O-1

Insertion of transgene in sex chromosomes in generating transgenic mice ○Naoki Ishikawa, Takayoshi Sasako, Katsuyoshi Kumagai, Naoto Kubota, Takashi Kadowaki Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

In generating transgenic (Tg) mice, the transgene is randomly inserted in the genome, including sex chromosomes, whose mechanism and possibility are still unclear. We examined 85 lines of 18 Tg mice which we have generated so far, focusing on those exhibiting sexually imbalanced genotype in the offspring, including 2 lines in which the transgene was potentially inserted in X chromosome, and 3 lines, in Y chromosome. We performed 3-color fluorescence in situ hybridization (FISH), except 1 line in which no mice were alive and available, confirming that the transgene was actually inserted in X chromosome in 2 lines, and in Y chromosome in 2 lines. All of the transgenes of these lines contained mouse-derived cDNAs, whereas promoters were derived from mice, rats and E. coli. The possibilities of insertion in X chromosome and Y chromosome are theoretically 5% and 1% respectively, since the mouse genome consists of autosomal chromosomes of 4.4 Gbps, X chromosome(s) of 1.7 Gbps, and Y chromosome of 0.9 Gbps. Our results were not necessarily compatible with this expectation, and examination of the transgenes is under way. To clarify what affects insertion of transgene in sex chromosomes in this way is expected to enable us to generate Tg mice more efficiently.

O-2

Improvement of mouse targeted transgenesis via pronuclear microinjection ○Masato Ohtsuka1, Hiromi Miura1, Masahiro Sato2, Minoru Kimura1 1

Basic Med. Sci. and Mol. Med., Sch. of Med., Tokai Univ., Kanagawa, Japan, 2Sec. of Gene Exp. Regul., FSRC, Kagoshima Univ., Kagoshima, Japan

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. However, it is always associated with random integration of multiple-copy transgenes that often causes unreliable transgene expression. Thus, typically three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them is/are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into the predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT); a similar method using PhiC31-attP/B system was developed by another group. Here, we developed an improved-PITT (i-PITT) system by combining Cre-loxP and PhiC31-attP/B systems directly under C57BL/6N inbred strain. The targeted Tg efficiency in the iPITT was improved up to 67%, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously: injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

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O-3

Viable chimeras created by injection of Mus caroli embryonic stem cells into tetraploid blastocysts ○Yasumitsu Nagao1, Yoshikazu Totsuka2, Takuro Horii3, Naomichi Moriya1, Kiyomi Kogusuri1, Izuho Hatada3, Tomoyuki Tokunaga4, Yutaka Hanazono5, Satoshi Kunita1, Hiroshi Imai6, Hitoshi Endo7 1

Center for Experimental Medicine, Jichi Medical University, Tochigi, Japan, 2Institute of Immunology Co., Ltd., Tochigi, Japan, 3Biosignal Genome Resource Center, Gunma University, Gunma, Japan, 4National Institute of Agrobiological Sciences, Ibaraki, Japan, 5Division of Regenerative Medicine, Jichi Medical University, Tochigi, Japan 6Graduate School of Agriculture, Kyoto University, Kyoto, Japan 7Department of Biochemistry, Jichi Medical University, Tochigi, Japan

Purpose) Mus caroli is another species of mouse closely related to Mus musculus (common laboratory mice). However, there are few reports about Mus caroli embryonic stem cells. To investigate the pluripotency of Mus caroli embryonic stem cells, we tried to produce chimeras by interspecific tetraploid complementation method. Methods) ES cells prepared from Car (BRC) random breeding strain were injected into tetoraploid blastocysts stage embryos (MCH). The embryos were transfered into the uterus of recipient mouse (MCH). Results) Injection of Mus caroli ES cells into Mus musculus tetoraploid blastocysts led to 100% chimeras of Mus caroli at E12.5. This result supports the pluripotency of Mus caroli embryonic stem cells and the utility of interspecific tetraploid complementation method between Mus caroli and Mus musculus .

O-4

Generation of knock-out rat using mouse←rat ES-chimera ○Ayako Isotani1, Masaki Ogawa3, Koudai Tanaka4, Takafumi Matsumura3, Kazuo Yamagata2, Masaru Okabe2, Masahito Ikawa1,2,3,4 1

World Premier International Research Center Immunology Frontier Research Center, Osaka University, Research Institute for Microbial Diseases, Osaka University, 3Faculty of Pharmaceutical Sciences, Osaka University, 4Faculty of Medicine, Osaka University

2

In a previous study, we produced mouse←rat chimera by injecting rat embryonic stem (ES) cells into mouse blastocysts. In this model, rat ES cells contributed to various organs, including spermatozoa, indicating the potential utility for the production of a knockout rat. Hence, we established new rat ES cell lines to visualize rat spermatozoa using green fluorescent protein (GFP) from green body-green sperm (GBGS) doubly transgenic rats. When GBGS-rat ES cells were used to produce mouse←rat chimera, rat spermatozoa were easily detected with GFP in chimeric testes. Subsequently, the germline potential of rat spermatozoa in the chimera were assessed by intracytoplasmic injections. Rat pups were generated from rat spermatozoa in chimeric testes, and normal germline potential of rat spermatozoa in the chimeric testes was demonstrated. Furthermore, we prepared a homologous recombined rat ES cell lines. Subsequently, we obtained rat pups from homologous recombined rat ES cell lines from chimeric mouse←rat ES cells. This study provides a novel method for the production of knockout rat.

