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After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.

Since the discovery of odorant receptors (ORs) in rodents, most ORs have remained orphan receptors. Even for deorphanized ORs in vitro, their in vivo properties are largely unknown. Here, we report odor response profiles of two highly homologous mouse ORs, MOR29A and MOR29B, both in vivo and in vitro. The BAC transgenic mouse was generated, in which olfactory sensory neurons (OSNs) expressing the transgenes MOR29A and MOR29B were differently tagged with IRES-gapECFP and IRES-gapEYFP, respectively. MOR29A- and MOR29B-expressing OSN axons converged on separate but nearby loci on the dorsal surface of the olfactory bulb (OB). Optical imaging of intrinsic signals in the OB identified five different phenyl ethers as candidate ligands for MOR29B. Based on in vitro calcium imaging with the isolated OSNs and luciferase assay with heterologous cells, only guaiacol and vanillin were found to be potent agonists for MOR29A and MOR29B. Because of its accessible glomerular locations in the dorsal OB and defined odor response profiles both in vivo and in vitro, the MOR29A/29B tagging mouse will serve as an excellent tool for studying both odor-signal processing and neural circuitry in the OB.

Infection with cagA -positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA -encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori -associated neoplasms.

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse β-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IκBα accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.

The preparation of gelatinous aluminium hydroxide from aqueous solutions of the formate, citrate or tartrate of aluminium on addition of aqueous sodium hydroxide has been investigated under different conditions. The precipitates from aqueous solutions of aluminium formate consist of amorphous aluminium hydroxide, pseudoboehmite, bayerite, hydrargillite or their mixtures, depending on the condition of preparation. A mixed solution of aluminium citrate and sodium hydroxide yields no solid-phase product without aging on heating to enhance the hydrolysis of aluminium citrate. The resulting precipitate exists as pseudoboehmite, independent of the preparation conditions. In contrast, aluminium tartrate is not hydrolysed to precipitate aluminium hydroxide even by aging on heating.

Aim: Apoptosis is involved in hepatic ischemia/reperfusion injury. The protein FNK, constructed from an anti-apoptotic protein Bcl-xL, exhibits the stronger anticell death activity. We evaluated the effect of FNK on apoptosis after hepatic ischemia and reperfusion, using FNK-overexpressing transgenic mice and the HIV/Tat protein-transduction-domain (PTD) that mediates the introduction of FNK into cells when fused with FNK (PTD-FNK). Methods: Mice were given hepatic ischemic insult for 90 min followed by reperfusion for 3 h. FNK overexpression was determined by immunohistochemistry and Western blot. PTD-FNK was intraperitoneally injected into wild mice 3 h before the insult. Liver injury was determined by the caspase activation, DNA fragmentation, and hematoxylin–eosin and terminal deoxynucleotidyl transferase-mediated dUTP- digoxigenin nick-end labelling (TUNEL) stainings. Results: In FNK-transgenic mice, FNK overexpression inhibited the activation of caspase 3/caspase 3-like activity and DNA fragmentation caused by the injury. In wild mice preinjected with PTD-FNK, PTD-FNK significantly inhibited the caspase activation and DNA fragmentation, reduced the area of liver vacuolization, and protected hepatic cells surrounding blood vessels, irrespective of central or portal veins, from the ischemia/reperfusion damage. Conclusions: FNK inhibits apoptotic death due to the ischemia/reperfusion injury. Our results provide the reasonable expectation of therapeutic protein PTD-FNK for clinical applications, such as transplantation, to protect against ischemia/reperfusion injury.

