Search

Cryopreservation of genetically modified mouse lines prevents the loss of specific mutants that are of enormous scientific value for both basic and applied research. Cryopreservation of spermatozoa or preimplantation embryos enables discontinuation of breeding as well as archiving of specific lines for future studies. Regarding active inter-laboratory exchange of mutants, cryopreserved material is more advantageous to transport than live animals. However, transportation stress should not be trivialized. Security scanning of transport boxes at airports and customs, in particular, as well as additional cosmic radiation, pose a threat to undefined dosages of irradiation exposure. To simulate this, cryopreserved samples of mouse spermatozoa and preimplantation embryos were exposed to an X-ray dosage of 1 mGy in an X-ray machine. For subsequent investigation of the cell integrity of irradiated spermatozoa and embryos, spermatozoa forward motility as well as embryo developmental capacity and apoptosis values were examined and compared with nonirradiated control samples. The percentage of forward-moving spermatozoa per sample appears to be significantly reduced after irradiation exposure. The in vitro developmental capacity of preimplantation embryos as well as their relative share of apoptotic cells do not seem to be influenced by irradiation exposure. This leads to the assumption that, at least in preimplantation embryos, X-ray dosages of 1 mGy do not induce sudden severe cellular harm. Nevertheless, stochastic effects of ionizing irradiation, such as mutations, do not have a dosage threshold and always represent the potential danger of alterations to cells and cellular components, especially the DNA. This could lead to undefined mutations inducing genetic drift, in the worst case to the loss of a mutant line. We therefore strongly recommend minimizing "transportation stress," in particular by irradiation exposure, to keep its potential consequences in mind, and to standardize shipping procedures.

Article 23(2) of the European Union Directive 2010/63/EU, which regulates welfare provisions for animals used for scientific purposes, requires that staff involved in the care and use of animals for scientific purposes be adequately educated and trained before they undertake any such work. However, the nature and extent of such training is not stipulated in the Directive. To facilitate Member States in fulfilling their education and training obligations, the European Commission developed a common Education and Training Framework, which was endorsed by the Member States Competent Authorities. An Education & Training Platform for Laboratory Animal Science (ETPLAS) Working Group was recently established to develop further guidance to the Learning Outcomes in the Framework, with the objective to clarify the levels of knowledge and understanding required by trainees, and to provide the criteria by which these Learning Outcomes should be assessed. Using the Framework document as a starting point, assessment criteria for the Learning Outcomes of the modules required for Function A persons (carrying out procedures on animals) for rats, mice and zebrafish were created with sufficient detail to enable trainees, providers and assessors to appreciate the level of knowledge, understanding and skills required to pass each module. Adoption and utilization of this document by training providers and accrediting or approving bodies will harmonize introductory education and training for those involved in the care and use of animals for scientific purposes within the European Union, promote mutual recognition of training within and between Member States and therefore free movement of personnel.

Genetically modified animals are unique models with enormous scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if a sufficient number of samples have been cryopreserved. To maintain the opportunity to recover a line, it is mandatory to assess the quality of the cryopreserved samples and to assure safe long-term storage conditions. Here, we investigated the revitalization rate of cryopreserved pre-implantation embryos stored in-house up to 158 months, of imported (and shipped) embryos, and of embryos received after in vitro fertilization. The storage period did not affect the revitalization rate, whereas the recovery of imported embryos was significantly reduced, possibly due to shipment conditions. The genotypes of genetically modified pups received following embryo-transfer were slightly smaller than expected by Mendelian laws. Intensive investigations of the hygienic state of the cryopreserved samples and the equipment used never showed microbiological contamination of a sample within a cryo-tube. However, environmental organisms were found frequently in the permanent freezers and dry shippers used. Since such contamination cannot be completely excluded and an embryo-transfer might not lead in all cases to a secure rederivation, foster mothers and revitalized pups should be housed in an intermediate facility and their health assessed before introducing them into the target facility.

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

Generation and cryopreservation of transgenic mice depend on reliable and continuous production of pre-implantation embryos. To suppress circannual and circadian rhythms driving the physiological and sexual behaviour of free living animals, laboratory animals are housed under standardized conditions. It remains to be elucidated if the artificial climate can cover all environmental effects. Here, we report that the humidity in an animal facility affects the embryo yield. The weather at the location of the facility, especially the temperature, influences the climate within an animal facility; weather peaks are obviously covered in part only, even if the facility is equipped with a powerful air-conditioning supply. Subsequently, external weather changes interact with the environment within the facility, influencing the production of embryos. Furthermore, noise and/or vibrations as generated by construction works, negatively affect the embryo yield.

Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance.

Neurodegenerative diseases often result in neuronal cell death, but the molecular mechanisms responsible are not fully understood. The expression and activation by phosphorylation of the c-Jun transcription factor plays an important role for the fate of affected neurons in response to injury. c-Jun is phosphorylated at serines 63 and 73 by the c-Jun N-terminal kinases and c-Jun N-terminal phosphorylation augments the transcriptional activity of c-Jun. Two approaches in neurodegeneration were investigated: The transection of the medial forebrain bundle to study neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads as a long-term reaction to degeneration of the affected SNC neurons. A central component of the axotomy-induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. The focal cerebral ischemia, induced by occlusion of the arteria cerebri media and the subsequent reperfusion, serves as a suitable in vivo model for stroke. Also, ischemia leads to upregulation and activation of c-Jun and its target genes. Here the key role of c-Jun for the fate of neurons following degeneration is discussed with data received from experiments performed in Manfred Zimmermann's department investigating the effects of c-Jun on its target genes and on factors influencing c-Jun expression and activation.

Naïve CD4+ T cells differentiate into effector T helper 1 (Th1) or Th2 cells, which are classified by their specific set of cytokines. Here we demonstrate that loss of JunB in in vitro polarized Th2 cells led to a dysregulated expression of the Th2-specific cytokines IL-4 and IL-5. These cells produce IFN-gamma and express T-bet, the key regulator of Th1 cells. In line with the essential role of Th2 cells in the pathogenesis of allergic asthma, mice with JunB-deficient CD4+ T cells exhibited an impaired allergen-induced airway inflammation. This study demonstrates novel functions of JunB in the development of Th2 effector cells, for a normal Th2 cytokine expression pattern and for a complete Th2-dependent immune response in mice.

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.

We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene.

Expression of the apoptosis-inhibitory protein Bcl-2 has frequently been detected in human cancer including mammary carcinoma. The functional significance of its expression has been well established in experimental tumors of the lymphoid system, however, remains to be elucidated for epithelial tumors. In order to assess the role of Bcl-2 in mammary tumorigenesis we have generated WAP-bcl-2 transgenic mice. The strong overexpression of Bcl-2 in lactating mammary glands was preserved during early postlactational involution and apoptosis of alveolar epithelial cells was prevented without influencing the dedifferentiation of the milk-producing epithelium. Although Bcl-2 overexpression was not sufficient to induce spontaneous tumors it, however, led to an accelerated development of MMTV myc transgene-induced mammary tumors. In the mammary glands of MMTV myc transgenic mice, a high proportion of apoptotic cells was detected which was significantly reduced in the mammary glands of WAP-bcl-2/ MMTV myc double transgenic mice. Taken together, these results suggest that Bcl-2 contributes to mammary tumorigenesis by inhibiting apoptosis.

The number of genetically modified mice is increasing rapidly. Several limitations when working with these animals are to be considered: small colonies, the continued danger of loss, often a limited breeding-success, the need to keep those mutants in stock, difficult and costly import-procedures, and also a major (scientific) value of those mutants often available only with major restrictions. To gather relevant information about all active and archived genetically modified mouse lines available in-house (>1.500) and to deal with a unique resource for several, quite different purposes, a data base was developed enabling optimum knowledge management and easy access. The data base covers also legal restraints and is being linked with the institutional publication repository. To identify the lines available detailed information is provided for each line, as the international designation, a short name, the characterization/description, and the genetic modification including the technique used therefore. The origin of the mutation (gene-ID# and donor organism), the origin of regulatory elements and their donors are listed as well as the genetic background, back-cross generation, phenotype, possible publications, keywords, and some in-house information. Also aspects of animal welfare, obligations to record genetically modified organisms, and technology transfer are displayed; the latter to make licenses possible (if legally permitted). Material transfer agreements, patents, or legal restrictions are listed. This data base helps to avoid double-imports, saves animals and costs since a redundant generation or import can be omitted. However, this is a contribution to the 3R principles developed by Russell and Burch.

