A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene.

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.

The multiple roles of macrophage scavenger receptors (MSR) in vivo: resistance to atherosclerosis and susceptibility to infection in MSR knockout mice.

Both type I and type II MSRs are integral membrane proteins containing a collagenous domain and elicit an extraordinarily wide range of ligand binding capability. They were found during the search for the molecule(s) responsible for the accumulation of modified LDL during atherogenesis. However, all prior the evidence relating to their physiological and pathophysiological roles in vivo had been indirect. Targeted disruption of the MSR gene results in a reduction in the size of atherosclerotic lesions in an apo E deficient animal. Macrophages from MSR deficient mice exhibit a marked decrease in modified LDL uptake in vitro, whereas modified LDL clearance from plasma remains normal, suggesting that there are alternative mechanisms for the uptake of modified LDL from the circulation. In addition, MSR knockout mice are more susceptible to L. monocytogenes and HSV-1 infection, indicating a role for MSR in host defense against various pathogens.

Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos.

Mice entirely derived from ES cells were obtained from aggregates of TT2 ES cells and cytochalasin B induced tetraploid embryos. Tetraploid embryos were cocultured with ES cells in a well on the Multiplate-Terasaki. After embryo transfer of the aggregates, the male newborns were recovered normally after Cesarean section and reached adulthood. The male mice exhibited complete pigmentation of the eye and coat, suggesting ES cell contributions alone. Alkaline phosphatase-1 analysis yielded no evidence of tetraploid cells in the kidney or liver. The TT2-derived males were fertile, produced normal offspring, and exclusively transmitted the TT2 genotype to their progeny. This result clearly shows that ES cells are able to support complete fetal development.

Germ-line contribution of embryonic stem cells in chimeric mice: influence of karyotype and in vitro differentiation ability.

The effect of the karyotype and the ability to differentiate in vitro upon germ-line transmission by A3-1 embryonic stem (ES) cells in chimeric mice were examined. Germ-line transmission was confirmed in ES cells exhibiting 38% and more of the normal karyotype, but no chimeric mice and/or germ-line transmitters were observed regardless of the karyotype when the cystic embryoid body (CEB) was formed on day 8 and later in the suspension culture. Germ-line transmission of the ES cells was not significantly influenced by formation of the simple embryoid body (SEB). Germ-line transmitters were preferentially observed in chimeras when the ES cell contribution to coat color was markedly increased, but this contribution to coat color varied regardless of the karyotype or in vitro differentiation ability. These results suggest that A3-1 ES cells which exhibit CEB at 7 days after suspension culture and approximately 40% of normal karyotype are capable of germ-line transmission in chimeric mice.

Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay.

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than -80 degrees C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and -80 degrees C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Alpha-tocopherol transfer protein is specifically localized at the implantation site of pregnant mouse uterus.

Alpha-tocopherol transfer protein (alpha-TTP) was first described to play a major role in maintaining alpha-tocopherol levels in plasma, while alpha-tocopherol was primarily reported to be a factor relevant for reproduction. Expression of alpha-TTP is not only seen in the liver, from where it was first isolated, but also in mouse uterus, depending on its state of pregnancy, stressing the importance of alpha-TTP for embryogenesis and fetal development. The cellular localization of alpha-TTP in mouse uterus is reported here. By immunohistochemistry, alpha-TTP could be localized in the secretory columnar epithelial cells of the pregnant uterus on Days 4.5 and 6.5 postcoitum as well as in the glandular epithelial cells and the inner decidual reaction zone surrounding the implantation site. On Days 8.5 and 10.5 postcoitum (midterm of mouse pregnancy), alpha-TTP could still be detected in the uterine secretory columnar epithelial cells, while in alpha-TTP knockout mice, no immunostaining was visible. It is suggested that alpha-TTP plays a major role in supplying the placenta and consecutively the fetus with alpha-tocopherol throughout pregnancy. We conclude that alpha-tocopherol plays a role in the process of implantation and that alpha-TTP may be necessary for adequate alpha-tocopherol status of the fetus.

