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The enumeration of the interaction partners of the cellular prion protein, PrPC, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrPC. When expressed in transgenic mice, PrPmyc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrPC. PrPmyc antagonized the toxicity of truncated PrP, restored prion infectibility of PrPC-deficient mice, and was physically incorporated into PrPSc aggregates, indicating that it possessed all functional characteristics of genuine PrPC. We then immunopurified myc epitope-containing protein complexes from PrPmyc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrPC and may represent component of a multiprotein complex. Selected PrPC interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance., PLoS ONE, 4 (2), ISSN:1932-6203

The synaptic serine protease neurotrypsin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90-kDa and a C-terminal 22-kDa fragment from the N-terminal moiety of agrin. Morphological analyses indicate that neurotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin-mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild-type and neurotrypsin-overexpressing mice revealed that neurotrypsin-dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.

金沢大学医薬保健研究域 医学系, Background. The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. Results. Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. Conclusion. Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog. © 2008 Cinelli et al; licensee BioMed Central Ltd.

Type I IFN (IFN-I) increase the sensitivity of cells and mice to lethal infection with Listeria monocytogenes. Therefore the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. Two commonly used strains of L. monocytogenes, EGD and LO28, were identified as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to trigger the IRF3 signalling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NFkappaB, MAPK, or inflammasome signalling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57/BL6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57/BL6 mice. Together our data suggest that isolates of wt L. monocytogenes differ in their ability to trigger the IRF3 signalling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence.

Lipopolysaccharide (LPS) is an integral structural component of the outer membrane of Gram-negative bacteria and the principal active agent in the pathogenesis of endotoxin shock. LPS is a potent inducer of a variety of cytokines and inflammatory agents that lead to a profound alteration of gene expression patterns in cells and organs. The gene coding for the inducible nitric oxide synthase (iNOS) is highly responsive to LPS in vitro and in vivo and accounts for the production of nitric oxide (NO). The Janus kinase (JAK) family member tyrosine kinase 2 (TYK2) is a constituent of the interferon (IFN) type I response pathway and an important effector in the progression of endotoxin shock. Macrophages deficient for IFNalphabeta receptor chain 1 (IFNAR1) or TYK2 were shown to have an impaired LPS-induced iNOS expression. Here we determined the contribution of IFNAR1 and TYK2 to iNOS expression in vivo in a lethal LPS challenge model. TYK2 and IFNAR1 were found to be crucial for the LPS-induced iNOS mRNA and protein expression in spleen and lung that could be attributed to the Mac3-positive population. In liver LPS-induced iNOS mRNA expression was only partially impaired in TYK2-deficient mice and was unimpaired in IFNAR1-deficient mice, indicating organ specificity. TYK2(-/-) and IFNAR1(-/-) mice also differ with respect to IFNgamma production upon LPS challenge in that TYK2(-/-) mice show a defect while IFNAR1(-/-) mice do not. Our data suggest that iNOS is induced through IFNAR1 and TYK2 in Mac3-positive cells which are the main source of iNOS in spleen and lung. The LPS-induced iNOS expression in liver is independent of IFNAR1 and partially dependent on TYK2, which is most likely due to the lack of IFNgamma production in the absence of TYK2.

The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23-128) hinged to a membrane-anchored globular domain (GD, aa 129-231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi"). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.

Confronting a recently mated female with a strange male can induce a pregnancy block ('Bruce effect'). The physiology of this effect is well studied, but its functional significance is still not fully understood. The 'anticipated infanticide hypothesis' suggests that the pregnancy block serves to avoid the cost of embryogenesis and giving birth to offspring that are likely to be killed by a new territory holder. Some 'compatible-genes sexual selection hypotheses' suggest that the likelihood of a pregnancy block is also dependent on the female's perception of the stud's and the stimulus male's genetic quality. We used two inbred strains of mice (C57BL/6 and BALB/c) to test all possible combinations of female strain, stud strain, and stimulus strain under experimental conditions (N(total) = 241 mated females). As predicted from previous studies, we found increased rates of pregnancy blocks if stud and stimulus strains differed, and we found evidence for hybrid vigour in offspring of between-strain mating. Despite the observed heterosis, pregnancies of within-strain matings were not more likely to be blocked than pregnancies of between-strain matings. A power analysis revealed that if we missed an existing effect (type-II error), the effect must be very small. If a female gave birth, the number and weight of newborns were not significantly influenced by the stimulus males. In conclusion, we found no support for the 'compatible-genes sexual selection hypotheses'.