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O-5

Generation of nonagouti (a) mutant MSM/Ms mice by the CRISPR/ Cas9 system and their characterization ○Michiko Hirose1, Ayumi Hasegawa1, Keiji Mochida1, Yuki Hatanaka1, Arata Honda1,2, Hideki Kaneda1, Ikuko Yamada1, Tamio Furuse1, Kuniya Abe1,3, Shigeharu Wakana1, Atsuo Ogura1,3 1

RIKEN BRC, Ibaraki, Japan, 2University of Miyazaki, Organization for Promotion of Tenure Track, Miyazaki, Japan, 3University of Tsukuba, Graduate School of Life and Environmental Sciences, Tsukuba, Japan

Generally, wild-derived mice have a higher resistance to tumor, diabetes, and obesity than conventional laboratory mice and they can provide invaluable models for the study of these human diseases. For this purpose, comparative analysis with laboratory strains as well as their genetic modification would enhance the efficiency of research using wild-derived strains. In this study, we employed CRISPR/Cas9 to produce knockout mice of inbred strain MSM/Ms derived from Japanese wild mice. We selected the nonagouti (a) gene, a coat-color gene also known as a domestication gene for a target. Plasmids prepared for the MSM nonagouti locus were injected into the pronucleus of the MSM/Ms zygotes. Of the 127 zygotes injected, 91 were cleaved and were transferred into pseudo-pregnant ICR recipients. Three pups were born at term, all of which carried homozygous mutants. These founder mice had different 4 mutant alleles, and 2 were propagated separately into 2 mutant MSM lines. Tame tests using an open field box indicated that homozygous mutant mice showed in significant reductions of locomotion and jumping behaviors as compared with wild-type littermates. Thus, we successfully generated knockout MSM lines having a tamer character.

O-6

Production of KO mice using various CRISPR/Cas9 vectors and freeze-thawed fertilized oocytes ○Yoshiko Nakagawa1, Tetsushi Sakuma2, Takuya Sakamoto2, Masaki Ohmuraya3, Takashi Yamamoto2, Naomi Nakagata1 1

Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan, 2Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan, 3Division of Transgenic Technology, CARD, Kumamoto University, Kumamoto, Japan

Genome editing using zinc-finger nucleases, TAL effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) permits the rapid production of genetically engineered mice (GEM). In this study, we investigated easier and more efficient methods of creating knockout mice using several CRISPR/Cas9 systems and freeze-thawed oocytes fertilized by in vitro fertilization (IVF). We constructed three types of CRISPR/Cas9 vectors expressing 1) single guide RNA (gRNA) and Cas9 nuclease, 2) two gRNAs and Cas9 nickase, and 3) two gRNAs and FokI-dCas9, targeting the same genomic locus. These vectors were microinjected into freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant mice. Cryopreservation and IVF technologies are applicable to the production of CRISPR/cas9mediated genome editing mice. Cas9 nuclease and nickase showed bias toward mutation and birth rates, whereas FokI-dCas9 represented well-balanced mutation and birth rates. We conclude that our study presents accessible platforms for CRISPR/Cas9-based generation of GEM.

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O-7

Efficient generation of knockin mice by CRISPR/Cas system ○Takuro Horii1, Miho Yamazaki1,2,3, Yuji Arai4, Sumiyo Morita1, Mika Kimura1, Masahiro Itoh3, Yumiko Abe2, Izuho Hatada1 1 Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan, 2Graduate School of Health Sciences, Gunma University, Gunma, Japan, 3Gunma CHUO Hospital, Gunma, Japan, 4National Cerebral and Cardiovascular Center, Osaka, Japan

Genome editing using CRISPR/Cas system enables to produce knockout (KO) mice within a month. In this method, both Cas9 nuclease and target-specific gRNA are necessary. CRISPR/Cas system have enabled to generate a lot of knockout mice by the non-homologous end joining (NHEJ) manner; however, there are few reports about knockin mice via homologous recombination, especially in Japan. Expression vector or in vitro transcribed RNA have been used to introduce Cas9 into zygotes; but, in this study, we introduced recombinant Cas9 protein in combination with gRNA specific to a Tet1 gene and donor DNA to produce knockin mice. First, we introduced only Cas9 protein and gRNA to examine NHEJ efficiency in this system. Results of PCR analysis showed that NHEJ occurred in all blastocysts, and more than 90% of them were biallelic mutation. Next, target+loxP (single-stranded oligonucleotides, 155 bp) or target+GFP (PCR products, 0.8 kb) as donor DNA was introduced into zygotes with Cas9 protein and gRNA. The results showed that knockin efficiency was 30-50 % in loxP and about 20% in GFP. Here we show the efficient generation of knockin mice by combination with recombinant Cas9 protein and donor DNA in CRISPR/Cas system.

