he most popular approach for generating transgenic mammals is the direct injection of transgenes into one pronucleus of a fertilized oocyte. In the past 15 years microinjection has been successfully applied in laboratory as well as in farm animals. The frequency of transgenic founders, although highly different between the species, is efficient enough to render this technique applicable to a wide range of mammals. The expression levels and patterns of a transgene are initially influenced by the construction of the transgene. However, the overall phenotype of a transgenic organism is influenced by several genetic and environmental factors. Due to the features of this technique not all of the genetic factors can be experimentally controlled by the scientist. In this article we will emphasize some peculiarities which have to be taken into account for the successful performance of transgenesis by pronuclear microinjection

The development of gene targeting technology, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination, and the application of this technique to murine embryonic stem cells has made it possible to alter the germ-line of mice in a predetermined way. Gene targeting has enabled researchers to generate mouse strains with defined mutations in their genome allowing the analysis of gene function in vivo. This review presents the essential tools and methodologies used for gene targeting that have been developed over the past decade. Special emphasis has been laid on the available embryonic stem cell lines and the importance of the genetic background. Also, the state-of-the art of gene targeting approaches in species other than mice will be discussed.

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

Increasingly sophisticated and precise molecular genetic tools are applied to mice in order to study the cellular mechanisms underlying higher brain functions, including learning and memory. However, several studies have produced unclear or conflicting results. One reason for this is that performance in the behavioural tests used to assess learning and memory is influenced by various non-cognitive phenomena and can thus easily be affected by mutations through mechanisms unrelated to memory function. We conducted principal component analysis on data from 3003 mice tested using a standardized protocol to demonstrate this for the Morris swimming navigation test, one of the most widely used paradigms to assess memory and hippocampal function. In addition, we present a meta-analysis showing that genetic background and environment alone produce sufficient variation to span the range of most, if not all, behavioural variables and can thus easily mask or fake mutation effects if genetic studies are not designed properly. We suggest that the chance of obtaining useful results is maximized if behavioural deficits are differentiated by combining complementary behavioural protocols and by analysing multiple complementary parameters in each of them. Mutation effects must be contrasted statistically against the influences of genetic background and environment. In many situations, this is most efficiently achieved if (i) mutations are backcrossed to and maintained in one or (preferably) two well-characterized, commonly available inbred strains and (ii) if mutant and wild-type littermates are analysed on a hybrid or mixed genetic background, that is in F1 or F2 generations derived from the inbred stocks

The most important tool for obtaining insight into the function of genes is the use of mutant model organisms. Homologous recombination in embryonic stem cells allows the systematic production of mouse mutants for any gene that has been cloned. Gene trap strategies have been designed to interrupt even unknown genes which are tagged by the inserted vector and can be characterised structurally and functionally. Complementary to such 'gene-driven' approaches, 'phenotype-driven' approaches are necessary to identify new genes or gene products through a search for mutants with specific defects, uncovering the function of genetic pathways in physiological and pathological processes. Mutagenesis using the alkylating agent N-ethyl-N-nitrosourea (ENU) is a powerful approach for the production of such mouse mutants. Since ENU induces mainly point mutations in premeiotic spermatogonia, this strategy allows the production of multiple alleles of a particular gene, which is pivotal for a fine tuned analysis of its function.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock-out in vitro, ES cells are differentiated into three-dimensional structures called embryoid bodies, because of their ability to mimick post-implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro

In the pathogenesis of autoimmune diseases, tolerance against self-determinants is lost and autoreactive lymphocytes are activated leading to pathological damage of single or multiple organs. Viral and other microbial infections have been implicated in these processes. Viruses may induce immunopathological damage by maintaining a chronic immune response against the locally persisting infectious agent. Alternatively, viruses may help to initiate anti-self immunoreactivity, e.g. by induction of an inflammatory milieu needed to overcome tolerance against self-antigens. Presentation of viral antigens and/or previously immunologically ignored self-antigens in secondary lymphoid organs is most probably the key event in the initiation of autoimmune diseases. Translocation of antigens to secondary lymphoid organs and primary induction of T cell responses is primarily mediated by dendritic cells (DCs). We discuss here two transgenic models of autoimmune diseases where DC-mediated antigen transport initiated autoimmune responses against microbial neoself antigens. In the first model, dose and timing of antigen delivery by DCs and turnover of antigenic peptides presented by DCs are the main parameters regulating the outcome of autoimmune diabetes. In the second model, chronic stimulation of organ-specific immune responses via DCs leads to severe cardiovascular immunopathology with arteritis, myocarditis and eventually dilated cardiomyopathy. Taken together, transgenic mouse models are valuable tools for delineating basic pathogenic mechanisms and evaluating therapeutic strategies to interfere with early detrimental processes that lead to manifest autoimmune diseases.