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and beta-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules.

The role of nonthymic epithelial (non-TE) MHC in T cell repertoire selection remains controversial. To analyze the relative roles of thymic epithelial (TE) and non-TE MHC in T cell repertoire selection, we have generated tetraparental aggregation chimeras (B6-nude↔BALB/c and B6↔BALB/c-nude) harboring T and B cells from both parents, whereas TE cells originated exclusively from the non-nude donor. These chimeras mounted protective virus-specific TE and non-TE MHC-restricted T cell responses. To further evaluate whether non-TE MHC alone was sufficient to generate a functional T cell repertoire, we generated tetraparental aggregation chimeras lacking MHC class II (B6-nude↔MHCII−/−) or both MHC molecules (B6-nude↔MHCI−/−II−/−) on TE cells, but not on cells of B6-nude origin. Chimeras with MHC-deficient TE cells mounted functional virus-specific CD8+but not CD4+T cell responses. Thus, maturation of functional CD4+T cell responses required MHC class II on thymic epithelium, whereas CD8+T cells matured in the absence of TE MHC.

Leukemic stem cells (LSCs) have recently been identified as an important target of therapy in various human leukemias and related blood cell disorders. Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of mast cells (MCs) in various organ systems, including the bone marrow (BM). Whereas patients with indolent SM (ISM) have a normal life-expectancy, patients with more advanced forms of SM have a poor prognosis. In these patients, neoplastic MCs are usually resistant against conventional drugs and various targeted drugs. MC leukemia (MCL) is the rare leukemic variant of advanced SM, defined by a rapidly devastating expansion of immature MCs in various hematopoietic organs and a poor prognosis with short survival times. Although MCL is considered a stem cell disease, little is known about the origin and phenotype of MCL-initiating LSCs. We examined the phenotypic and functional characteristics of putative LSCs in patients with aggressive SM (ASM, n=12) and MCL (n=6). Putative LSCs were identified and characterized phenotypically by flow cytometry. Highly enriched, sorted LSCs were injected into NOD-SCID-IL-2Rγ-/- mice exhibiting a 220 amino acid isoform of human membrane-bound hSCF (NSGSCF). We found that disease-initiating and propagating LSCs reside within a CD34+ fraction of the MCL clone. Whereas cell fractions containing CD34+ cells as well as highly enriched CD34+ cells produced engraftment in NSGSCF mice with a MCL-like disease (43-77% human MCL cells in mouse BM after 10-22 weeks), no substantial engraftment was produced by MC-rich but stem cell-depleted, KIT+/CD34─ cell fractions obtained from the same patients ( Disclosures Valent: Novartis: Consultancy, Honoraria, Research Funding.

Metal-responsive transcription factor-1 (MTF-1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryogenesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Here we studied Mtf1 knockout mice at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild-type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild-type rat embryo hepatocytes, indicating a cell-autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non-stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF-1 in coping with heavy metal stress. An additional MTF-1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF-1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis.