The POU domain transcription factor Brn-2 is required for the determination of specific neuronal lineages in the hypothalamus of the mouse.

We generated mice carrying a loss-of-function mutation in Brn-2, a gene encoding a nervous system specific POU transcription factor, by gene targeting in embryonic stem cells. In homozygous mutant embryos, migratory precursor cells for neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SO) of the hypothalamus die at approximately E12.5. All homozygous mutants suffered mortality within 10 days after birth, possibly because of a complete deficiency of these neurons in the hypothalamus. Although neither developmental nor histological abnormalities were observed in heterozygous mice, the levels of expression of vasopressin and oxytocin in the hypothalamus of these animals were half these of wild-type mice. These results strongly suggest that Brn-2 plays an essential role in the determination and development of the PVN and SO neuronal lineages in the hypothalamus.

Genetic and physical delineation of the region of the mouse deafness mutation shaker-2.

A total of 951 backcross progeny have been obtained from a backcross segregating for the mouse deafness mutation, shaker-2(sh-2). Linkage analysis provides a detailed genetic map in the vicinity of sh-2 which comprises 40 backcross mice identified as recombinant within a 4 cM region. This allows construction of a contig consisting of 21 BAC clones across an approximately 700-kb region of sh-2. This covers the entire nonrecombinant region of sh-2 and is therefore useful to facilitate the identification of genes in the sh-2 region.

Accelerated rejection of Fas ligand-expressing heart grafts.

The Fas/Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Recently, there has been renewed interest in immune privilege, as it was shown that two privileged sites (the eye and testes) constitutively express FasL, which kills lymphoid cells that invade these areas. We have established murine FasL-transgenic mice (B6) under the control of the cardiac alpha-myosin heavy chain promotor, and transplanted FasL-expressing F1(B6 x C3H/HeJ) heart grafts into syngeneic (F1) and allogeneic (C3H/HeJ) recipients. FasL-expressing F1 heart allografts placed in C3H/HeJ recipients as well as FasL-expressing F1 isografts placed in nontransgenic and FasL-transgenic F1 were more rapidly rejected, and their survival was much shorter than that of nontransgenic control F1 allografts placed in C3H/HeJ. Native control and FasL-expressing hearts looked normal in mice up to 8 wk of age on hematoxylin-eosin staining. Control heart allografts undergoing ordinally acute rejection showed moderate focal lymphocyte infiltrates, while FasL-expressing F1 allografts and isografts showed massive hemorrhage, edema, and massive neutrophil infiltration as early as 1 day after transplantation. In conclusion, FasL expression and surgical procedure (ischemia/reperfusion) were synergistic in the induction of accelerated heart graft rejection, while allogenicity was not necessary. It may be necessary to find ways of controlling neutrophilic reaction/apoptosis in infiltrating lymphocytes to use FasL in clinical organ transplantation.

Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes.

Streptozotocin (STZ), a glucose analogue known to induce diabetes in experimental animals, causes DNA strand breaks and subsequent activation of poly(ADPribose) polymerase (Parp). Because Parp uses NAD as a substrate, extensive DNA damage will result in reduction of cellular NAD level. In fact, STZ induces NAD depletion and cell death in isolated pancreatic islets in vitro. Activation of Parp therefore is thought to play an important role in STZ-induced diabetes. In the present study, we established Parp-deficient (Parp-/-) mice by disrupting Parp exon 1 by using the homologous recombination technique. These mice were used to examine the possible involvement of Parp in STZ-induced beta-cell damage in vivo. The wild-type (Parp+/+) mice showed significant increases in blood glucose concentration from 129 mg/dl to 218, 370, 477, and 452 mg/dl on experimental days 1, 7, 21, and 60, respectively, after a single injection of 180 mg STZ/kg body weight. In contrast, the concentration of blood glucose in Parp-/- mice remained normal up to day 7, slightly increased on day 21, but returned to normal levels on day 60. STZ injection caused extensive necrosis in the islets of Parp+/+ mice on day 1, with subsequent progressive islet atrophy and loss of functional beta cells from day 7. In contrast, the extent of islet beta-cell death and dysfunction was markedly less in Parp-/- mice. Our findings clearly implicate Parp activation in islet beta-cell damage and glucose intolerance induced by STZ in vivo.

Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disease of humans caused by an unidentified infectious agent, the prion. To determine whether there was an involvement of the host-encoded prion protein (PrPc) in CJD development and prion propagation, mice heterozygous (PrP+/-) or homozygous (PrP-/-) for a disrupted PrP gene were established and inoculated with the mouse-adapted CJD agent. In keeping with findings of previous studies using other lines of PrP-less mice inoculated with scrapie agents, no PrP-/- mice showed any sign of the disease for 460 days after inoculation, while all of the PrP+/- and control PrP+/+ mice developed CJD-like symptoms and died. The incubation period for PrP+/- mice, 259 +/- 27 days, was much longer than that for PrP+/+ mice, 138 +/- 12 days. Propagation of the prion was barely detectable in the brains of PrP-/- mice and was estimated to be at a level at least 4 orders of magnitude lower than that in PrP+/+ mice. These findings indicate that PrPc is necessary for both the development of the disease and propagation of the prion in the inoculated mice. The proteinase-resistant PrP (PrPres) was undetectable in the brain tissues of the inoculated PrP-/- mice, while it accumulated in the affected brains of PrP+/+ and PrP+/- mice. Interestingly, the maximum level of PrPres in the brains of PrP+/- mice was about half of the level in the similarly affected brains of PrP+/+ mice, indicating that PrPres accumulation is restricted by the level of PrPc.

Delayed-onset ataxia in mice lacking alpha -tocopherol transfer protein: model for neuronal degeneration caused by chronic oxidative stress.

alpha-Tocopherol transfer protein (alpha-TTP) maintains the concentration of serum alpha-tocopherol (vitamin E), one of the most potent fat-soluble antioxidants, by facilitating alpha-tocopherol export from the liver. Mutations of the alpha-TTP gene are linked to ataxia with isolated vitamin E deficiency (AVED). We produced a model mouse of AVED by deleting the alpha-TTP gene, which showed ataxia and retinal degeneration after 1 year of age. Because the brain alpha-TTP functions in maintaining alpha-tocopherol levels in the brain, alpha-tocopherol was completely depleted in the alpha-TTP(-/-) mouse brain, and the neurological phenotype of alpha-TTP(-/-) mice is much more severe than that of wild-type mice when maintained on an alpha-tocopherol-deficient diet. Lipid peroxidation in alpha-TTP(-/-) mice brains showed a significant increase, especially in degenerating neurons. alpha-Tocopherol supplementation suppressed lipid peroxidation and almost completely prevented the development of neurological symptoms. This therapy almost completely corrects the abnormalities in a mouse model of human neurodegenerative disease. Moreover, alpha-TTP(-/-) mice may prove to be excellent animal models of delayed onset, slowly progressive neuronal degeneration caused by chronic oxidative stress.

A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection.

Macrophage type-I and type-II class-A scavenger receptors (MSR-A) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins (mLDL). MSR-A can bind an extraordinarily wide range of ligands, including bacterial pathogens, and also mediates cation-independent macrophage adhesion in vitro. Here we show that targeted disruption of the MSR-A gene in mice results in a reduction in the size of atherosclerotic lesions in an animal deficient in apolipoprotein E. Macrophages from MSR-A-deficient mice show a marked decrease in mLDL uptake in vitro, whereas mLDL clearance from plasma occurs at a normal rate, indicating that there may be alternative mechanisms for removing mLDL from the circulation. In addition, MSR-A-knockout mice show an increased susceptibility to infection with Listeria monocytogenes or herpes simplex virus type-1, indicating that MSR-A may play a part in host defence against pathogens.