O-8

Complex genome editing in mouse ES cells using the CRISPR/Cas system ○Asami Oji, Yoshitaka Fujihara, Masahito Ikawa Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Recently, CRISPR/Cas system has opened a new era for targeted mutagenesis. The combination of the Cas9 protein with a single guide RNA (sgRNA) causes a double stranded break (DSB) at the sgRNA target site. Whereas subsequent non-homologous end joining (NHEJ) would result in small insertions or deletions (indels), designed mutations could be introduced by homology dependent repair (HDR). Targeted genome editing in mammalian zygotes has already been demonstrated using this system.Previously, when we injected pX330 plasmids expressing Cas9/sgRNA into zygotes, about a half of the pups (100/196) carried an indel mutation. We next co-injected pX330 plasmids along with ssODN or dsDNA to introduce desined mutations such as a point mutation, tag insertion, or reporter knockin. The designed mutants were achievable but only 5-10% of the pups carried the desired mutation. Therefore we needed to increase the number of zygotes to be injected and subsequently had to sacrifice more of the undesired offspring. In the present study, we transfected ES cells with the same plasmids and examined the mutation frequencies. With a single Cas9/sgRNA pX330 plasmid, more than 80% of the clones had indels . HDR mediated mutations were achieved in the ES cell genomes (about 50% or more) when co-transfected with a dsDNA. Therefore, we conclude that the combination of ES cells with the CRISPR/Cas system provides a robust tool for the generation of genome edited mice, especially for the more complex genome editing.

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O-9

New production method of genetically engineered rats by electroporation ○Takehito Kaneko1, Tetsushi Sakuma2, Takashi Yamamoto2, Tomoji Mashimo1 1

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan 2

Production of genetically engineered animals become more simply and rapidly by genome editing techniques. Genetically engineered rats have been produced using ZFN, TALEN and CRISPR-Cas system. However, the microinjection of nucleic acids into embryos requires a high skill level. This study showed the production method of genetically engineered rats by introduction of ZFN, TALEN and CRISPR-Cas mRNA using electroporation. ZFN, TALEN and CRISPR-Cas mRNA could be introduced efficiently into intact rat embryos, and genetically engineered rats were obtained in all experiments. Electroporation provides a simple and effective method to produce genetically engineered animals.

O-10

Effective superovulation method in inbred mouse strain by synchronization of estrus cycles ○Ayumi Hasegawa, Keiji Mochida, Atsuo Ogura RIKEN BRC

Although superovulation of oocytes is one of the important techniques of assisted reproduction, conventional treatment with PMSG followed by hCG has not been fully optimized for inbred mice yet. It is known that the number of collected oocytes is also affected by the estrus cycle of females. The average numbers of oocytes collected from C57BL/6JJcl (10 < weeks old) females after injection with 100 μl of anti-inhibin serum (AIS) or 5 IU eCG followed by 5 IU hCG 48 h apart were 41.3 and 21.4 per female, respectively. The numbers of oocytes from females at estrus or metestrus were larger than those from females at proestrus in the AIS treatment group, but not in the eCG treatment group. Successful synchronization of the estrus cycle by consecutive injections of progesterone (P4; 2 mg/animal) for two days was confirmed by 90% females showing metestrus at the second day after P4 injection. By treatment with AIS after synchronization of estrus, we obtained 55.9 oocytes per female. We confirmed the practically high fertilizing ability of ovulated oocytes and full developmental ability of embryos derived from IVF and vitrification at the 2-cells. Thus, superovulation by AIS-induced endogenous FSH in combination with synchronization of the estrus cycle of females is effective method to collect a large number of oocytes with high quality in inbred mouse strains. These techniques are expected to be applied to poor responder strains to eCG treatment.

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O-11

Effect of cysteine analogs on the fertilizing ability of sperm and oocytes in mice ○Toru Takeo1, Yuka Horikoshi1, Satohiro Nakao1, Ayumi Mukunoki1, Kiyoko Fukumoto1,2, Tomoko Kondo1,2, Yukie Haruguchi1,2, Yumi Takeshita1,2, Yuko Nakamuta1,2, Tomoko Umeno1,2, Shuuji Tsuchiyama1, Naomi Nakagata1 1 Division of Reproductive Engineering, Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan, 2Kyudo Co. Ltd., Tosu, Japan

Reduced glutathione (GSH) greatly improves the fertilization rate on in vitro fertilization (IVF) in mice. Application of GSH to IVF techniques is widely accepted as a standard protocol for the production of embryos of genetically engineered mice. Previously, we revealed cysteine analogs (L-cysteine: L-Cys, D-cysteine: D-Cys and N-acetyl-L-cysteine: NAC) have similar ability to enhance the fertilization rate in IVF. However, the detailed mechanism of the enhancement of fertilization mediated by cysteine analogs in vitro is not fully understood. Here we examined effect of the cysteine analogs on the fertilization of sperm or oocytes. We show here that pretreatment of cysteine analogs increased the rate of fertilization. But pretreated sperm with cysteine analogs did not affect the rate. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by cysteine analogs. These results suggest that cysteine analogs remarkably increase fertilizing ability of oocytes with dissecting disulfide bonds in ZP.

O-12

N-acetyl cysteine is useful for the cold storage of 2-cell mouse embryos ○Yuka Horikoshi, Satohiro Nakao, Hidetaka Yoshimoto, Ayumi Mukunoki, Toru Takeo, Naomi Nakagata Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan

The cold storage of 2-cell embryos is a useful means of transporting genetically engineered mice. We have previously developed a transport system for genetically engineered mice which incorporates the cold storage of 2-cell embryos at 4oC. Recently this system has been used to efficiently transport genetically engineered mice. However, the longer the embryos remained in cold storage, the lower their developmental ability became. In this study, we examined the effect of adding N-acetyl cysteine (NAC) to the cold-storage medium on the developmental ability of cold-stored 2-cell mouse embryos. Two-cell embryos were prepared via in vitro fertilization using oocytes and sperm collected from female and male C57BL/6 mice respectively. The 2-cell embryos were stored in M2 media containing NAC for 96 hours at 4oC. After cold storage, the embryos were incubated in KSOM and their developmental ability to blastocysts was evaluated. NAC at 2 mM enhanced the developmental rate of the cold-stored 2-cell embryos after storage for 96 hours (control: 28.3%, NAC: 68.9%). In summary, we demonstrated that adding NAC to M2 medium improved the developmental ability of cold-stored 2-cell embryos. Hereafter, cold-storage medium containing NAC will be useful for the stable transportation of genetically engineered mouse embryos for 96 hours at 4oC.