Within the CNS, the normal form of cellular prion protein (PrPC) is expressed on neurons, oligodendrocytes, and astrocytes. The contribution of these cell types to prion replication and pathogenesis is unclear. To assess the role of oligodendrocytes, we expressed PrPCunder the control of the myelin basic protein (MBP) promoter in mice lacking endogenous PrPC. PrPCwas detected in oligodendrocytes and Schwann cells but not in neurons and astrocytes. MBP-PrP mice never developed scrapie after intracerebral, intraperitoneal, or intraocular challenge with scrapie prions. Transgenic brains did not contain protease-resistant prion protein and did not transmit scrapie when inoculated into PrPC-overexpressing indicator mice. To investigate whether prion spread within the CNS depends on oligodendrocytic PrPC, we implanted PrPC-overexpressing neuroectodermal grafts into MBP-PrP brains. After intraocular prion inoculation, none of the grafts showed spongiform encephalopathy or prion infectivity. Hence oligodendrocytes do not support cell-autonomous prion replication, establishment of subclinical disease, and neural spread of prions. Prion resistance sets oligodendrocytes aside from both neurons and astrocytes.

PrP knockout mice with disruption of only the PrP-encoding region (Zurich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zurich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.

Because recent studies have indicated that tissue plasminogen activator (tPA) aggravates neurodegenerative processes in many neural pathologies, we studied whether the endogenous tPA antagonist neuroserpin has a neuroprotective effect in an animal model of focal ischemic stroke. After induction of a focal ischemic stroke in the mouse by occlusion of the midddle cerebral artery, we found that microglial cells accumulated in the marginal zone of the infarct are the most important source for both plasminogen activators, tPA and uPA. To investigate the effect of neuroserpin on the size and the histology of the infarct we produced transgenic mice overexpressing neuroserpin approximately sixfold in the nervous system. In the brain of these mice the total tPA activity in the uninjured tissue was strongly reduced. After induction of a focal ischemic stroke in the transgenic mice by a permanent occlusion of the middle cerebral artery (MCA), the infarcts were 30% smaller than in the wild-type mice. Immunohistochemical analyses and in situ hybridization revealed an attenuation of the microglial activation in the reactive zone. Concomitantly, the microglial production of tPA and uPA, as well as the PA-activity in the infarct region was markedly reduced. Thus, our results indicate that neuroserpin reduces microglial activation and, therefore, the PA activity and has a neuroprotective role after focal ischemic stroke.

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. We conclude that splenic B lymphocytes are not prion-replication competent and that they acquire prions from other cells, most likely follicular dendritic cells with which they closely associate and whose maturation depends on them.

PrP knockout mice in which only the open reading frame was disrupted ('Zurich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zurich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zurich I/Zurich II heterozygotes had half the Dpl of Zurich II mice and developed symptoms 6 months later. Zurich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.

Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro . To delineate potential beneficial effects of NO in preventing vascular disease in vivo , we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAME-treated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2−/−/APLP1−/−and APLP2−/−/APP−/−mice proved lethal early postnatally. Surprisingly, APLP1−/−/APP−/−mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1−/−/APP−/−/APLP2+/−mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicityin vitro.

We studied the reactivity of T and B cells against a soluble form of the glycoprotein of vesicular stomatitis virus (VSV-G) which was expressed in a transgenic mouse (line 23) under the control of the hormone regulated beta-lactoglobulin promoter. Transgenic mice expressed VSV-G in the thymus, spleen, mammary gland and lung. VSV-G transcripts in the thymus varied with age, i.e., expression was high early in life and decreased with age. VSV-G transgenic mice immunized with recombinant vaccinia virus expressing VSV-G exhibited normal VSV-G-specific IgM levels, but a 30-fold reduction in IgG response, indicating functional VSV-G-specific B cell activity but impaired T helper cell responses. Interestingly, VSV-G-specific T helper cell activity was reduced only early (4-10 weeks) and late in life (>40 weeks) but was normal in between. Double transgenic mice expressing VSV-G and a VSV-G-specific TCR (line 23x7) demonstrated that TCR transgenic CD4(+) T cells were partially deleted in early life, but then gradually repopulated the periphery and remained constant. These findings suggest that in line 23 two different mechanisms regulated levels of the immune response: clonal reduction/deletion of VSV-G-specific T cells during early life followed by peripheral anergy at a later stage.

