PL
Regenerative medicine for spinal cord injury ○Masaya Nakamura Keio University School of Medicine, Tokyo, Japan
1) Hepatocyte growth factor: Intrathecal administration of recombinant human HGF protein also reduced damaged area and promoted functional recovery after SCI even in common marmoset, suggesting the therapeutic efficacy of HGF for SCI and its possibility for clinical application. 2) Cell transplantation focusing on induced pluripotent stem cells: There is an increasing interest in the iPS cells and reprogramming technologies in medical science. While iPS cells are expected to open new era providing enormous opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine, safetyrelated concerns for iPS cell-based cell therapy should be resolved prior to the clinical application of iPS cells. In this lecture, the pre-clinical investigations of cell therapy for SCI using neural stem/progenitor cells derived from iPS cells, and their safety issues in vivo are outlined. 3) Diffusion tensor tractography: Recent advances in MRI technology have led to the development of diffusion tensor tractography (DTT) as a potential modality to perform in vivo tracing of axonal fibers. We demonstrated the effectiveness of DTT to visualize both intact and surgically disrupted spinal long tracts in common marmosets using hemisection and contusion injury model. DTT clearly illustrated spinal projections such as the corticospinal tract and afferent fibers in control animals and depicted the severed long tracts in the injured animals. Histology of the spinal cords was consistent with DTT findings, verifying the accuracy of DTT.
L-1
Development of reproduction engineering techniques for bioresources and their research use. ○Atsuo Ogura RIKEN BioResource Center, Tsukuba, Japan
Laboratory animals are important bioresources that greatly contribute to advancements of medicine and biology. Especially, the laboratory mouse (Mus musculus) is the mammalian species that is most widely used because of the availability of the abundant genetic information and the technologies for gene modifications. Since the establishment of RIKEN BioResource Center in 2001, the Bioresource Engineering Division, my laboratory, is the sole technology division at BRC. Since then the division has been in charge of the development of bioresource-related techniques, especially reproductive engineering techniques applied to mice and stem cell technologies, and of maintaining and distributing these bioresources at a high quality. The specific functions of the division are: (1) cryopreservation of embryos and gametes; (2) microinsemination (sperm injection); (3) nuclear transfer; and (4) the establishment of new stem cell lines and generation of new animal models. Our laboratory may be the only one in the world that uses all four technologies at the highest level, thanks to the big efforts of laboratory members. Additionally, I would like to stress that the high achievements of my laboratory depended largely on highly recognized collaborators outside RIKEN. Our team’s strong point is that we can combine our techniques with genetic and biochemical analyses, so that we may solve important biological questions in a unique and refined way.
—S 3—
L-2
Identifying of the causative genes in spontaneous mutant mouse strains by forward-reverse genetics ○Seiya Mizuno Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan
The forward genetic analysis with spontaneous mutant gives us new insights of gene function in vivo. I also had found three mutant strains (TAS, Moja, and WS) in our laboratory.The TAS is a transgenic mouse carried rtTA and tTS. Although these transgenes does not affect brain development, TAS mouse showed agenesis of the corpus callosum (ACC). The FISH analyses revealed that both rtTA and tTS were inserted between 11 and 13 Mb on Chr. 18. I then found abnormal Cables1 gene expression in TAS. The exon4 of Cables1 was deleted by transgene insertion. Interestingly, spliced exon 1-3 of Cables1 was fused to transgene in mRNA level. I concluded ACC in TAS is caused by effect of mutated Cables1 fused to transgenes.The Moja mouse is spontaneous mutant which showed long hair. Because the phenotypes of Maja were very similar to Fgf5 null mutant mouse, we analyzed Fgf5 gene locus in Moja. As expected, Fgf5 was not expressed in Moja skin caused by lack of exon3 of Fgf5 by natural LTR insertion mutation.The WS mutant embryos failed to implantation. FISH and SNPs analyses revealed that approximately 1.2 Mb genomic region containing 9 genes (from Kit to Cep135) was deleted in WS. Since the Exoc1, one of these 9 genes, was highly expressed in early embryos, I selected the Exoc1 as first candidate of causative gene for early embryonic death in WS. As expected, Exoc1 null mutant showed embryonic death same as WS. These results indicate lethal phenotype in WS is caused by Exoc1 deletion.
S1-1
Germfree and gnotobiotic animals ○Kazuhiro Hirayama Department of Veterinary Public Health, The University of Tokyo, Tokyo, Japan
Intestinal microbiota plays important roles in health and diseases. However, it is often very difficult to study the role of intestinal microbiota, partly due to its extremely diverse composition and very special environment in the intestine including highly anaerobic condition. By comparing conventional and germfree animals, with and without intestinal microbiota, role of microbiota and their effects on hosts, both beneficial and harmful, can be studied in vivo. At the very beginning of the germfree animal research, major interest on the great efforts to establish germfree animal colonies was whether life without bacteria would be possible or not. Since germfree technology has been established, germfree animals have been used to study physiology and pathology of the host without interference of indigenous microbiota. Infectious diseases have been one of the most important subjects in germfree research. Germfree animals have also been playing significant contributions to the understanding of nutrition and immunological phenomena. Germfree animals inoculated with known bacterial strain(s), gnotobiotic animals, have revealed the role of particular bacterial strain(s) or species. Recently, germfree genetically modified rodents which are used as disease models demonstrated important role of intestinal microbiota for expression of pathophysiology.