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O-13

Study for in vitro maturation of immature rat oocytes ○Hiroaki Taketsuru, Takehito Kaneko Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan

The rat shows high reproductive ability, and can be superovulated as well as mouse. However, some strain is difficult to obtain the oocytes by superovulation. In these strains, it is possible to obtain oocytes by in vitro maturation of immature oocytes collected from ovary. This study investigated the in vitro maturation of the immature oocytes collected from ovary and their developmental potential. More than 8 weeks Wistar female rats were injected 150 IU/kg PMSG. At 48h after the PMSG injection, germinal vesicle (GV) oocytes were collected from ovaries. GV oocytes were cultured in HTF, αMEM, HTF+αMEM or TYH+αMEM, respectively. After in vitro maturation, more than 80% of oocytes were showed the MII-like formation in all culture medium. These oocytes were activated by 10 mM strontium chloride (SrCl2) in HTF medium. The pronuclear formation of oocytes cultured with αMEM (76%) and TYH+αMEM (75%) were higher than that cultured with other medium. Furthermore, 10% of oocytes cultured with αMEM were developed to offspring. These results indicated that the oocytes collected from rat ovary could be matured in αMEM medium, and some of these oocytes have developmental potential to offspring. This system can be used to maintain the rat strains which are difficult to obtain enough oocytes and offspring.

O-14

Cryoinjury after vitrification in canine ovarian tissues ○Hiroshi Suzuki, Madoka Hariya Obihiro Univ. of Agri. and Vet. Med., Obihiro, Japan

Cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of companion and working dogs. Although possible vitrification and follicular survival subsequent xenotransplantation of canine ovarian tissues has been reported, the degree of cryoinjury after the vitrification has not yet been fully examined. Since cryoinjury after vitrification of ovary has been reported in bovine and humans, in this study, canine ovarian tissues after vitrification were evaluated by immunohistochemistry and TUNEL assay. Ovaries were collected from inmature bitches. The ovarian slices were pretreated with 1M DMSO. Subsequently, DAP 213 solution was added. After placing the cryotubes containing the ovarian tissues on ice for 5 min, they were plunged directly into liquid nitrogen. After warming with 0.25M sucrose, cryopreserved ovarian tissues were fixed and subsequently stained with hematoxylin-eosin, proliferation cell nuclear antigen (PCNA) antibody or active caspase-3 (AC-3) antibody, and evaluated by TUNEL assay. There was no difference of number of primordial, primary, secondary and antral follicles/mm2 between fresh and vitrified-warmed ovarian tissues. Percentages of PCNA and AC-3 antibodies positive cells were not different regardless of developmental stages of follicles and experimental groups. Few TUNEL positive cells were detected in both experimental groups. These results suggest that vitrification by DAP214 do not induce cryoinjury in ovarian tissues from immature bitches.

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O-15

Robust self-renewal of ESCs is defined by LIF responsiveness ○Satoshi Ohtsuka, Hitoshi Niwa RIKEN CDB, Lab. for Pluripotent Stem Cell Studies, Kobe, Japan

Robust self-renewal of ESCs is highly dependent on culture context and genetic background, which discriminates permissive and non -permissive genetic backgrounds for ESCs derivation. However, serumcontaining culture allows ESCs self-renewal from limited strains of mice, such as 129. Recently, it has reported that a presence of two inhibitors (2i) in culture allows rodents derived ESCs self-renewal. We previously reported that permissiveness for ESCs maintenance are correlated with intra-cellular LIF signaling. Dominant activation of Stat3 by LIF is a pivotal cue for ESCs self-renewal in FCS/LIF. However, it is still unknown which factor is crucial for sustaining self-renewal of 129-ESCs in FCS/LIF. We tried to identify the genetic factor for selfrenewal of 129- ESCs in FCS/LIF. Comparing gene expression between 129 and NOD ESCs revealed a set of transcription factors, which is quickly down-regulated in NOD-ESCs. Among them, Tfcp2l1, a member of CP2like transcription factors, expression enables to sustain NOD-ESCs self-renewal in FCS/LIF. Exogenous Tfcp2l1 restored LIF-responsiveness in NOD-ESCs with increased expression of LIF signaling factors such as Lifr and Stat3. Deletion of endogenous Stat3 abrogated ESCs self-renewal in FCS/LIF, and ectopic Tfcp2l1 did not bypass this. Collectively, we conclude that Tfcp2l 1 supports self-renewal ESCs is Stat3-depedent. We are now analyzing detailed mechanism of how Tfcp2l1 supports robust self-renewal in NOD-ESCs in FCS/L IF. We would like to discuss further in our presentation.