遺伝子改変マウス（トランスジェニックマウス，ノックアウトマウス）の作製および使用は，遺伝子組換え実験（注1）の動物使用実験（動物作成実験）に該当するので，「遺伝子組換え生物等の使用等の規制による生物の多様性の確保に関する法律」（注2）（以下，カルタヘナ法）が適用される．このカルタヘナ法は「生物の多様性に関する条約のバイオセーフティーに関するカルタヘナ議定書」（注3）（以下，カルタヘナ議定書）の的確かつ円滑な実施を確保することを目的としている．すなわち，遺伝子改変マウスの作製および使用は，生物多様性への悪影響を防止するために，環境中への拡散を防止しつつ行われなければならない．具体的なルールは「研究開発等に係る遺伝子組換え生物等の第二種使用等に当たって執るべき拡散防止措置等を定める省令」（注4）（以下，研究開発二種省令）などに規定されている．遺伝子組換え実験の手続きは，実験責任者が，執るべき拡散防止措置があらかじめ定められているかどうかを確認の上，実験計画書（機関内実験）または大臣確認申請書（大臣確認実験）を作成し，所属機関内の遺伝子組換え実験安全委員会等に申請して，承認または確認を得なければならない．カルタヘナ法に違反した場合には，最も重いもので，1年以内の懲役もしくは100万円以内の罰金またはこれの併科とされているので，実験者は，事前にカルタヘナ法を熟知して実験を行わなければならない． 注1）遺伝子組換え実験：遺伝子組換え技術により得られた核酸またはその複製物（組換え核酸）を有する遺伝子組換え生物等の使用等をいう． 注2）遺伝子組換え生物等の使用等の規制による生物の多様性の確保に関する法律 注3）生物の多様性に関する条約のバイオセーフティーに関するカルタヘナ議定書 注4）研究開発等に係る遺伝子組換え生物等の第二種使用等に当たって執るべき拡散防止措置等を定める省令 注1，2，3，4）の詳細は，文部科学省ホームページを参照． http://www.lifescience-mext.jp/bioethics/anzen.html#kumikae

SummaryIn the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.

水硬性を有する高炉水砕スラグの硬化に及ぼす粒度(粒子破砕)，拘束圧，アルカリ刺激，温度等の影響を室内試験で確認するとともに，粒子接触点数と一軸圧縮強さの関係，液相中のイオン濃度に着目して調べた．また，地盤改良工法への適用性の確認のため試験施工を行い，硬化・透水性の経時変化を調べた.その結果，粒径と間隙比から求めた単位体積あたりの接触点数と一軸圧縮強さには相関性が認められること，サンドコンパクションパイル(SCP)工法におけるスラグ杭は締固めに伴う粒子破砕により，施工後約3か月で著しい硬化と透水性の低下が生じることを確認した．

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca(2+) current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.

SUMMARY Human serum amyloid P component (SAP) binds avidly to DNA, chromatin and apoptotic cells in vitro and in vivo. 129/Sv 3 C57BL/6 mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoantibodies and immune complex glomerulonephritis. SAP-deficient animals, created by backcrossing the 129/Sv SAP gene deletion into pure line C57BL/6 mice and studied here for the first time, also spontaneously developed broad spectrum antinuclear autoimmunity and proliferative immune complex glomerulonephritis but without proteinuria, renal failure, or increased morbidity or mortality. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP –/– mice than in wild-type mice. In contrast, SAP-deficient pure line 129/Sv mice did not produce significant autoantibodies either spontaneously or when immunized with extrinsic chromatin or apoptotic cells, indicating that loss of tolerance is markedly strain dependent. However, SAP deficiency in C57BL/6 mice only marginally affected plasma clearance of exogenous chromatin and had no effect on distribution of exogenous nucleosomes between the liver and kidneys, which were the only tissue sites of catabolism. Furthermore, transgenic expression of human SAP in the C57BL/6 SAP knockout mice did not abrogate the autoimmune phenotype. This may reflect the different binding affinities of mouse and human SAP for nuclear autoantigens and/or the heterologous nature of transgenic human SAP in the mouse. Alternatively, the autoimmunity may be independent of SAP deficiency and caused by expression of 129/Sv chromosome 1 genes in the C57BL/6 background.

The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZreporter genes nearly covering the Fugu Otx2 locus, from –30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor–one neuron rule.

