT2 receptor was expressed in brain areas implicated in learning and memory led to the hypothesis that it may also be implicated in cognitive functions. However, linking signaling pathways to physiological effects has always proven challenging since information relative to its physiological functions has mainly emerged from indirect observations, either from the blockade of the AT1 receptor or through the use of transgenic animals. From a mechanistic standpoint, the main intracellular pathways linked to AT2 receptor stimulation include modulation of phosphorylation by activation of kinases and phosphatases or the production of nitric oxide and cGMP, some of which are associated with the Gi-coupling protein. The receptor can also interact with other receptors, either G protein-coupled such as bradykinin, or growth factor receptors such as nerve growth factor or platelet-derived growth factor receptors. More recently, new advances have also led to identification of various partner proteins, thus providing new insights into this receptor’s mechanism of action. This review summarizes the recent advances regarding the signaling pathways induced by the AT2 receptor in neuronal cells, and discussed the potential therapeutic relevance of central actions of this enigmatic receptor. In particular, we highlight the possibility that selective AT2 receptor activation by non-peptide and selective agonists could represent new pharmacological tools that may help to improve impaired cognitive performance in Alzheimer’s disease and other neurological cognitive disorders.

5HT2C Receptors in the Pathophysiology of CNS Disease

I remember the debate in the 1960s and 1970s as to whether there were two or three 5-HT (serotonin) receptors and was an interested spectator to the subsequent explosion in molecular pharmacology that led to our current tally of 14 receptors for 5-HT reliably characterized by expression cloning. The maturity of this field of study today is well illustrated by this book, where 557 pages are devoted to a summary of our state of knowledge of just one of these receptors. The book is well presented, with good use of diagrams, some in colour, although micrographs suffer from being reproduced on matt rather than glossy paper. In the main it is suitably up to date, with many citations from the 2010 literature, although a few chapters had clearly been longer in gestation. There is a good mix of international authors, with acknowledged leaders in the field being responsible for some key chapters. The book opens with a chapter on the history of the discovery of the 5HT2C receptor some 25 years ago by a traditional pharmacology approach at the then Sandoz laboratories in Basle. Initially called the 5-HT1C receptor on the basis of ligand-binding studies, it was renamed 5HT2C after its second messenger was found to be the phospholipase C cascade and expression cloning revealed a family resemblance to the other 5-HT2 receptors. Subsequent chapters describe the widespread but heterogeneous distribution of the 5HT2C receptor in the brain, with good evidence emerging for a role in modulating the dopamine pathways. The special features of the receptor are given suitable prominence, revealing that this is one of the small number of G-protein-coupled receptors that displays constitutive activity and is subject to RNA editing during its expression. The hot topic of receptor dimerization also receives a mention, although it is not yet clear whether this is a property of the 5HT2C receptor in vivo. It is pleasing to find a chapter devoted to the medicinal chemistry of 5HT2C ligands, explaining the challenge of making molecules that adequately distinguish between 5HT2A, 5HT2B and 5HT2C receptors. The use of transgenic animals, in particular 5HT2C−/− mice, in order to help elucidate the diverse physiological functions of this receptor is also comprehensively described. There is a succession of interesting chapters dealing with the possible role of the receptor in depression, schizophrenia, addiction, eating behaviour, dyskinesias, sleep, epilepsy, cognition, Alzheimer's disease and ocular hypotension. For the non-expert, it would have been valuable to have followed these with an expert summary that attempted to put all of this in context and point out the areas of most likely clinical relevance. As it was, the book was left open ended, which may not be an issue if it is intended only for the bookshelves of the cognoscenti. In summary, this is a useful book describing the properties of a receptor that may well turn out to be a therapeutically important drug target, with selective ligands already being evaluated in phase II trials for control of appetite. However, the high price of the book and the lack of a concluding summary chapter may restrict it to use by experts in the field.

Use of transgenic animals in understanding molecular mechanisms of toxicity.