O-16

Efficient iPSC reprogramming by Jarid2 overexpression ○Hiroyoshi Iseki1,2, Yutaka Nakachi2, Tomoaki Hishida2, Yzumi Sugahara2, Yoko Tanimoto1, Saori Iijima1, Fumihiro Sugiyama1, Ken-ichi Yagami1, Satoru Takahashi1, Akihiko Okuda2, Yasushi Okazaki2 1

Laboratory Animal Resource Center, University of Tsukuba, Ibaraki, Japan, 2Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan

Somatic nuclei can be reprogrammed by fusion with ESCs, nuclear transfer into an enucleated oocyte, or combined expression of transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM). Reprogramming by OSKM is a less efficient and slower process than that by cell fusion or nuclear transfer, suggesting that additional factors are required for efficient reprogramming. Here we show that forced expression of Jarid2, a highly expressed gene in both ESCs and oocytes, promotes induced pluripotent stem cell (iPSC) reprogramming. First, we selected highly expressed genes in both ESCs and oocytes, compared with those in somatic tissues, from the BioGPS microarray database, and evaluated the reprogramming-promoting activity of selected genes. We found that Jarid2 promoted reprogramming from MEFs to iPSCs most efficiently among selected genes. Deletion mutation analysis revealed that the N-terminal half of Jarid2 was sufficient to enhance iPSC reprogramming. Notably, retroviral Klf4 transgene silencing was observed at an early passage in iPSCs generated with Jarid2 compared with that in control iPSCs, suggesting that Jarid2 accelerates the generation of completely reprogrammed iPSCs. These findings suggest that Jarid2 is a potent reprogramming factor expressed in both ESCs and oocytes.

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O-17

Immunohistochemical of dental pulp cell cultured on amnion and transplanted into lumbar of nude mice ○Ken-ichi Honjo1, Toshiro Yamamoto1, Takeshi Amemiya1, Narisato Kanamura1, Masakazu Kita2 1

Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Laboratory Animal Center, Kyoto Prefectural University of Medicine, Kyoto, Japan

Paying attention to the usefulness of the amnion (AM) as a cell culture matrix, we have established methods to prepare oral mucoepithelium-derived and periodontal ligament-derived cell cultured on it. Then, we prepared a dental pulp-derived cell (DPC) cultured on the AM because they contain many stem cells. In this study, we induced the bone differentiation of this cell sheet, subcutaneously transplanted it into the lumbar region of nude mice, and immunohistologically investigated it. Dental pulp tissue was collected from wisdom teeth extraction, and subjected to primary culture. DPCs were seeded on the AM and cultured for about 4 weeks in control medium or osteoinductive medium. The skin of the lumbar region was incised and dissected in 7-weekold male BALB/C nude mice, and the cell sheet was placed in the wound and sutured. At about 4 weeks after transplantation, the mice were examined by imaging using a soft X-ray device. The transplants were collected from the mice and immunohistologically investigated. Radio-opacity was noted in the cell sheet induced for bone differentiation. The sheet showed high-level stainability with alizarin red S and von Kossa, and it was positive for osteocalcin, suggesting that calcified tissue was maintained after transplantation, and that the sheet is applicable for periodontal tissue regeneration.

O-18

Phylogenetic study of ‘CAR bacillus’ ○Fumio Ike1, Ayako Kajita1, Mitsuo Sakamoto2, Toshiaki Kokubo3 1

Experimental Animal Division, RIKEN BRC, 2Japan Collection of Microorganisms, RIKEN BRC, Tsukuba, Japan, 3Fundamental Technology Center, National Institute of Radiological Sciences, Chiba, Japan

The cilia-associated respiratory bacillus (CAR bacillus), an unclassified, extracellular, gram-negative filamentous bacterium, colonizes the ciliated respiratory epithelium of rodents and causes persistent respiratory disease (CRD). The present study was performed to determine the taxonomic status of ‘CAR bacillus’ rodentisolates by using strain SMR-C. Strain SMR-C, which was originally isolated as strain SMR from the respiratory tract of laboratory rats showing CRD, sequentially maintained by inoculation in mice because of growth inability by classical agar-using methods, and nowadays successfully cultured in Vero E6 conditioned medium. SMR-C grew at 37 oC in 5% CO2/ 95% air humidified chamber, showed gliding activity without locomotive apparatus such as flagella and pili. The dominant cellular fatty acids detected were C14:0, C16:0, iso-C15:0 and anteiso-C15:0. The DNA G+C content calculated from its full genome sequence was 47.7%. 16S rRNA gene sequence analysis revealed SMR-C and other ‘CAR bacillus’ rodent-isolates were all belonged to cytophaga-flavobacteriabacteroides (CFB) phylum but phylogenetically novel. The nearest known type strain, with 86% sequence identity, was Chitinophaga pinensis DSM 2588T in the family Chitinophagaceae.