Previous studies have indicated that the Undulated short-tail deletion mutation in mouse Pax1 (Pax1Un-s) not only ablates Pax1, but also disturbs a gene or genes nearby Pax1. However, which gene(s) is involved and how the Pax1Un-s phenotype is confined to the Pax1-positive tissues remain unknown. In the present study, we determined the Pax1Un-s deletion interval to be 125 kb and characterized genes around Pax1. We show that the Pax1Un-s mutation affects four physically linked genes within or near the deletion, including Pax1, Nkx2-2, and their potential antisense genes. Remarkably, Nkx2-2 is ectopically activated in the sclerotome and limb buds of Pax1Un-s embryos, both of which normally express Pax1. This result suggests that the Pax1Un-s deletion leads to an illegitimate interaction between remotely located Pax1 enhancers and the Nkx2-2 promoter by disrupting an insulation mechanism between Pax1 and Nkx2-2. Furthermore, we show that expression of Bapx1, a downstream target of Pax1, is more strongly affected in Pax1Un-s mutants than in Pax1-null mutants, suggesting that the ectopic expression of Nkx2-2 interferes with the Pax1-Bapx1 pathway. Taken together, we propose that a combination of a loss-of-function mutation of Pax1 and a gain-of-function mutation of Nkx2-2 is the molecular basis of the Pax1Un-s mutation.

We previously reported close physical linkage between Pax9 and Nkx2-9 in the human, mouse, and pufferfish (Fugu rubripes) genomes. In this study, we analyzed cis-regulatory elements of the two genes by comparative sequencing in the three species and by transgenesis in the mouse. We identified two regions including conserved noncoding sequences that possessed specific enhancer activities for expression of Pax9 in the medial nasal process and of Nkx2-9 in the ventral neural tube. Remarkably, the latter contained the consensus Gli-binding motif. Interestingly, the identified Pax9 cis-regulatory sequences were located in an intron of the neighboring gene Slc25a21. Close examination of an extended genomic interval around Pax9 revealed the presence of strong synteny conservation in the human, mouse, and Fugu genomes. We propose such an intersecting organization of cis-regulatory sequences in multigenic regions as a possible mechanism that maintains evolutionary conserved synteny.

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33°C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39°C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39°C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33°C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39°C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.

The CDR3 regions of T cell receptor (TCR)-alpha and -beta chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-alpha and -beta to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-alpha or -beta chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-beta or -alpha chains. Surprisingly, one-third of such TCR-beta containing random CDR3 beta from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-alpha. A similar dominant function of TCR-alpha in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus-specific TCR. Subsequently, we generated TCR-alpha and/or -beta transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-alpha Tg generated CTLs with heterogeneous TCR-beta, whereas TCR-beta Tg-induced CTLs bearing a single TCR-alpha. These findings of Ag recognition with minimum involvement of CDR3 beta expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-alpha chain in determining the preimmune repertoire of Ag-specific TCR.

Parvalbumin (PV) is expressed in fast-twitch fibers in skeletal muscles and a subpopulation of inhibitory neurons in the CNS. We generated transgenic mice that expressed the human interleukin-2 receptor alpha-subunit-green fluorescent fusion protein (hIL-2R-GFP) using two types of PV transgene. One contained the hIL-2R-GFP gene downstream of a 16.5-kb 5'-upstream PV genomic sequence (PV line). The other comprised the hIL-2R-GFP gene in bacterial artificial chromosome (BAC) with either a 180-kb (PA line) or 155-kb (PB line) insert encompassing the PV gene. Independent lines of all transgenic mice showed a faithful hIL-2R-GFP expression in fast-twitch muscle fibers. However, appreciable hIL-2R-GFP expression in the CNS occurred only in the PA transgenic lines. In one line of PA transgenic mice, hIL-2R-GFP was properly expressed in PV-containing neurons in the cerebellum, thalamic reticular nucleus, globus pallidus and cerebral cortex, though ectopic expression was observed in a particular subset of cerebellar astrocytes. Another line of PA transgenic mice showed a selective and mosaic expression of hIL-2R-GFP in PV-containing Purkinje, basket and stellate cells in the cerebellum. These results indicate that the 16.5-kb PV genomic sequence is sufficient for fiber-type-selective transcription but additional regulatory sequences comprised in BAC DNA are required for proper expression in PV-containing neurons.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for fibroblasts, chondrocytes, and vascular endothelial cells. In the present study, we established transgenic (Tg) mice that overproduce CTGF/Hcs24 under the control of mouse type XI collagen promoter. Tg mice could develop and their embryonic and neonatal growth occurred normally. But they showed dwarfism within a few months of birth. X-ray analysis revealed that their bone density was decreased compared with normal mice. The femurs in the hindlimbs in particular showed an apparent low density. These results indicated that overexpression of CTGF/Hcs24 affects certain steps of endochondral ossification. In addition, the testes were much smaller than normal and fertility was affected in Tg mice, indicating that CTGF/Hcs24 may also regulate the embryonic development of the testis.