Genetic methodologies have provided powerful means for investigating the cellular and molecular mechanisms of biological functions. Cell-cell and cell-extracellular matrix interactions in particular have been studied in different functional systems with genetically modified animals. In the peripheral and central nervous system, many aspects of specific processes based on such interactions, including myelination, synaptic transmission and plasticity, have been elucidated at the cellular and molecular level. Importantly, genetic approaches have greatly advanced the understanding of pathologies resulting from impaired cellular interactions in the brain and the periphery. In this review, some of the most relevant genetic mouse models in cell biology and the methodologies employed for their production will be described. In addition their usefulness for studies of the mechanisms of hereditary neuropathies, learning and memory, and tumorigenesis will be illustrated.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are able to lyse suitable target cells. CTLs recognize a specific peptide epitope presented by major histocompatibility complex (MHC) class I molecules on the target cells, whereas NK cells lyse targets that express no or low MHC class I molecules. Using perforin-deficient mice, we provide evidence that both NK cells and CTLs exclusively use perforin-dependent cytolysis as an effector mechanism in vitro, as well as in vivo. This review summarizes the most important role of perforin-dependent cytolysis in a wide variety of bacterial and viral infections, in tumour surveillance, in immunopathology and in autoimmunity.

Genetically modified mice have become an invaluable tool in modern biomedical and basic research. This review provides an overview of knockout and transgenic mice studied with regard to their cutaneous wound healing properties. In addition, several gene transfer studies are briefly introduced, which have further highlighted our knowledge on individual gene function in wound healing

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrPC. Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrPC is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.

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.

Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3',5'-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism

There is a great need for closer integration between physiologists and molecular biologists. These two research fields might soon form a single research discipline under the umbrella of molecular physiology. One of the areas where this interaction may be particularly fruitful is with somatic gene transfer using replication-deficient viral vectors. We applied this approach to study the central control of the cardiovascular system at the level of the nucleus of the solitary tract (NTS). We assess critically this new methodology as applied to experiments in an integrative environment (such as the whole animal). The usefulness of in vivo gene transfer is illustrated by an experiment where viral gene delivery helped to circumvent the problem imposed by an absence of a specific pharmacological blocker of the enzyme, endothelial nitric oxide synthase (eNOS). The pros and cons of using adenoviral vectors as opposed to conventional pharmacological approaches are discussed. We conclude that the use of adenoviruses to manipulate genes offers a new avenue for physiologists studying neuronal mechanisms in integrative models.

In the past several years, the progress in the development of molecular biology and the elucidation of gene sequences has created new approaches for studying biological functions and developing new diagnostic and therapeutic strategies. One of the most exciting advances has been the development of antisense technology, which represents a new strategy allowing modulation of protein synthesis with high specificity by preventing protein expression at the level of RNA or DNA. Many classical pharmacological approaches in neurobiological research are often based on the inhibition of biologically active proteins, such as receptors for neurotransmitters, or enzymes involved in neurotransmitter synthesis or degradation. The use of antisense oligodeoxynucleotides offers an alternative tool to manipulate selectively the expression of neurotransmitters or their receptors in neuronal tissue. This approach is especially useful when selective, high-affinity antagonists are not available. As a result, this technology has gained acceptance in the study of cell signalling mechanisms and the molecular basis of neuronal function. This paper provides a brief background to the antisense technique and explores methodological aspects, particularly in the whole animal. The use of the antisense technology in studies focused on central mechanisms regulating the cardiovascular system is then discussed.