Transgenic animal models for neurocarcinogenesis have provided significant insights into the molecular mechanisms underlying carcinogenic processes, including those which affect the nervous system. In view of the very rapid pace of acquisition of knowledge, it is not possible to cover all transgenic mouse models for neural tumors. Instead, this article discusses some of the most important technical innovations for manipulation of the mammalian genome (notably the various methods for targeted genome modifications, as well as the technology for introducing large DNA fragments into the germ line of mice), and presents a selection of the transgenic mouse models which are proving most promising for furthering our understanding of the pathogenetic basis of cancer in the nervous system.

The promoter of the GABA A receptor δ subunit gene was analyzed in transgenic mice and in cultured cells to study sequences involved in neuronal subtype-specific gene expression. A 6.4-kb genomic fragment faithfully directed neuron-specific transcription of a lacZ reporter gene in the central nervous system. The transgene expression pattern in the cerebral cortex, hippocampal formation, thalamus, and brainstem was consistent with the regional and neuronal subtype-specific expression of the endogenous δ subunit protein in both developing and mature brain. In the cerebellum, however, the transgene was ectopically expressed in Purkinje cells and silent in granule cells, where the endogenous δ subunit is abundantly expressed. These mice provide a useful tool for investigating activity-dependent regulation of GABA A receptor expression under physiological and pathological conditions. Transfection studies using primary cortical neurons and astroglial cells revealed that the δ subunit gene promoter was selectively active in neurons even when truncated to a 267-bp core fragment. In conclusion, the δ subunit gene promoter/upstream region contains the information for neuronal subtype-specific expression in the entire brain except in the cerebellum and is selectively active in primary cortical neurons in vitro.

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.

We have generated a transgenic mouse model for astrocytoma by expressing the v-src kinase under control of the glial fibrillary acidic protein (GFAP) gene regulatory elements in astrocytes. Abnormal astrogliosis was observed in all transgenic animals already at 2 weeks postnatally, frequently followed by the development of dysplastic changes. Later, small proliferative foci arose, and overt astrocytoma developed in the brain and spinal cord in 14.4% of mice after a follow up time of 65 weeks. While early lesions were histologically consistent with low-grade astrocytoma, at later stages most tumors were highly mitotic and frankly malignant. Vascular endothelial growth factor (VEGF) was expressed by tumor cells already at early stages, suggesting induction by v-src, and it was most pronounced in pseudopalisading cells surrounding necrotic areas, implying additional upregulation by hypoxia. In larger lesions, mitotic activity and expression of flk-1, the cognate receptor of VEGF were induced in endothelial cells. Therefore, end-stage tumors mimicked the morphological and molecular characteristics of human glioblastoma multiforme. Time course and stochastic nature of the process indicate that v-src did not suffice for malignant transformation, and that astrocytomas were the result of a multistep process necessitating co-operation of additional genetic events.

One evolutionary explanation for the success of sexual reproduction assumes that sex is an advantage in the coevolutionary arms race between pathogens and hosts. Accordingly, an important criterion in mate choice and maternal selection thereafter could be the allelic specificity at polymorphic loci involved in parasite-host interactions, e.g. the MHC (major histocompatibility complex). The MHC has been found to influence mate choice and selective abortions in mice and humans. However, it could also influence the fertilization process itself, i.e. (i) the oocyte's choice for the fertilizing sperm, and (ii) the outcome of the second meiotic division after the sperm has entered the egg. We tested both hypotheses in an in vitro fertilization experiment with two inbred mouse strains congenic for their MHC. The genotypes of the resulting blastocysts were determined by polymerase chain reaction. We found nonrandom MHC combinations in the blastocysts which may result from both possible choice mechanisms. The outcome changed significantly over time, indicating that a choice for MHC combinations during fertilization may be influenced by one or several external factors.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

The amyloid precursor protein (beta APP) gene of the mouse was disrupted by homologous recombination; however, contrary to expectation, brain and other tissues still contained beta APP-specific RNA, albeit at a level 5-10 fold lower than wild-type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.