Understanding molecular mechanisms of chemical toxicity and the potential risks of drugs to man is a pivotal part of the drug development process. With the dramatic increase in the number of new chemical entities arising from high throughput screening, there is an urgent need to develop systems for the rapid evaluation of potential drugs so that those agents which are most likely to be free of adverse effects can be identified at the earliest possible stage in drug development. The complex mechanisms of action of chemical toxins has made it extremely difficult to evaluate the precise toxic mechanism and also the relative role of specific genes in either potentiating or ameliorating the toxic effect. This problem can be addressed by the application of genetic strategies. Such strategies can exploit strain differences in susceptibility to specific toxic agents or, with the rapidly developing technologies, can exploit the use of transgenic animals where specific genes can be manipulated and subsequent effects on chemical toxicity evaluated. Transgenic animals can be exploited in a variety of ways to understand mechanisms of chemical toxicity. For example, a human gene encoding a drug metabolizing enzyme can be directly introduced and the effects on toxic response evaluated. Alternatively, specific genes can be deleted from the mouse genome and the consequences on toxicological response determined. Many toxic chemical agents modulate patterns of gene expression within target cells. This can be used to screen for responses to different types of toxic insult. In such experiments the promotor of a stress-regulated gene can be ligated to a suitable reporter gene, such as lacZ, or green fluorescent protein, and inserted into the genome of an appropriate test species. On administration of a chemical agent, cells which are sensitive to the toxic effects of that chemical will express the reporter, which can then be identified using an appropriate assay system. This latter strategy provides the potential for screening a large number of compounds rapidly for their potential toxic effects and also provides information on tissue and cellular specificity. Experiments using transgenic animals can be complex, and care must be taken to ensure that the results are not affected by background activities within the species being used. For example, the introduction of a specific human cytochrome P450 gene may have no effect on the metabolic disposition of a drug or toxin because of the background activity within the mouse. As the toxicity of a chemical agent is determined by a wide range of different factors including drug uptake, metabolism, detoxification and repair, differences between man and the species being used could potentially generate a toxic response in the animal model whereas no toxicity may be observed in man. In spite of these confounding factors, the application of transgenic animals to toxicological issues has enormous potential for speeding up the drug discovery process and will undoubtedly become part of this process in the future.

Optimizing a Rodent Model of Parkinson's Disease for Exploring the Effects and Mechanisms of Deep Brain Stimulation

Deep brain stimulation (DBS) has become a treatment for a growing number of neurological and psychiatric disorders, especially for therapy-refractory Parkinson's disease (PD). However, not all of the symptoms of PD are sufficiently improved in all patients, and side effects may occur. Further progress depends on a deeper insight into the mechanisms of action of DBS in the context of disturbed brain circuits. For this, optimized animal models have to be developed. We review not only charge transfer mechanisms at the electrode/tissue interface and strategies to increase the stimulation's energy-efficiency but also the electrochemical, electrophysiological, biochemical and functional effects of DBS. We introduce a hemi-Parkinsonian rat model for long-term experiments with chronically instrumented rats carrying a backpack stimulator and implanted platinum/iridium electrodes. This model is suitable for (1) elucidating the electrochemical processes at the electrode/tissue interface, (2) analyzing the molecular, cellular and behavioral stimulation effects, (3) testing new target regions for DBS, (4) screening for potential neuroprotective DBS effects, and (5) improving the efficacy and safety of the method. An outlook is given on further developments of experimental DBS, including the use of transgenic animals and the testing of closed-loop systems for the direct on-demand application of electric stimulation.

Synthetic peptide vaccines

This review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modelling human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. Examples of synthetic peptide vaccine developments against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease) are given.

Synthetic peptide vaccines

This review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modelling human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. Examples of synthetic peptide vaccine developments against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease) are given.

Synthetic peptide vaccines

The review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modeling of human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. The review contains examples of the developments of synthetic peptide vaccines against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease).

Introduction

Introduction

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals (Fig. 1). These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or threedimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified (“humanised”) animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with “negative” outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use.The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

FDA to regulate the use of transgenic animals

FDA to regulate the use of transgenic animals

The transgenic mouse assay as an alternative test method for regulatory carcinogenicity studies—Implications for REACH

REACH, an EU regulation that requires the submission of safety data in support of the protection of human and environmental health, mandates that registration should be achieved with the minimum amount of animal testing possible. Under REACH, a two-year carcinogenicity assay may be required for certain chemicals produced at >1000 metric tonnes per year. In addition, some chemicals that are found to be genotoxic will also require testing. Alternative methods have been explored in an attempt to improve the predictivity of this bioassay as well as to reduce the number of animals used for such testing. This research has focused on the use of transgenic/knockout mouse models. Study results from selected models indicate that they are useful in hazard identification, even if they are not entirely suitable for risk assessment on their own. Carcinogenic hazard assessment can be greatly enhanced and animal use reduced if the traditional two-year rat bioassay is combined with a well conducted transgenic mouse assay. Importantly, the use of transgenic animals to supplement a traditional two-year carcinogenicity study may help reduce the number of false negatives, one of the unstated goals of REACH via the precautionary principle.