Activation of mature lymphocytes requires in addition to the TCR contact with the corresponding antigen the binding of the CD8 or CD4 co-receptors to MHC class I or class II proteins respectively. To investigate the contribution of the CD8-class I interaction to the elimination of autoreactive T cells during negative selection in the thymus we generated two types of transgenic mice. One set expressed a modified Kb molecule which contained a human HLA-A2 alpha 3 domain, thereby missing the binding residues for the murine CD8 molecules. The second set of mice expressed an anti-Kb specific TCR. Both lines were crossed and in the resulting double transgenic mice the development of Kb-reactive T cells was followed with an anti-clonotypic antibody. Surprisingly, efficient clonal deletion in the thymus was still observed, although the reduced CD8-class I adhesion abrogated effector functions in vivo and in vitro. These results imply that even T cells with intermediate affinity for self are negatively selected in the thymus despite the fact that they are not able to react against self antigens in the periphery. Thus a safety window is created which decreases the risk of autoaggression.

BCL-2 overexpression is frequently detected in nonmelanoma skin cancer. In normal skin, BCL-2 expression is restricted to the basal cell layer and the hair follicle bulge. Both contain stem cells targeted by carcinogens upon initiation of mouse skin carcinogenesis. It is unknown whether the anti-apoptotic activity of BCL-2 is involved in the susceptibility of this cell type to malignant transformation. If so, extending the pool of BCL-2-expressing cells to suprabasal skin layers should increase the likelihood of skin tumour formation.To resolve this issue, we generated a novel transgenic mouse line overexpressing BCL-2 in suprabasal layers of the epidermis. The influence of suprabasal BCL-2 on tumour formation was then tested by chemically inducing skin cancer using the two-stage initiation-promotion protocol.Bcl-2 expression neither influenced the incidence nor the multiplicity of papillomas upon chemical tumour induction with 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), nor their progression to carcinomas.Suprabasal expression of BCL-2 in skin does not increase the formation of papillomas or their malignant progression to squamous cell carcinomas in two-stage mouse skin carcinogenesis.

The E2 early protein of human papillomaviruses (HPV) has been found associated with the mitotic spindle therefore being implicated in the partition of the replicated viral DNA to daughter cells. In addition, E2 proteins bind to the upstream regulatory region of the virus and to cellular promoters modulating thereby cellular transcription and differentiation. In many cervical cancers, the E2 reading frame is interrupted upon incorporation of the viral genome into the host DNA. This results in the loss of the E2 mediated transcriptional repression and uncontrolled expression of the viral oncogenes. All these results have been obtained in transfected cells but no information is available on the E2 effects in the context of the entire organism. Transgenic mice were generated expressing the E2 protein of HPV11 under the control of the Ubiquitin C promoter. E2 mRNA is present in all mice tissues analysed and the E2 protein expressed in the skin (the target tissue of HPV11) was shown by Western blotting, albeit at a very low level. Analysis of the transgenic mice shows no major histological changes in the skin or all other tissues investigated. These data indicate that in transgenic mice the human papillomavirus type 11 E2 does not grossly modulate cellular proliferation or differentiation events.

Stroke therapy aims to save penumbral tissue from apoptosis that is activated in response to the ischemic injury. Since the c-Jun transcription factor plays a crucial role in promoting apoptosis, inhibition of its activation might reduce the final infarct size and thus increase functional outcome. To test this hypothesis we made use of four genetically modified mouse lines influencing the c-Jun pathway at various steps. Upon transient middle cerebral artery occlusion for 90 min and 24 h of reperfusion, infarct volume and number of ATF-2-, TUNEL- and cleaved Caspase-3-positive cells were determined in conditional c-Jun knock-out mice (cond. c-Jun), mice overexpressing JunB (JunBtg), mice lacking the phosphoacceptor serines 63 and 73 of c-Jun (JunAA) and in mice overexpressing Bcl-2 (Bcl-2tg). Cond. c-Jun as well as JunAA mice did not show significant differences in the infarct size when compared to their non-mutant controls. By contrast smaller infarct volumes were detected in transgenic mice merely attenuating c-Jun action (JunBtg and Bcl-2tg). ATF-2, TUNEL or cleaved Caspase-3 staining revealed no significant differences between the experimental groups. A complete lack of functional c-Jun might be compensated by other cellular mechanisms, in contrast to its reduced function. Thus, our data suggest that attenuation rather than a complete block of c-Jun action appears to be more promising for therapy of stroke.