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O-19

Helicobacter sp. MIT01-6451 infection during fetal and neonatal life of laboratory mice ○Hitoki Yamanaka, Tai Nakanishi, Toshikazu Takagi, Makiko Ohsawa, Noriaki Kubo, Naoto Yamamoto, Takahira Takemoto, Kazutaka Ohsawa Division of Comparative Medicine, Life Science Support Center, Nagasaki University, Nagasaki, Japan

To characterize Helicobacter sp. MIT01-6451 which has been frequently detected in SPF mice, we investigated the possibility of vertical transmission of it to fetuses and newborns using BALB/c, C57BL/6N, and SCID mice. The mice were exposed to the mixture with soiled bedding or kept with mice carrying MIT01-6451 in same cage to lead infection. Helicobacter genes were detected in tissue samples by nested PCR using genus specific primers following DNA extraction. The specific IgA and IgG titers in serum to MIT01-6451 were determined by ELISA using whole cell proteins as antigen.Although MIT01-6451 genes were undetectable in uteruses, vaginas, and mammary glands of all immunocompetent mice, that was present in 50% of SCID mice in these tissues. All fetuses including SCID mice were not infected with MIT01-6451 at 16-18 days after pregnancy, whereas MIT01-6451 genes were detected in placentas of a SCID mouse. In newborns, MIT01-6451 was detected in gastrointestinal tissue of C57BL/6 and SCID mice by 9-11 days after birth, but not in those of BALB/c mice. The IgA and IgG titers to MIT01-6451 in sera of C57BL/6 were significantly lower than those of BALB/c mice. Therefore, MIT01-6451 infects in newborns after birth, but not transmits vertically to fetus via placenta. The maternal immune responses may affect the infection with MIT01-6451 in newborns through breast milk.

O-20

Evaluation of the novel treatment for influenza using the senescence-accelerated mice ○Eriko Ohgitani1, Masakazu Kita1, Osam Mazda1, Jiro Imanishi2 1

Graduate School of Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Meiji University of Integrative Medicine, Kyoto, Japan

Objective: The duration of influenza virus shedding following antiviral drug treatment is prolonged in children and elderly people. This prolonged virus shedding increases the risk of the emergence of drug-resistant mutations. Here, a novel treatment was developed, i.e., the combined administration of antiviral drug and immune-stimulating Japanese herbal (KAMPO) medicine, to reduce the viral load more rapidly, and its effect was evaluated using the senescence-accelerated mice. Methods: SAMP1 mice were orally administered Hochuekki-to (HET) for 2 weeks and intranasally infected with influenza virus. Four hours after infection, mice were administered oseltamivir by oral gavage twice daily for 5 days. Viral load, cytokine production in the lungs, and the phagocytosis activity of alveolar macrophages (AMs) were subsequently investigated. Results and Discussion: Viral load of the combined administration of oseltamivir and HET group was considerably reduced as compared with those of oseltamivir stand-alone administration group. HET significantly increased the induction of IL-12, IL-1β and TNF-α and stimulated the phagocytosis of AMs; these functions possibly contribute to the reduction of viral load. Conclusion: The results from this study suggest that the combined administration of oseltamivir and HET is a potentially beneficial therapy that is based on the empirical data obtained using SAMP1 mice.

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O-21

Preparation of the monoclonal antibody against swine SLA-1 allotype ○Shino Ohshima1, Asuka Miyamoto1, Atsuko Shigenari1, Masaki Takasu2, Noriaki Imaeda2, Kazutaka Kitaura3, Masahumi Tanaka1, Hitoshi Kitagawa2, Noriaki Hirayama4, Ryuji Suzuki3, Asako Ando1, Yoshie Kametani1 1 Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan, 2Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 3Department of Rheumatology and Clinical Immunology, Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, National Hospital Organization, 4Tokai University Institute of Glycoscience

As the expression of major histocompativility complex (MHC) is closely related to various hereditary diseases, the regulation of MHC expression is an important issue. In order to examine the swine MHC (SLA) expression of the protein along with mRNA, we prepared an anti-SLA-1 allotype specific monoclonal antibody.BALB/c mice were immunized with swine PBMC pre-treated with mytomicin C, followed by the boosters of SLA-1/B2M transfected A20 cells. After the conventional spleen cells/P3X fusion, specific antibody-secreting hybridomas were selected using Array Scan and FACS analyses. A clone named X2F6 reacted with only SLA-1 transfectants and no other SLA molecules were recognized by this antibody. SLA-1 molecules of five different allotypes SLA1 showed the distinct crossreactivity. The cDNA and amino acid sequences were determined. The simulation of the 3D-structure suggested that the antigen determination sites might be located on outer regions of the SLA-1 molecules.

O-22

HCV based chimeric virus of which envelope is derived from GBV-B infects tamarin persistently ○Saori Suzuki1, Atsunori Higashino1, Ken-ichi Mori2, Yuko Katakai3, Noboru Maki2, Hirofumi Akari1 1

Primate Research Institute, Kyoto University, Inuyama, Japan, 2Advanced Life Science Institute, Wako, Japan, 3Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan

Majority of Hepatitis C virus (HCV) infected-patients suffer from liver pathologies including fibrosis, cirrhosis and hepatocellular carcinoma. But development of vaccines has been hampered by the lack of appropriate animal models. GBV-B is closely related to HCV genetically and GBV-B-infected red-handed tamarins (Saguinus midas) and common marmosets (Callithrix jacchus) show similar symptom with HCV infection. In this study, we report that a tamarin infected with HCV/GBV-B chimeric virus (HCV/GB) containing GBV-B structural genes coding for envelope proteins (E1-p6) substituted for the counterparts of HCV. And marmosets infected with GBV-B/HC chimeric virus (GBV-B/HC-1, 2) containing HCV genes coding for either envelope proteins (E1-p6) or proteins (5’UTR-E2) substituted for the counterparts of GBV-B respectively. HCV/GB infected tamarin persisted chronic infection for over three years but GBV-B/HC-1, 2 caused acute infection and were excluded in marmosets. This is the first report demonstrating that HCV based chimeric virus caused chronic infection in New World Monkeys. This primate model has a possibility that it could evaluate efficacy of new vaccine candidates against HCV in the future.