Transgenic mice expressing rat parvalbumin under the control of the human metallothionein IIA (MTII A), SV-40 early, and neuron-specific enolase (NSE) promoters were produced. Ectopic expression was analyzed by RNA polymerase chain reaction and RNase protection in combination with immunohistochemistry. From a total of 25 transgenic lines 18 were found to express the transgene. Expression strength and tissue specificity were dependent upon the promoter used and varied considerably among animal lines produced with the same construct. Highest constitutive MT IIA-driven expression was found in lung, liver, heart, and kidney, as well as in brain, and lower amounts of transgene expression were found in spleen, testis, and muscle. Immunohistochemistry of tissue sections of metallothionein-parvalbumin transgenic strain 29 in the noninduced state revealed that ectopic PV mRNA is translated into protein. Short-term induction of the MT IIA promoter by CdSO 4 or CdCl 2 leads to a shift in tissue specificity and does not increase ectopic expression in tissues where the transgene is active in the noninduced state. As expected the NSE promoter showed highest activity in brain. However, NSE-driven expression could also be detected to various degrees in all investigated tissues. SV-40-dependent PV expression showed no tissue preference and varied considerably among different strains. Except for the observation that the SV-40-PV construct showed lower yields in transgenic production and reduced numbers of positive offspring no obvious impairment of growth or behavior as a consequence of transgenic PV expression could be detected.

The paracortex of lymph nodes (LNs) harbors a sponge-like scaffold of stromal cells known as fibroblastic reticular cells (FRCs). A major function of FRCs is to build and enwrap the conduit system of collagen fibers that directs interstitial fluids from the afferent lymph through the T-cell zone to HEVs. Furthermore, FRCs regulate T-cell migration and survival by producing the chemokines CCL19 and CCL21. To gain further knowledge on the biology of FRCs and possibly other stromal cell subsets, we have generated a bacterial artificial chromosome (BAC)-transgenic mouse model that utilizes the CCL19 promoter to direct the Cre-recombinase to LN stromal cells. Crossing of CCL19-Cre mice to the R26-EYFP reporter revealed that transgene expression in LNs was almost exclusively confined to podoplanin+CD31- cells. Likewise, transgene activity in spleens and Peyer’s patches of CCL19-Cre mice was largely restricted to FRC-like cells, i.e. stromal cells within the T cell zone and the T-B border. Selective ablation of the lymphotoxin-beta receptor on CCL19-Cre-positive cells resulted in profound changes in the development and organization of secondary lymphoid organs. Taken together, stromal CCL19-Cre expression is well-suited (i) to characterize the development of LN FRCs in vivo, (ii) to molecularly dissect the contribution of stromal cells to lymphoid organogenesis, and (iii) to address the function of LN FRCs in the generation of innate and adaptive immune responses.