The t(2;5)(p23;q35) translocation is associated with a high percentage of anaplastic large-cell lymphomas (ALCL) of T- or null-cell phenotype. The translocation produces an 80 kDa hyperphosphorylated chimeric protein (p80) derived from the fusion of the anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM). The NPM-ALK chimeric protein is an activated tyrosine kinase that has been shown to be a potent oncogene and presumably plays a causative role in lymphomagenesis.A transgenic mouse line was generated, where the human NPM-ALK cDNA is driven by the lck promoter conferring transgene expression to early T-cells.Mice rapidly developed large cell lymphoblastic lymphomas with a median latency of 8 weeks, primarily involving the thymus, with lymph node as well as histologically evident extranodal organ infiltration by large tumor cells.The transgenic approach described provides direct evidence for the strong transforming potential of NPM-ALK in T-cells and furthermore represents a system for the analysis of the oncogenic events mediated by NPM-ALK in vivo, which might be instrumental in the development of tyrosine kinase inhibitor therapies of potential clinical use.

Transection of the medial forebrain bundle is a well established approach to investigate neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads in mouse within 50 days post transection to degeneration of up to 70% of the affected SNC neurons. A central component of the axotomy induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. However, the role of c-Jun in the process of neuronal degeneration is not fully understood. Since null mutations of c-Jun cause embryonic lethality, this study was designed to investigate the impact of two c-Jun modulating proteins on neuronal survival after axotomy in transgenic mice: JunB, a Jun family member affecting c-Jun expression, and Bcl-2, an antiapoptotic protooncogene interacting among others with the c-Jun N-terminal kinases. In JunB as well as in Bcl-2 transgenic mice the long term survival rate of transected SNC neurons was remarkably increased when compared to wildtype controls. These effects were obviously achieved by cellular modulations directly following axotomy: Whereas JunB overexpression attenuated c-Jun induction and simultaneously led to a higher phosphorylation rate of c-Jun in SNC neurons, Bcl-2 overexpression did not influence c-Jun expression, but resulted in a reduced phosphorylation state of c-Jun in transected SNC neurons. We therefore conclude that the early phosphorylation rate of c-Jun might play an important role for the long term fate of transected neurons.

Hereditary hyperekplexia is caused by disinhibition of motoneurons resulting from mutations in the ionotropic receptor for the inhibitory neurotransmitter glycine (GlyR). To study the pathomechanisms involvedin vivo, we generated and analyzed transgenic mice expressing the hyperekplexia-specific dominant mutant human GlyR α1subunit 271Q. Tg271Q transgenic mice, in contrast to transgenic animals expressing a wild-type human α1subunit (tg271R), display a dramatic phenotype similar to spontaneous and engineered mouse mutations expressing reduced levels of GlyR. Electrophysiological analysis in the ventral horn of the spinal cord of tg271Q mice revealed a diminished GlyR transmission. Intriguingly, an even larger reduction was found for GABAA-receptor-mediated inhibitory transmission, indicating that the expression of this disease gene not only affects the glycinergic system but also leads to a drastic downregulation of the entire postsynaptic inhibition. Therefore, the transgenic mice generated here provide a new animal model of systemic receptor interaction to study inherited and acquired neuromotor deficiencies at different functional levels and to develop novel therapeutic concepts for these diseases.