c-Jun has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to c-Jun activation in AD is not known. Activation of c-Jun N-terminal kinase (JNK) is obviously a candidate for the upstream event. We tested this possibility focusing on PS1-linked AD. First, we observed that JNK is actually activated in cerebral neurons of PS1-linked AD patients, using immunohistochemistry and Western blot analyses with anti-activated JNK antibodies. We analyzed the relationship between beta-amyloid (beta A) and JNK activation by using aged transgenic mice overexpressing mutant (M146L) PS1 and human AD brains. The mice showed no neuronal loss but a very few diffuse beta A deposits, corresponding to the early stage of PS1-linked AD brain. Some neurons were reactive for anti-beta A antibodies in the cerebral cortex. Interestingly, JNK activation was observed in neurons showing intracellular beta A immunoreactivity in transgenic mice. Association between intracellular beta A and JNK activation was confirmed in cortical neurons of sporadic and PS1-linked AD patients. Furthermore, introduction of beta A peptides into the primary culture cortical neurons induced JNK activation and cell death. Collectively, these results suggested that intracellular beta A accumulation might trigger JNK activation leading to neuronal death.

At around embryonic day 9, the primitive heart of a mouse embryo undergoes spectacular alterations within 24 hours. We created mice harboring an nls-lacZ gene in place of connexin45, which encodes the only known gap junction protein in the primitive heart before embryonic day 9, using the Cre-loxP system. Connexin45-deficient mice died of heart failure at around embryonic day 10. They initiated heart contractions, but conduction block appeared within 24 hours after the first contractions. Their cardiac walls displayed an endocardial cushion defect, while the cardiac jelly was present. These abnormalities were caused by impairment of the epithelial-mesenchymal transformation of the cardiac endothelium. Activation of the cardiac endothelium depended on the presence of the connexin45 gap junctions since signaling through Ca2+/calcineurin and NF-ATc1 (originally named NF-ATc) was disrupted in the mutant hearts. These results indicate a requirement for gap junction channels during early cardiogenesis and hence implicate connexin45 in congenital heart diseases. Movies available on-line: http://www.biologists.com/Development/movies/dev4369.html

At least two types of interleukin (IL)-15 mRNA isoforms are generated by alternative splicing at the 5' upstream of exon 5 in mice. To elucidate the potential roles of IL-15 isoforms in immune responses in vivo, we constructed two groups of transgenic mice using originally described IL-15 cDNA with a normal exon 5 (normal IL-15 transgenic [Tg] mice) and IL-15 cDNA with an alternative exon 5 (alternative IL-15 Tg mice) under the control of an MHC class I promoter. Normal IL-15 Tg mice constitutionally produced a significant level of IL-15 protein and had markedly increased numbers of memory type (CD44(high) Ly6C(+)) of CD8(+) T cells in the LN. These mice showed resistance to Salmonella infection accompanied by the enhanced interferon (IFN)-gamma production, but depletion of CD8(+) T cells exaggerated the bacterial growth, suggesting that the IL-15-dependent CD8(+) T cells with a memory phenotype may serve to protect against Salmonella infection in normal IL-15 Tg mice. On the other hand, a large amount of intracellular IL-15 protein was detected but hardly secreted extracellularly in alternative IL-15 Tg mice. Although most of the T cells developed normally in the alternative IL-15 Tg mice, they showed impaired IFN-gamma production upon TCR engagement. The alternative IL-15 transgenic mice were susceptible to Salmonella accompanied by impaired production of endogenous IL-15 and IFN-gamma. Thus, two groups of IL-15 Tg mice may provide information concerning the different roles of IL-15 isoforms in the immune system in vivo.