Abstract 961 In Philadelphia-positive (Ph+) chronic myeloid leukemia (CML), leukemic stem cells (LSC) supposedly reside in a CD34+/CD38−/Lin− fraction of the leukemic clone. However, little is known about phenotypic properties of LSC in CML. We screened for novel LSC markers and targets in CML by gene chip studies and extensive flow cytometry analyses using monoclonal antibodies against various surface antigens (n=50). A total number of 240 bone marrow or peripheral blood samples (CML, n=95; AML, n=103; CMML, n=10, control marrow, n=32) were examined. In common with normal SC, CD34+/CD38− CML LSC were found to co-express the homing-receptor CD44, G-CSF-R (CD114), KIT (CD117), FLT3 (CD135), and CXCR4 (CD184). Similar to LSC in AML and CMML, CML LSC were found to display higher levels of Siglec-3 (CD33) and IL-3RA (CD123). Most significantly, however, we found that in contrast to normal CD34+/CD38− stem cells, CD34+/CD38− CML LSC aberrantly express IL-2RA (CD25), dipeptidylpeptidase IV (DPPIV=CD26), and IL-1RAP. In other myeloid leukemias (AML, CMML), CD34+/CD38− LSC also co-expressed CD25, but usually did not express CD26 or IL-1RAP. Whereas CD26 was expressed almost invariably on CD34+/CD38− cells in all CML patients tested, the surface enzyme was neither detectable in more mature CD34+/CD38+ progenitor cells nor on CD34+/CD38− stem cells in reactive bone marrow or healthy controls. During successful treatment with imatinib or nilotinib (patients examined at CCyR and/or MMR), CD34+/CD38− stem cells invariably showed a ‘normal' phenotype (CD25−, CD26−, IL-1RAP−), whereas in relapsing CML, CD34+/CD38− cells were again found to co-express CD25 and CD26. Sorted Lin−/CD26− stem cells obtained from CML patients (at diagnosis) engrafted irradiated NOD-SCID IL-2Rγ−/− (NSG) mice with normal multilineage BCR/ABL1− hematopoiesis, whereas Lin−/CD26+ stem cells were found to engraft NSG mice with BCR/ABL+ cells. We next examined the regulation of expression of CD25 and CD26 on CML LSC. Whereas expression of CD25 was found to depend on BCR/ABL1 and STAT5-activity, CD26 expression was found to be expressed independent of BCR/ABL1 and independent of STAT5-signaling. In a next step, we examined the potential function of CD26 on CML LSC. In these studies, CD26 was identified as a target-enzyme disrupting the niche-related SDF-1α/CXCR4 axis by degrading SDF-1α. Correspondingly, CD26-targeting gliptins (sitagliptin, 1 μM; vildagliptin, 1 μM) were found to revert recombinant DPPIV/CD26-induced or cellular CD26-induced inhibition of SDF-1α-mediated in vitro migration of CD26+ leukemic cells. Finally, we found that in a CML patient treated with nilotinib, in whom uncontrolled diabetes mellitus required therapy with saxagliptin, BCR/ABL1 levels (in percent of ABL according to IS) that were found to increase before the start of saxagliptin (IS before saxagliptin: 1.6 [-4 months], 2.3 [-3 months], and 2.4 [at therapy-start]), decreased over time during saxagliptin-therapy (IS: 1.0 [+1 month], 1.0 [+3 months], 0.8 [+5 months]). Together, the CML-initiating LSC is a CD34+/CD38− cell that exhibits aberrant expression of IL-1RAP, CD25, and DPPIV/CD26. All three markers may be useful for purification of CML LSC. DPPIV/CD26 appears to be a functionally and pathogenetically relevant antigen that may facilitate niche-independent uncontrolled redistribution and thus extramedullary spread of LSC and LSC-derived progenitor cells in CML. Whether CD26 can be developed as a novel therapeutic target in CML is currently under investigation. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

Abstract 3399 The Jak/Stat signalling pathway is essential for survival and proliferation of haematopoietic cells. The Stat5 transcription factors (Stat5a and Stat5b) play a crucial role in the development of various lineages of the hematopoietic system. Stat5 is activated by many cytokines and growth factors that regulate hematopoiesis. Persistent Stat5 activity is frequently found in hematopoietic cancers due to aberrant tyrosine kinase signalling. We have previously characterized a constitutively activated version of Stat5 (called oncogenic Stat5; cS5F), which mimics persistent tyrosine kinase signalling and promotes multi-lineage leukaemia development in a bone marrow transplantation model. The aim of the study is to generate a novel inducible mouse model for oncogenic Stat5a using the BAC recombineering technology. A cassette expressing cS5F (with a C-terminal FLAG tag) and a reporter gene (truncated human CD2; hCD2) was cloned, sequenced and biochemically verified for persistent tyrosine phosphorylation in absence of cytokines or growth factors. The cassette is flanked by loxP sites in opposite orientation and was used to replace the original Stat5a gene in a BAC carrying the endogenous Stat3/5 locus using BAC recombineering. Therefore, the expression of the transgene is regulated by the endogenous Stat5a promoter. The transgene is in an inverted (off) orientation and can be switched into the transcribing (on) orientation by Cre activity. Pronuclear injections were performed with the linearized and purified BAC. We obtained 5 transgenic founder lines which showed germ line transmission of the transgene. The copy number of the BAC was estimated to be 2 by Southern blot analysis. The transgenic founder lines were bred with the Rosa CreERT2 mice, and the Cre activity was induced by treating the mice with 1 mg of tamoxifen every day, for 5 consecutive days. The recombination of the transgene into the ‘on' orientation in liver, lungs, kidney, spleen and bone marrow was demonstrated by Southern blotting. The expression of the hCD2 marker was detected in various hematopoietic lineages by FACS and the expression of the transgenic protein in liver was confirmed by Western blotting with anti-Stat5a and anti-FLAG antibodies. One week after induction of the transgene, all the mice induced with tamoxifen (n=6) developed atrophic thymii with nearly a 4-fold reduction in total thymocyte numbers (p=0.0003). However, the percentage of CD8+ cells was increased more than 3 fold in the thymus (p=0.0002). Moreover, there was a 2-fold reduction in the number of early hematopoietic progenitors; defined as lin−, Sca1+ and c-kit+, in the bone marrow (n=5, p=0.0217). We have established an inducible mouse model for expression of constitutively active Stat5a under the endogenous promoter. We are currently monitoring these mice for development of cancer as they provide a competent tool to study the molecular mechanisms triggered by persistent Stat5a activity, including identification of other cooperating signalling pathways that contribute to generation of cancer. Moreover, it would allow one to test interference strategies, which could lead to potential therapeutics. Disclosures: No relevant conflicts of interest to declare.