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined,in vivoandin vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cellsin vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands bothin vivoandin vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, inlprmice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Transgenic mice provide one of the best experimental systems to study gene function in vivo. Although transgenic mice carry the additional genetic material in every cell of the body, expression of the transgenes under the control of constitutive promoter/enhancer units, such as the murine major histocompatibility complex promoter H2-Kb, the CMV enhancer/promoter, the murine Pgk-1 promoter or the chicken cytoplasmatic β-actin promoter is often restricted to a limited number of tissues (1–5). However, for most experimental dominant gain-of-function approaches addressing the role of a given protein in a multicellular organism an ubiquitous expression of the transgene might be highly desired. Here we report the application of a powerful expression vector using the 5′-flanking region of the human ubiquitin C gene that allows very efficient expression of a given transgene in a broad range of tissues. The structure of the ubiquitin proteins is highly conserved during evolution. In human, there exist several ubiquitin proteins which are encoded by a multigene family (6). According to the general requirement of these proteins for ATP-dependent, non-lysosomal intracellular protein degradation ubiquitin proteins have been found in all eukaryotic cells examined so far (for review see 7,8). Previous work has demonstrated that the human ubiquitin C promoter is very active in conferring expression of exogenous genes following transient transfection of the appropriate expression vectors in various cell lines (9; Schorpp and Angel, unpublished). In view of its powerful and ubiquitous activity in tissue culture cells we have used the human ubiquitin C promoter as regulatory unit to drive overexpression of two cellular genes, junB and bcl-2α, in transgenic mice. As shown in Figure 1A, Ubi-JunB and Ubi-Bcl-2α carry 1.2 kb of the human ubiquitin C promoter region (position –1225 to –6) fused to either the coding region of the mouse junB gene (position +240 to +1485; 10) or the human bcl-2α cDNA (11) respectively. 3′ of the coding sequences, 150 bp of 3′ nontranslated sequences of the human c-jun gene (12) and the splice and poly(A) sequences of SV40 were inserted. These gene constructs were used to generate four independent Ubi-junB and two bcl-2α transgenic mouse lines. Two out of four Ubi-junB lines (1598 and 1605) and both bcl-2α lines (2266 and 2272) showed high level expression of the transgene in all tissues which were examined (Fig. 1B and C). Efficient expression from the Ubi-JunB construct could be detected at early stages of development (10.5, 11.5, 12.5, 14.5 and 15.5 d.p.c.; data not shown) and in tissues of adult animals (Fig. 1B). The levels of expression of junB and bcl-2α transgenes in some tissues were up to 20-fold higher than of the endogenous transcript (e.g. liver and brain, and brain; Fig. 1B and C respectively). The ubiquitous presence of the bcl-2α transgene product was also confirmed by western blot analysis (data not shown). The human ubiquitin C sequences described here allow expression of transgenes in an even wider range of tissues compared to the promoter/enhancer units that are routinely used. Transgenes driven by H2-Kb, CMV or Pgk-1 regulatory sequences are highly expressed in spleen, lung and salivary glands (H2-Kb; 1), in spleen, stomach and heart (CMV; 2,3), or in heart, kidney and brain (Pgk-1; 4). Only very low expression has been found for each regulatory unit in certain tissues, such as stomach and brain (H2-Kb), liver (CMV and Pgk-1) and spleen (Pgk-1). Most importantly, the human ubiquitin C promoter is able to confer high expression also in those tissues in which the other promoter/enhancer sequences are hardly active. Therefore, the human ubiquitin C 5′-flanking sequences appear to be a powerful transcriptional control unit useful for experiments in which transgene expression in a very broad range of tissues is required.

Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses.

Functional rescue of the glomerulosclerosis phenotype in Mpv17 mice by transgenesis with the human Mpv17 homologue. The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.

Antigen-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. A liver-specific inducible promoter was used to drive the expression of the major histocompatibility complex antigen Kb in transgenic mice. Minute amounts of Kb, expressed exclusively on hepatocytes, induced tolerance by partial down-regulation of the T cell receptor (TCR) on the self-reactive CD8+ cells. Contact of these tolerant T cells with high concentrations of Kb after induction led to complete down-regulation of TCR. Thus, tolerant T cells are susceptible to further tolerogenic signals and reach different levels of tolerance depending on antigen dose.

A lambda gt11 cDNA library was prepared from activated guinea pig T-lymphocyte blasts. cDNA clones coding for the alpha-chain and beta-chain of the constant region of the guinea pig T-cell receptor were isolated by means of crosshybridization using the corresponding mouse cDNA genes. The guinea pig and the corresponding mouse cDNA genes hybridized in Northern blots with mouse mRNA of the same size. Guinea pig T-cell receptor mRNA showed the same size like its mouse counterpart. In contrast to the alpha-chain clone, genomic Southern blot analysis showed that the identified beta-chain gene fragment undergoes genomic rearrangement during T-cell differentiation.

To generate antibodies to the constant region of the mouse T-cell-receptor beta chain, the corresponding cDNA clone 86T5 was subcloned into the bacterial expression vector pEX2. The induced hybrid protein consisting of a 30 kDa T-cell-receptor segment and a beta-galactosidase carrier moiety was used for immunization. In Western blots using lysates of a T-cell-receptor-positive T-cell hybrid, the antisera obtained reacted with a 42 kDa protein under reducing and a 85 kDa protein under nonreducing conditions. The antisera did not react in binding assays with intact T lymphocytes, i.e. membrane-associated T-cell-receptor protein.