Altering [K+]o might have different effects on action potential duration (APD) in myocytes from different regions. Therefore, the effects of [K+]o on regional differences in action potential characteristics were investigated in sub-endocardial, mid-myocardial and sub-epicardial myocytes isolated from the base of guinea-pig left ventricular free wall using three different [K+]o (2.7, 5.4 and 8.1 mM KCl). Action potentials were recorded using the switch-clamp technique at 0.5 Hz. Increasing [K+]o from 2.7 to 8.1 mM shortened the action potential duration to 90 % repolarization (APD90; mean APD90 values in sub-endocardial, mid-myocardial and sub-epicardial myocytes were, respectively, 295 +- 9, 286 +- 9 and 266 +- 8 ms in 2.7 mM [K+]o, 270 +- 7, 255 +- 7 and 215 +- 7 ms in 5.4 mM [K+]o, 234 +- 7, 212 +- 10 and 155 +- 8 ms in 8.1 mM [K+]o), depolarized the resting potential, and reduced the amplitude of the action potential. The effect of increasing [K+]o on action potential characteristics was more pronounced in sub-epicardial myocytes than in sub-endocardial and mid-myocardial myocytes. The regional differences in APD90 in 5.4 mM [K+]o were increased in 8.1 mM [K+]o and abolished in 2.7 mM [K+]o. In conclusion, changing [K+]o produces more pronounced effects on action potentials in sub-epicardial myocytes than in sub-endocardial myocytes, modifying the normal heterogeneity of action potentials. The differences in the response of sub-epicardium and sub-endocardium to [K+]o may contribute to the flattening or inversion of the T wave commonly seen in patients presenting with hypokalaemia and the upright and tall T waves observed in electrocardiograms recorded during hyperkalaemia, although the underlying ionic currents remain to be determined.

This study was performed to compare glucose metabolism in anaesthetised spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) in an attempt to clarify whether this animal model of hypertension approximates the insulin-resistant state seen in human hypertension. With this aim the minimal model of glucose kinetics was applied to glucose and insulin data derived from a 12-sample, 120 min intravenous glucose tolerance test (IVGTT) performed in ten SHR and nine WKY rats under pentobarbital anaesthesia. This method provided two metabolic indices: the glucose effectiveness, SG, which quantifies the ability of glucose per se to enhance its rate of disappearance and to inhibit hepatic glucose production, and the insulin sensitivity, SI, which measures the ability of insulin to enhance plasma glucose disappearance and to inhibit hepatic glucose production. Systolic and diastolic arterial pressures in the SHR group were significantly higher (P < 0.0005) than in the WKY group. Mean SG and SI estimates from the SHR group (SG = 16.2 (± 2.0) × 10-2 dl min-1 kg-1 and SI = 12.5 (± 1.9) × 10-4 dl min-1 kg-1 (µU ml-1)-1) were not significantly different (P > 0.05) from mean estimates that characterised the WKY group (SG = 13.1 (± 1.5) × 10-2 dl min-1 kg-1 and SI = 15.8 (± 4.3) × 10-4 dl min-1 kg-1 (µU ml-1)-1). This result is in contrast with reported findings from humans in which insulin sensitivity is significantly reduced in the presence of hypertension.

In addition to its known effect on renal tubular transport, frusemide (furosemide) has been shown to affect renal circulation. This study in the anaesthetised rat examined the influence of frusemide (bolus 0.25 or 0.5 mg kg-1 I.V., then infusion delivering the same dose over 1 h) on renal cortical and medullary circulation measured as laser-Doppler blood (cell) flux. The responses were compared with simultaneously measured changes in renal excretion and in the tissue admittance, an index of medullary ionic hypertonicity of the interstitium. Renal vascular responses to frusemide were significant but not dose dependent. During low-dose frusemide infusion cortical flux decreased 11.5 ± 0.9 % and medullary flux decreased 32.3 ± 3.5 % (difference significant at P < 0.001). During high-dose infusion the decreases were by 13.5 ± 1.4 and 29.3 ± 3.8 %, respectively (difference significant at P < 0.001). Sodium excretion increased 15-fold (by 3.7 ± 0.4 µmol min-1) and 30-fold (by 5.9 ± 1.1 µmol min-1) during low- and high-rate infusion of frusemide, respectively. By contrast, medullary tissue admittance decreased similarly with the two doses: maximally by 13.4 ± 1.4 and 10.9 ± 0.9 %, respectively. The observations that an exaggerated post-frusemide decrease in blood flow within the medulla coincided with decreasing tissue admittance in this zone and that neither medullary blood flow nor admittance changes were related to the dose suggest a causal relationship between interstitial ionic hypertonicity and vascular resistance. We propose that the post-frusemide decrease in medullary tissue NaCl depressed medullary circulation by inhibiting local generation of vasodilator prostaglandins.