During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events., Author Summary Cytomegaloviruses (CMVs) infect the majority of the human population and persist lifelong via latency. CMV latency is thought to be a dynamic state, characterized by stochastic viral reactivation events coupled to CMV-derived antigen presentation. In support of this hypothesis is the exceptionally large CMV-specific CD8 T cell response which constitutes an integral part of immune surveillance of CMV reactivation. Conversely, it may also contribute to immune senescence as it significantly shapes the overall CD8 T cell pool in bias of CMV-specificity. In mice, only a subset of CMV-specific CD8 T cells, also called ‘inflationary CD8 T cells’, contribute to this large response. The mechanism leading to the selective accumulation and persistence of memory CD8 T cells during MCMV latency is largely unknown. Here, we unraveled the mechanisms of memory CD8 T cell inflation using a newly generated TCR transgenic mouse with specificity for an immunodominant inflationary MCMV epitope. We show that antigen presentation on non-hematopoietic cells is essential for memory inflation and that memory inflation in peripheral tissues is fueled by lymph node-resident central memory CD8 T cells, being locally reactivated by non-hematopoietic cells, inducing their local expansion and migration to peripheral tissues where they control viral reactivation events.

Many viral infections are efficiently defeated by a vigorousy cytotoxic T cell mediated early immune response. Before T cells in the periphery can be activated they have to undergo thymic positive and negative selection. During positive selection T cells acquire the capacity to H-2 restrictedly recognize the foreign antigens whereas negative selection prevents the development of self reactive T cells. However not all self antigens are presented to the developing T cells in the thymus to establish the so called central tolerance by physical elimination of self reactive T cells (Kappler et al. 1988; MacDonald et al. 1988). Therefore potentially autoreactive T cells may reach the periphery. These cells are presumable involved in many autoimmune disease processes (Sinha et al. 1990; Zamvil and Steinman, 1990). Several studies have shown that potentially self reactive T cells may be rendered tolerant to self antigens not expressed in the thymus by extrathymic mechanisms of tolerance induction (Webb and Sprent, 1990; Hanahan et al. 1990; Ramsdell and Fowlkes, 1990; Schonrich et al. 1991; Schoenrich et al. 1992).