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Contributing factor to facilitate viral growth of macaque-tropic HIV-1 in vivo ○Yohei Seki1, Takeshi Yoshida1,5, Akatsuki Saito1, Kazuhiro Matsuoka2, Hirotaka Ode2, Yasumasa Iwatani2, Yasuhiro Yasutomi3, Tetsuro Matano4, Tomoyuki Miura5, Wataru Sugiura2, Hirofumi Akari1,5 1

Primate Research Institute, Kyoto University, Inuyama, Japan, 2National Hospital Organization, Nagoya Medical Center, Nagoya, Japan, 3National Institute of Biomedical Innovation, Tsukuba, Japan, 4National Institute of Infectious Diseases, Tokyo, Japan, 5Institute for Virus Research, Kyoto University, Kyoto, Japan

Macaque monkeys serve as important animal models for understanding the pathogenesis of lentiviral infections. Since HIV-1 hardly replicates in macaque cells, the development of a macaque-tropic HIV-1 (HIV1mt) having the ability to replicate efficiently in macaques has long been desired. In addition, since HIV-1 in human population usually uses CCR5 as a co-receptor during transmission, it will be straightforward to develop an R5-tropic HIV-1mt in order to reproduce the transmission, latency, and pathogenicity of HIV-1 in macaques. Therefore, we constructed a new R5-tropic HIV-1mt named AS38 having the env gene of the R5-tropic chimeric viruses between HIV-1 and simian immunodeficiency virus (SHIV), MK38. Furthermore, we performed a serial in vivo passage of AS38 in the cynomolgus monkey in order to adapt to cynomolgus monkeys. Subsequently, we analyzed the mutation of viral genomes using next-generation sequencing to determine that the mutations are involved in facilitating viral growth. The results indicated that characteristic amino acid substitutions were observed in Vif, Nef, MA, p6, RT, and, Tat.

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The advantages of primary cultured rat hepatocytes to assess anti-inflammatory effects of drugs ○Mikio Nishizawa1, Tetsuya Okuyama1, Hiroyuki Inaba1,2, Yukinobu Ikeya3, Tadayoshi Okumura4,5 1

College of Life Sciences, Ritsumeikan University, Kusatsu, Japan, 2R&D Headquarters, Lion Corporation, Odawara, Japan, 3College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Japan, 4Research Org. of Science and Technology, Ritsumeikan University, Kusatsu, Japan, 5Dept of Surgery, Kansai Medical University, Hirakata, Japan

We have been using primary cultured hepatocytes that are isolated from rat liver to assess the anti-inflammatory effects of drugs, herbal medicines, and functional foods. The primary cultured hepatocytes produce albumin and inflammatory mediators, such as nitric oxide (NO). Alternatively, cell lines are used. Here, we measured the expression of inducible nitric oxide synthase (iNOS) gene in the hepatocytes and cell lines and analyzed the effects of antipyretic analgesic drugs on the iNOS gene expression. Hepatocytes were isolated from Wistar rat livers using the collagenase perfusion method. More than 100 dishes were obtained from a rat. NO, iNOS protein, and iNOS mRNA were induced In IL-1β-treated hepatocytes, whereas the LPS-induced NO levels varied by macrophage and hepatoma lines, and RAW264.7 cells showed the highest efficacy. Four antipyretic analgesic drugs suppressed NO induction. The potency of NO suppression of each drug in hepatocytes did not always correlate with that observed in RAW264.7 cells. Because the use of cell lines is limited, primary cultured rat hepatocytes are feasible to analyze the anti-inflammatory effects of antipyretic analgesic drugs.

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Effects of royal jelly supplementation on hyperglycemia and body weight in KK-Ay mice ○Risa Watadani, Daiki Sasaki, Jun Koto, Yu Seto, Ayaka Tanida, Kohei Honda, Kozo Matumoto

Kyoto Sangyo University, Kyoto, Japan

Royal jelly (RJ) has several pharmacological activities, such as anti-inflammatory activity, and antiaging activity. Moreover, it has been reported that RJ increases insulin sensitivity and decrease blood glucose level in nondiabetic human. However, effects of RJ are not fully understood under condition of diabetes or hyperglycemia. Therefore we investigated whether RJ exerts protective effect on hyperglycemia or body weight using KK-Ay mice. To examine effects of RJ in diabetic or hyperglycemic situation, we administered RJ orally to female KK-Ay mice at 8-week-old for 3 weeks. We showed that the body weight and the blood glucose levels were significantly lower in the RJ treated mice than in PBS treated mice. However, there was no difference in the plasma insulin levels. Then, we examined gene expression levels involving obesity and blood glucose in liver and fat tissues from KK-Ay mice using real-time PCR. The expression of Pparg1 in RJ treated mice was significantly increase in the livers than those in PBS treated mice, and Pparg2 was significantly increased in the abdominal fats. The reduction of Pparg1 expression in liver may suppress fat accumulation and gluconeogenesis. Moreover, elevation of the Pparg2 expression may normalize lipid metabolism and glucose homeostasis. In summary, these date suggest that RJ has may pharmacological effects on reduction of body weight and high plasma glucose through Pparg expression under diabetogenic condition.