Crk family adapter proteins including Crk-II, Crk-I, and Crk-L consist mostly of SH2 and SH3 domains. Through the interactions between SH2 domain and phosphotyrosine residues and/or between SH3 domain and proline-rich motifs, they are involved in a variety of signaling cascades. Despite their essential roles in the signal transductions, knock-out mice of these molecules have not been reported yet. We performed the gene trap insertional mutagenesis with a trap vector, pU-Hachi, and generated a mutant mice line, Ayu 8104, in which the trap vector was inserted into the c-crk gene. Homozygous Ayu 8104 mice lacked Crk-II and Crk-I transcripts but expressed the truncated Crk proteins retaining one SH2 and one SH3 domain. Since the structure of the truncated proteins was similar to that of Crk-I, the insertion was considered to cause Crk-II-specific disruption. Homozygous mutant mice, however, did not exhibit any obvious abnormalities, suggesting that Crk-family adapters, Crk-II, Crk-I, and Crk-L would redundantly function in the signaling cascades and Crk-II was not apparently essential for embryonic development.

HSP47 is a collagen-binding heat shock protein and is assumed to act as a molecular chaperone in the biosynthesis and secretion of procollagen. As the synthesis of HSP47 is closely correlated with that of collagen in various cell lines and tissues, we performed a promoter/reporter assay using HSP47-producing and nonproducing cells. 280 base pairs (bp(s)) of upstream promoter were shown to be necessary for the basal expression but not to be enough for the cell type-specific expression. When the first and the second introns were introduced downstream of this 280-bp region, marked up-regulation of the reporter activity was observed in HSP47-producing cells but not in nonproducing cells. This was confirmed in transgenic mice by staining the lacZ gene product under the control of the 280-bp upstream promoter and the introns. Staining was observed in skin, chondrocytes, precursor of bone, and other HSP47/collagen-producing tissues. A putative Sp1-binding site at -210 bp in the promoter, to which Sp3 and an unidentified protein bind, was shown to be responsible for this up-regulation when combined with the introns. However no difference in the binding to this probe was observed between HSP47-producing and nonproducing cells. The responsible region for cell type-specific up-regulation was found to be located in a 500-bp segment in the first intron. On electrophoresis mobility shift assay using this 500-bp probe, specific DNA-protein complexes were only observed in HSP47-producing cell extracts. These results suggest that two separate elements are necessary for the cell type-specific expression of the hsp47 gene; one is a putative Sp1-binding site at -210 bp necessary for basal expression, and the other is a 500-bp region within the first intron, required for cell type-specific expression.

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken β-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development. Mol. Reprod. Dev. 52:376–382, 1999. © 1999 Wiley-Liss, Inc.

A mouse model of Rubinstein-Taybi syndrome (RTS) was generated by an insertional mutation into the cyclic AMP response element-binding protein (CREB)-binding protein (CBP) gene. Heterozygous CBP-deficient mice, which had truncated CBP protein (residues 1-1084) containing the CREB-binding domain (residues 462-661), showed clinical features of RTS, such as growth retardation (100%), retarded osseous maturation (100%), hypoplastic maxilla with narrow palate (100%), cardiac anomalies (15%) and skeletal abnormalities (7%). Truncated CBP is considered to have been acting during development as a dominant-negative inhibitor to lead to the phenotypes of RTS in mice. Our studies with step-through-type passive avoidance tests and with fear conditioning test showed that mice were deficient in long-term memory (LTM). In contrast, short-term memory (STM) appeared to be normal. These results implicate a crucial role for CBP in mammalian LTM. Our CBP +/- mice would be an excellent model for the study of the role of CBP in development and memory storage mechanisms.

The role of inhibitory Golgi cells in cerebellar function was investigated by selectively ablating Golgi cells expressing human interleukin-2 receptor α subunit in transgenic mice, using the immunotoxin-mediated cell targeting technique. Golgi cell disruption caused severe acute motor disorders. These mice showed gradual recovery but retained a continuing inability to perform compound movements. Optical and electrical recordings combined with immunocytological analysis indicated that elimination of Golgi cells not only reduces GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells. These results demonstrate that synaptic integration involving both GABA inhibition and NMDA receptor activation is essential for compound motor coordination. Furthermore, this integration can adapt after Golgi cell elimination so as not to evoke overexcitation by the reduction of NMDA receptors.