Abstract 4936 Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organ systems, resistance to conventional cytoreductive drugs, and a poor prognosis. In most patients, transforming mutations in the KIT proto-oncogene are detectable and are considered to contribute to resistance. MC lines are an important model for analyzing drug resistance in neoplastic MC. We have established a novel canine mastocytoma cell line, NI-1 from a canine patient suffering from mast cell leukemia. NI-1 cells were found to harbour several homozygous KIT mutations including two single nucleotide mutations, one at nucleotide 107 (C to T, leading to a missense mutation, P36L) and one at nucleotide 1187 (A to G, leading to a missense mutation, Q396R), a 12 bp duplication at nucleotide 1263, and a 12 bp deletion at nucleotide 1550, the latter reflecting a transcriptional variant. NI-1 cells contained histamine and formed tumors in NOD-SCID IL-2Rgammanull (NSG) mice. As assessed by 3H-thymidine uptake, a number of targeted drugs were found to inhibit the proliferation of NI-1 cells at pharmacologically relevant concentrations. Among these drugs were the KIT kinase blockers midostaurin (IC50 2 μM). However, the HDAC inhibitor vorinostat was found to block growth of NI-1 cells (0.1-0.5 μM). Drug response profiling also revealed that NI-1 cells differ markedly from canine C2 mastocytoma cells harbouring an exon 11 KIT mutation, and the human mast cell lines HMC-1.1 (exon 11 mutation in KIT) and HMC-1.2 (exon 11 and exon 17 KIT mutations). In contrast to C2 cells, NI-1 cells were found to be largely resistant against the growth-inhibitory effects of masatinib, sorafenib, and sunitinib. By contrast, NI-1 cells were found to be even more sensitive against the growth-inhibitory effects of the mTOR blocker RAD001 and the PI3-kinase/mTOR inhibitor NVP-BEZ235. Together, our data suggest that certain KIT mutations may be associated with relative resistance of neoplastic MC against KIT-targeting drugs, a phenomenon that has also been described for human MC and the KIT mutant D816V that mediates resistance against masatinib and imatinib. The novel MC line NI-1 may serve as a novel tool to investigate the mechanisms of resistance against TKI in neoplastic MC. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

We and others have shown that transgenic expression of the transcription factor PLZF in the T cell lineage induces the appearance of CD44-high memory-phenotype (MP) T-cells (Savage et al. Immunity 2008; Raberger et al. PNAS, 2008). These MP T cells arise already in the thymus and produce IFNã upon PMA/ionomycin stimulation, thus sharing certain features with innate-like T cells. Furthermore, we observed that PLZF expression is up-regulated in Itk-deficient T cells, which are enriched with innate-like T cell subsets. To study the PLZF-induced MP T cell population in more detail, we are investigating whether these cells, like other innate-like T cells, are selected on classical or non-classical MHC molecules. Furthermore, PLZF transgenic mice are intercrossed with several knockout mice (e.g. Itk-/- or IL-15-/- mice) to determine signaling pathways/molecules required for their development and maintenance. Our studies will provide more insight into transcription pathways controlling the development of MP T cells with "innate-like" functions. Supported by the Austrian Science Fund FWF (SFB-F23, P19930).

The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner.

Transuterine migration is the passage of fertilised ova from one uterine horn into the other. The phenomenon has been described for animals of different species with a bicornuate type of uterus. Whether or not it occurs in rodents is questionable, but could have an impact on the way embryo transfers are carried out, i.e. unilaterally or bilaterally. The aim of this study was to examine the occurrence of transuterine migrations in nulliparous and multiparous mice after unilateral embryo transfer. Sixteen two-cell embryos were transferred into either the left or the right oviduct of mice with different genetic origin. With the exception of one reabsorption site in the opposite uterine horn, we never found evidence for the occurrence of transuterine migration. This is also true for embryo transfers carried out after parturition of the surrogate mother. Even the successful development of up to 13 embryos in one uterine horn did not result in transmigration but may be the reason for the widespread assumption that transuterine migration occurs after unilateral embryo transfers. The separation of the uterine body and the prevaginal portion of the uterine cervix into two canals by a septum seems to be the main reason for the absence of successful transuterine migration in mice.

Nat. Med. 11, 146–152 (2005) In the original version of this article, Fig. 5b was incorrect. The correct panel appears below.

The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32–121 or 32–134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1–3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.