The effects of nitric oxide synthase (NOS) inhibition (effected using L-NAME, 14 mg (kg body mass (BM))-1, administered intravenously) on systemic and renal circulation and renal excretory function has been investigated in anaesthetized Wistar rats subjected to one of two different degrees of isotonic extracellular (EC) volume expansion (40 and 60 ml kg-1 (240 min)-1). The administration of L-NAME resulted in an increase in mean arterial blood pressure and total peripheral vascular resistance (TPR), and a significant reduction in cardiac output (CO) and the kidney fraction of CO in both experimental groups. The total renal blood flow (RBF) dropped from 557 ± 43.4 to 149 ± 13.1 ml min-1 (100 g BM)-1 and from 592 ± 45.9 to 191 ± 16.3 ml min-1 (100 g BM)-1 in the 40 and 60 ml kg-1 (240 min)-1 experimental volume expansion groups, respectively. A redistribution of the intrarenal circulation from the medulla of the kidney toward the cortex may have occurred. The NOS inhibition induced a significant decrease in the glomerular filtration rate (GFR; from 1.18 ± 0.10 to 0.53 ± 0.08 ml min-1 (100 g BM)-1 and from 1.26 ± 0.07 to 0.73 ± 0.08 ml min-1 (100 g BM)-1 in the 40 and 60 ml kg-1 (240 min)-1 experimental volume expansion groups, respectively), and the filtration fraction increased. The urine excretion dropped in parallel with the GFR, while the reduction in sodium and potassium excretion was more marked than that of the GFR, raising the possibility of a direct effect on the kidney tubules. The difference in EC volume expansion (the calculated increases in the EC volume in the last 90 min were 1.30 and 5.44 % in the two time control groups and 3.66 and 7.45 % in the two L-NAME-treated groups) did not induce any significant modification of the L-NAME effect.

It is generally accepted that pheromones act by stimulating of the dendritic receptors of the olfactory neurones massed in the olfactory epithelium. This study was designed to ascertain whether it is possible for the boar pheromone androstenol (5alpha-androst-16-en-3-ol) to be transported from the nasal cavity of anaesthetized gilts to the brain and hypophysis via local transfer from the blood in the perihypophyseal vascular complex. The experiment was performed on days 18-21 of the porcine oestrous cycle (crossbred gilts, n = 6). Tritiated androstenol (3H-A; total amount 108 d.p.m. (758 ng)) was applied for 1 min onto the respiratory part of the nasal mucosa, 4-6 cm from the opening of the nares. Arterial blood samples from the aorta and from the carotid rete were collected every 2 min during the 60 min period following administration of the steroid. Total radioactive venous effluent from the head was removed and an adequate volume of homologous blood was transfused into the heart through the carotid external vein. At the end of the experiment gilts were killed and tissue samples of the hypophysis and some brain structures were collected to measure radioactivity. In addition, corresponding control tissues were collected from three untreated gilts and from three heads of gilts 60 min after 3H-A was applied post mortem into the nasal cavity. The concentration of 3H-A was significantly higher (P < 0.0001) in the arterial blood of the carotid rete than that of aorta. The mean rate of 3H-A counter current transfer from venous to arterial blood in the perihypophyseal vascular complex, expressed as the ratio of the 3H-A concentration in arterial blood of the carotid rete to the 3H-A concentration in blood sampled simultaneously from the aorta, was 1.96 ± 0.1. The concentration of 3H-A in plasma from the venous effluent from the head ranged from 1.3 to 1.8 pg ml-1. During the 60 min period of the experiment, 0.68 % of the total applied dose of 3H-A was resorbed from the nasal cavity into the venous blood. Moreover, we found that 3H-A was present in the olfactory bulb (P < 0.01), amygdala, septum, hypothalamus, adenohypophysis, neurohypophysis (P > 0.05) and perihypophyseal vascular complex (P < 0.01). These results demonstrate that, in anaesthetized gilts, the boar pheromone androstenol may be resorbed from the nasal mucosa, transferred in the perihypophyseal vascular complex into arterial blood supplying the brain and hypophysis, and then arrested in the hypophysis and certain brain structures. We suggest that in addition to the standard neural pathway for signalling pheromones, another pathway exists whereby androstenol, as a priming pheromone, may be resorbed from the nasal cavity into the bloodstream and then pass locally from the perihypophyseal vascular complex into the arterial blood supplying the brain and hypophysis, thus avoiding the first passage metabolism in the liver.