T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4+ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4+ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4+ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4+ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4+ T cell decline in patients.

Hepatitis C virus (HCV), a major causative agent of post transfusion non-A, non-B hepatitis (NANBH), can only infect humans and chimpanzees. We produced nine transgenic mouse lines carrying a full-length HCV cDNA with the human serum amyloid P component (hSAP) promoter that can direct liver-specific expression. In one of these lines HCV mRNA and HCV core protein were detected in the liver of the transgenic mouse, although the levels of expression were very low. In addition, HCV-related antibody was detected in the serum.

Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes straightepsilon-(gamma-glutamyl) lysine cross-links of proteins to form the cell envelope at the periphery of cornified cells. A transient expression assay using a luciferase reporter gene linked to the 2.5 kb 5' upstream region of the human TGase 1 gene (TGM1) showed phorbol ester-responsive promoter activity in cultured normal human keratinocytes. To assess its promoter activity in vivo, we generated transgenic mice expressing the Escherichia coli beta-galactosidase gene (lacZ) directed by the 5' upstream region. beta-Galactosidase histochemistry revealed that the TGM1-lacZ transgene was expressed in terminally differentiating keratinocytes in upper layers of stratified squamous epithelia in embryonic, neonatal and adult transgenic mice. The expression pattern was similar to that of endogenous TGase 1 mRNA detected by in situ hybridization. Furthermore, topical application of a phorbol ester to adult tail skin enhanced expression of the transgene as well as TGase 1 mRNA in the epidermis. Thus, the 2.5 kb 5' upstream sequence of TGM1 includes elements regulating tissue- and terminal differentiation-specific gene expression in stratified squamous epithelia.

Previous studies suggested that the Otx2 gene plays an essential role in the development of cranial skeletons and nerves of mesencephalic neural crest origin. To clarify this role, we have identified the cis-acting elements in mouse and pufferfish Otx2 genes responsible for the expression in the crest cells using a transgenic approach with the lacZ reporter gene. In mouse, 49 bp sequences in the proximal 5′ region upstream were essential and sufficient to direct the transgene expression in the cephalic mesenchyme. In pufferfish, the 1.1 kb distal region, located far downstream (from +14.4 to +15.5 kb), had almost identical activity. Between them, several DNA sequences were conserved, and mutational analyses indicated that motif A was critical for the transgene expression in the premandibular region while motif B was critical in both premandibular and mandibular regions. Motif B, CTAATTA, contains the core motif for binding of homeodomain proteins while motif A, TAAATCTG, does not match any known consensus binding sequences for transcriptional factors. The cephalic mesenchyme that expressed β-galactosidase under these cis-elements is most likely to correspond to mesencephalic crest cells. Thus the molecular machinery regulating Otx2 expression in these cells appears to be conserved between mouse and fish, implying a crucial role of the Otx2 gene in development of the neural-crest-derived structures of the gnathostome rostral head.

In order to study the regulatory mechanism of developmental and tissue-specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZ gene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.

Background: The c-myc protooncogene has been implicated in the control of cell proliferation, differentiation and/or apoptosis in various cellular systems. However, the role of c-myc in germ cell lineage is largely unknown. Results: We have produced transgenic mouse lines carrying the rat c-myc protooncogene under the control of human metallothionein promoter (hMT-c-myc). It was found that the male transgenic mice were sterile. In contrast, all of the female transgenic mice were completely fertile and transmitted the transgene to the next generation. However, male transgenic mice from the female transgenic founders were also found to be sterile. This sterility was due to a defect in spermatogenic cell differentiation, since virtually no sperm were seen within the seminiferous tubules or the cauda epididymis. Histological examination revealed that germ cell death occurred approximately 7 days after birth and, consequently, spermatogenesis was arrested at an early stage in meiotic division in the transgenic mice. Moreover, this germ cell death was found to be caused by apoptosis. Conclusion: We conclude that an excess level of c-myc expression in differentiating spermatogenic cells is responsible for the apoptotic death of germ cell, and that a decrease in c-myc level would be an obligatory step for the completion of normal spermatogenesis.