The pathogenesis of pleural space loculation and fibrosis

Organization of parapneumonic effusions may complicate pneumonia, and, annually, thousands of patients require procedures to treat intrapleural loculation and fibrosis. Surgical procedures are often used for the treatment, as fibrinolytic therapy is now not a routine and is undergoing reassessment. Investigation of mechanisms that underlie intrapleural loculation and fibrosis is therefore timely, as are studies on new strategies to medically address these problems with improved efficacy and safety. Contributions made over the past year include basic and translational studies unified by their broad focus on mechanisms by which the pleural compartment undergoes repair. Intrapleural single-chain urokinase was reported to effectively reverse intrapleural loculation when compared with commercially available agents in rabbits with tetracycline-induced pleurodesis. The ability of exogenous sclerosants to produce intrapleural loculation and fibrosis was compared. Overexpression of transforming growth factor beta in the pleural mesothelium promoted subpleural fibrosis, implicating the mesothelial cell in the pathogenesis of this lesion. A new model of pleurodesis in mice was reported, which could facilitate the use of transgenic animals to study the pathogenesis of pleural injury. New findings consolidate and extend the view that common mechanisms by which intrapleural organization occurs can be exploited to either generate pleurodesis or effectively reverse intrapleural loculation and fibrosis.

Animales genéticamente modificados, primates no humanos. (La visión europea)

European citizens think that more needs to be done to improve the level of welfare/protection of animals used in experiments. In recent years, it has become increasingly apparent that the existing legislation for the protection of animals used in experiments (Directive 86/609/EEC). needs to be revised in order to promote improvements in the welfare of laboratory animals and to further foster the development of alternative methods. The UE citizens’ consultation received the third largest number of responses to a Commission internet consultation ever. Since 1986 important progress has been made in science and new techniques have become available, such as use of transgenic animals, xenotransplantation and cloning. These require specific attention, which the current Directive does not provide for. Nor is the use of animals with a higher degree of neurophysiological sensitivity specifically regulated, such as in the case of non-human primates. This essay wants to reflect about experiments with primates and transgenic animals from the perspective of the institutional european ethics.

Adenylyl Cyclase Isoforms as Novel Therapeutic Targets: An Exciting Example of Excitotoxicity Neuroprotection

The use of transgenic animals lacking one or multiple adenylyl cyclase (AC) isoforms has provided significant information on the roles of AC-dependent signaling in the central nervous system. A recent study provides evidence that AC type 1 (AC1) might be important in glutamate-induced neuronal toxicity. However, the absolute AC specificity revealed in this study contrasts earlier work examining other forms of neurodegeneration. Nonetheless, these observations suggest that specific AC isoforms may represent novel targets for the treatment of central nervous system disorders. It is anticipated that such findings will help catalyze new drug discovery efforts to identify small-molecule modulators of individual AC isoforms.

Liver fibrosis: cellular mechanisms of progression and resolution

Liver fibrosis represents a major worldwide health care burden. The last 15 years have seen a rapid growth in our understanding of the pathogenesis of this clinically relevant model of inflammation and repair. This work is likely to inform the design of effective antifibrotic therapies in the near future. In this review, we examine how the innate and adaptive immune response interacts with other key cell types in the liver, such as the myofibroblast, regulating the process of hepatic fibrosis and, where relevant, resolution of fibrosis with remodelling. Emphasis is placed on the increasing knowledge that has been generated by the use of transgenic animals and animals in which specific cell lines have been deleted. Additionally, we review the increasing evidence that, although significant numbers of wound-healing myofibroblasts are derived from the hepatic stellate cell, significant contributions may occur from other cell lineages, including those from distant sites such as bone marrow stem cells.