—S 4—
Regenerative medicine for spinal cord injury ○Masaya Nakamura Keio University School of Medicine, Tokyo, Japan
1) Hepatocyte growth factor: Intrathecal administration of recombinant human HGF protein also reduced damaged area and promoted functional recovery after SCI even in common marmoset, suggesting the therapeutic efficacy of HGF for SCI and its possibility for clinical application. 2) Cell transplantation focusing on induced pluripotent stem cells: There is an increasing interest in the iPS cells and reprogramming technologies in medical science. While iPS cells are expected to open new era providing enormous opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine, safetyrelated concerns for iPS cell-based cell therapy should be resolved prior to the clinical application of iPS cells. In this lecture, the pre-clinical investigations of cell therapy for SCI using neural stem/progenitor cells derived from iPS cells, and their safety issues in vivo are outlined. 3) Diffusion tensor tractography: Recent advances in MRI technology have led to the development of diffusion tensor tractography (DTT) as a potential modality to perform in vivo tracing of axonal fibers. We demonstrated the effectiveness of DTT to visualize both intact and surgically disrupted spinal long tracts in common marmosets using hemisection and contusion injury model. DTT clearly illustrated spinal projections such as the corticospinal tract and afferent fibers in control animals and depicted the severed long tracts in the injured animals. Histology of the spinal cords was consistent with DTT findings, verifying the accuracy of DTT.
L-1
Development of reproduction engineering techniques for bioresources and their research use. ○Atsuo Ogura RIKEN BioResource Center, Tsukuba, Japan
Laboratory animals are important bioresources that greatly contribute to advancements of medicine and biology. Especially, the laboratory mouse (Mus musculus) is the mammalian species that is most widely used because of the availability of the abundant genetic information and the technologies for gene modifications. Since the establishment of RIKEN BioResource Center in 2001, the Bioresource Engineering Division, my laboratory, is the sole technology division at BRC. Since then the division has been in charge of the development of bioresource-related techniques, especially reproductive engineering techniques applied to mice and stem cell technologies, and of maintaining and distributing these bioresources at a high quality. The specific functions of the division are: (1) cryopreservation of embryos and gametes; (2) microinsemination (sperm injection); (3) nuclear transfer; and (4) the establishment of new stem cell lines and generation of new animal models. Our laboratory may be the only one in the world that uses all four technologies at the highest level, thanks to the big efforts of laboratory members. Additionally, I would like to stress that the high achievements of my laboratory depended largely on highly recognized collaborators outside RIKEN. Our team’s strong point is that we can combine our techniques with genetic and biochemical analyses, so that we may solve important biological questions in a unique and refined way.
—S 3—
L-2
Identifying of the causative genes in spontaneous mutant mouse strains by forward-reverse genetics ○Seiya Mizuno Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan
The forward genetic analysis with spontaneous mutant gives us new insights of gene function in vivo. I also had found three mutant strains (TAS, Moja, and WS) in our laboratory.The TAS is a transgenic mouse carried rtTA and tTS. Although these transgenes does not affect brain development, TAS mouse showed agenesis of the corpus callosum (ACC). The FISH analyses revealed that both rtTA and tTS were inserted between 11 and 13 Mb on Chr. 18. I then found abnormal Cables1 gene expression in TAS. The exon4 of Cables1 was deleted by transgene insertion. Interestingly, spliced exon 1-3 of Cables1 was fused to transgene in mRNA level. I concluded ACC in TAS is caused by effect of mutated Cables1 fused to transgenes.The Moja mouse is spontaneous mutant which showed long hair. Because the phenotypes of Maja were very similar to Fgf5 null mutant mouse, we analyzed Fgf5 gene locus in Moja. As expected, Fgf5 was not expressed in Moja skin caused by lack of exon3 of Fgf5 by natural LTR insertion mutation.The WS mutant embryos failed to implantation. FISH and SNPs analyses revealed that approximately 1.2 Mb genomic region containing 9 genes (from Kit to Cep135) was deleted in WS. Since the Exoc1, one of these 9 genes, was highly expressed in early embryos, I selected the Exoc1 as first candidate of causative gene for early embryonic death in WS. As expected, Exoc1 null mutant showed embryonic death same as WS. These results indicate lethal phenotype in WS is caused by Exoc1 deletion.
S1-1
Germfree and gnotobiotic animals ○Kazuhiro Hirayama Department of Veterinary Public Health, The University of Tokyo, Tokyo, Japan
Intestinal microbiota plays important roles in health and diseases. However, it is often very difficult to study the role of intestinal microbiota, partly due to its extremely diverse composition and very special environment in the intestine including highly anaerobic condition. By comparing conventional and germfree animals, with and without intestinal microbiota, role of microbiota and their effects on hosts, both beneficial and harmful, can be studied in vivo. At the very beginning of the germfree animal research, major interest on the great efforts to establish germfree animal colonies was whether life without bacteria would be possible or not. Since germfree technology has been established, germfree animals have been used to study physiology and pathology of the host without interference of indigenous microbiota. Infectious diseases have been one of the most important subjects in germfree research. Germfree animals have also been playing significant contributions to the understanding of nutrition and immunological phenomena. Germfree animals inoculated with known bacterial strain(s), gnotobiotic animals, have revealed the role of particular bacterial strain(s) or species. Recently, germfree genetically modified rodents which are used as disease models demonstrated important role of intestinal microbiota for expression of pathophysiology.
—S 4—