New Chemical Modification of Polymers with Pendant Chloromethyl Groups Using 1,8-Diazabicyclo-[5.4.0]-7-undecene

To evaluate the lateral resistance of single piles for dolphins under repeated loads and large deformations, lateral loading tests were carried out for model piles embedded in sandy soil in a laboratory. The model piles were made of high-tensile steel plates 2500mm in length and 200mm in width. The conclusions of this study are as follows: 1) the test results of the relationship between soil reaction p and pile deflection y show that Kubo's formula (pocy0.5) is applicable to large deflection under the initial loading phase of each package of cycles. 2) the deflections and inclination angles of the pile increase with the loading cycles. Increasing ratios have linear relations to the logarithmic number of loading cycles when the magnitude of the load is kept constant.

region in wild-type mouse, we found that the pattern was different in the left half of the insert (Fig. 1), suggesting the presence of a junction in the middle of the insert. We then subcloned a BglIIKpnI fragment that might contain the integration junction sequence. DNA sequence analysis of the fragment showed that one end of the insert contained a part of the transgene, while the other end corresponded to the exon 8 of the Brachyury gene. This finding clearly indicated that the #137 mutation represents a new disrupted allele of the Brachyury locus. We hence referred to #137

Mapping the Neuronal Map In vertebrates, sensory information is topographically represented as a neural map in the brain. How is the neural map formed in the brain? Nearly a half-century ago, Roger Sperry proposed the “chemoaffinity” model, in which the positional cues on the target determine the axonal projection site, thereby establishing the topographic neural map. However, molecular mechanisms of topographic map formation remain controversial. Imai et al. (p. 585 , published online 9 July; see the Perspective by Miyamichi and Luo ) now report that the topographic map is formed by axon-axon interactions before the axons reach the target. In the mouse olfactory system, the topography of the map is determined by the relative expression levels of a guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, expressed in axons. Topographic organization occurs even in the absence of the target, the olfactory bulb. These findings require that Sperry's model, which suggests that only the targets determine the topography of neural maps, needs to be reconsidered.

To establish cell lines exhibiting differentiation phenotypes, the immortalized cell lines were rapidly established from the primary culture of different tissues of transgenic mice harboring SV40 temperature-sensitive large T-antigen gene. The established cell lines grew at permissive temperature (33 degrees C), but not at nonpermissive temperature (39 degrees C). Several different cell types could be rapidly immortalized and cloned from the adult transgenic mice tissues. Among those cell lines, the established hepatocyte cell lines (TLR cell lines) exhibited liver-specific morphological and biochemical properties, but their properties were not coupled with the growth condition modified by temperature. The hepatocyte cell lines showed an inducibility of P450IA1 by 3-methylcholanthrene as observed in rat livers and this liver-specific function was stable even after 6 months of culture by continuous passages.

Transthyretin (TTR) associated amyloidosis is an autosomal dominant disorder characterized by peripheral and autonomic neuropathy. Both genetic and environmental factors are thought to be involved in development of TTR associated amyloidosis. Previously, we demonstrated that amyloid deposition was observed in various tissues of transgenic mouse lines carrying a human mutant TTR (Met30) gene. To analyze the influence of environmental factors on TTR amyloidosis, these amyloidogenic transgenic mouse models were kept under conventional (CV) or specific pathogen free (SPF) conditions. Although the serum levels of Met30 for mice housed in the CV and SPF conditions were similar, amyloid deposition was observed in CV conditions, but not in SPF conditions. In addition, the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. There were significant differences in proportion of amyloid deposition in several tissues between CV and SPF conditions. Maintenance of these mice at 30 degrees C did not induce amyloid deposition in SPF conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder.

Transgenic mice are very useful for analyzing the functions of a gene either at the tissue level or at the whole body level. Here we describe the production of transgenic mice by the microinjection of DNA into the pronuclei of fertilized mouse eggs.