Creation and Use of Transgenic Animals in Pharmaceutical and Biomedical Research

The creation of transgenic animals has application in the following areas of pharmaceutical and biomedical research: the production of biopharmaceuticals for human use; the production of organs for xenotransplantation; and the generation of animal models for human genetic diseases. Nuclear transfer technology offers a more precise and efficient way of performing genetic modification and creating transgenic animals than the more traditional method of pronuclear microinjection. This paper will review nuclear transfer as ameans of producing transgenic animals; introduce advantages nuclear transfer technology offers in the field of animal transgenesis; and highlight some of the animal welfare issues and ethical concerns raised by the generation and use of transgenic animals in the aforementioned fields of study. Finally, the influence of objectifying language and terminology used to describe transgenic animals will be considered, and the impact of phrases such as “living bioreactor” and “spare part supplier” examined.

Analysis of the effects of cannabinoids on identified synaptic connections in the caudate‐putamen by paired recordings in transgenic mice

CB(1) cannabinoid receptors are expressed in many neurons in the caudate-putamen. However, it is not known how the activation of these receptors influences synaptic transmission between different neuron classes. The aim was to establish a method for studying identified synaptic connections in the caudate-putamen, and to determine the effects of cannabinoids on these connections. Brain slices were prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) in parvalbumin-positive fast spiking interneurons (PV-FSNs). PV-FSNs were identified based on their fluorescence. Non-fluorescent medium-sized neurons were considered to be medium spiny neurons (MSNs). Synaptic transmission was studied by simultaneous patch-clamp recording from identified neuron pairs. In the case of PV-FSN --> MSN neurotransmission, the synthetic cannabinoid receptor agonist WIN55212-2 lowered the success rate of transmission and the amplitude of successful postsynaptic events. Analysis of miniature inhibitory postsynaptic currents indicated that WIN55212-2 inhibited synaptic transmission presynaptically. WIN55212-2 did not elicit somatodendritic effects in PV-FSNs: membrane potential, membrane current and evoked firing were not changed. WIN55212-2 also depressed the MSN --> MSN neurotransmission. The inhibitory synaptic input to MSNs was only weakly suppressed by endocannabinoids released by depolarized postsynaptic MSNs. The results show that the combined use of transgenic animals and paired-recording techniques allows the study of synaptic connections between rare neurons. Using these techniques, we showed that activation of CB(1) receptors on axon terminals of (i) PV-FSNs and (ii) MSNs leads to presynaptic inhibition of GABAergic synaptic transmission between these axons and their postsynaptic targets, the MSNs. The cannabinoids acted preferentially on axon terminals without effects on the somatodendritic region of the neurons.

New insights into the physiological actions of selenoproteins from genetically modified mice

Selenium (Se) is an essential trace element in mammals. Dietary Se restriction or conditions of Se malabsorption lead to deficiency syndromes or exacerbate established diseases in humans and in many animal models. It is assumed that most, if not all, physiological actions of Se are mediated by selenocysteine (Sec) containing proteins. However, the exact role of particular selenoproteins for certain molecular pathways, for the metabolism of nutrients, hormones or cellular components and for the development and adaptive responses of the organism have often remained elusive. Through the use of transgenic animals, it becomes increasingly feasible to interfere specifically with the expression of single selenoproteins in certain tissues or at certain times. While some transgenic animals exhibit phenotypes that were expected from biochemical studies, in other instances the observed effects were a surprise in view of earlier hypotheses.

Use of Transgenic Animals to Improve Human Health and Animal Production

ContentsTransgenic 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.

Application of Emerging Technologies in Toxicology and Safety Assessment: Regulatory Perspectives

Emerging technologies applied in the regulatory field encompass a group of technologies that are used in addition to or in replacement of the standard toxicology studies conducted to support an Investigational New Drug Application (IND) or New Drug Application (NDA). The standard package includes general toxicology studies of various duration, safety pharmacology studies, genetic toxicology studies, and reproductive toxicology studies. New and emerging technologies applied to the regulation of new drugs include the use of novel biomarkers, transfected cells and transgenic animals, and the "omics" technologies (toxicogenomics, proteomics, and metabonomics). These technologies are at various stages of regulatory development and acceptance. For example, the use of transgenic animals have gained acceptance by regulatory authorities to replace a 2-year carcinogenicity assay. Alternatively, the "omics" technologies are not sufficiently advanced to achieve regulatory acceptance as replacements, although these assays have a role early in drug development and they may prove useful as supplements to standard studies. Data from these assays have been used to address specific mechanistic questions in combination with standard toxicology assays.