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Analysis of autoinflammation mechanisms caused by abnormal innate immunity of Ali18 and Ali14 mice ○Koichiro Abe1, Satoshi Nunomura2, Chisei Ra2, Atsushi Tajima3 1

Department of Molecular Life Science, Tokai University School of Medicine, 2Nihon University School of Medicine, 3Kanazawa University, Graduate School of Medical Sciences

The Ali18 and Ali14 mutants were isolated in mouse ENU (N-ethyl-N-nitrosourea) mutagenesis because of spontaneous inflammation on the peripheral paws. The Ali18 and Ali14 mutations are thought to be a gain-offunction mutation of cell signaling molecules, and trigger autoinflammation through the innate immune cells by unknown mechanisms. We assumed mast cells as a candidate initiator of autoinflammation, and the mutants were crossed with W/Wv mice which lack whole mast cells. Double mutants with Ali18 homozygous and W/Wv (Ali18/ Ali18;W/Wv) did not show autoinflammation without mast cells, but Ali14/+;W/Wv did show inflammation on paws. These results suggest that mast cells can be an initiator of autoinflammation in Ali18 mutant mice but not in Ali14 mice. We also analyzed degranulation of cultured mast cells derived from bone marrow of Ali18 and Ali14 mice. In addition, intravenous injection of the cultured mast cells from each mutant mice separately into W/Wv mice showed autoinflammation on auricles of ears but not on paws. These results indicate that mast cells contribute limitedly to autoinflammation in the mutants. Rather, interaction with other myeloid cells, such as granulocytes and macrophages, could be important for initiation and development of autoinflammation in Ali18 and Ali14 mice.

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Establishment of two double congenic strains for the QTLs of the stroke susceptibility in SHRSP ○Hiroyuki Matsuo, Kaoru Niiya, Hitomi Hoshino, Hasan Zahid, Batbayar Odongoo, Toru Nabika Department of Functional Pathology, Shimane University, Izumo, Japan

[Background] In the previous studies, we identified QTLs for stroke-latency on chromosome 1 and 18 in SHRSP, and narrowed down the candidate regions to 1.8Mbp and 1.5Mbp on chromosome1 and 18, respectively. In the present study, we constructed two double subcongenic strains harboring the two candidate regions simultaneously, and effects of these chromosomal regions on the stroke latency and blood pressure (BP) were evaluated. [Methods] Two double subcongenic strains were constructed; SPrch1.1_18.5 harboring the maximal range of the QTL regions and SPrch 1.31_18.3 the shortest candidate regons according to analyses of the subcongenic rats. BP was measured at 12 weeks old by the tail-cuff method and the stroke latency was evaluated as days until appearance of signs of stroke after the salt loading with 1 % salt water was started. [Results] A significant difference in BP was observed between SHRSP (191 ± 7 mmHg, n=14) and SPrch1.1_18.5 (183 ± 8 mmHg, n=18) at 12 weeks of age (P=0.005). The evaluation of stroke latency in the two double subcongenic strains newly constructed is now on going. [Conclusion] Because significant difference was seen in BP, it was suggested that the gene(s) associated with BP was located in the chromosomal regions harbored by Pr1.1_18.5. We would like to report the stroke latency of the two double subcongenic strains, which will largely narrow down the candidate region of the stroke susceptibility genes.

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Obesity-related gene is located in rat Chromosome 10 ○Shimizu Yukiko1, Takanashi Rieko1, Okamura Tadashi1,2 1

Department Laboratory Animal Medicine, Research Institute, National Institute for Global Health and Medicine, Tokyo, Japan, 2Section of Animal Models

The LEA rats, a new rat model of non-obese type 2 diabetes, have been established at Tohoku University Graduate School of Medicine. Diabetes mellitus was observed only male rats, but glucosuria was appeared at 5 months of age in male and female. In LEA rats, the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in renal proximal tubules, resulting in renal glucosuria. We previously reported that causative gene for renal glucosuria was located on rat Chr.10 and LEA rats had a 13 bp deletion within exon 7 of Ctns gene, which is the causative gene of cystinosis. During the process to identify the Ctns mutation, we established a congenic strain (LB-Ctns(+/+)) carrying the BN allele (D10rat34-D10rat243) on the LEA rat genetic background. The body weight and the accumulation of intraabnominal fat of the LBCtns(+/+) were significantly increased compared with those of the LEA rat, suggesting obesity-related gene(s) were located in rat Chr.10.

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Selective breeding and simulation for identification of genetic loci associated with tameness ○Yuki Matsumoto1, Jo Nishino3, Tatsuhiko Goto1,4, Tsuyoshi Koide1,2 1 Mouse Genomics Resource Laboratory, National Institute of Genetics, Shizuoka, Japan, 2School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Kanagawa, Japan, 3Graduate School of Medicine, Nagoya University, Nagoya, Japan, 4College of Agriculture, Ibaraki University, Ibaraki, Japan

To identify genes associated with tameness in mice, we established the combination method using selective breeding and computer simulation using the Wild-derived heterogeneous stock (WHS), which has genetic variation because the stock derived from 8 wild strains (MSM, HMI, BLG2, PGN2, KJR, CHD, NJL, and BFM/2) originated from various geographic regions. The basic idea of the method hypothesized the allele frequency associated with tameness will increase through the selective breeding for tameness. At first we conducted selective breeding for tameness using the stock. Second, we obtained 37,714 single nucleotide polymorphism (SNP) data in the WHS founders and 378 WHS mice in the selection line at second generation of selection. Third, using computer simulation based on non-selection model, we determined thresholds (p6 mm) and identified Stmm3 (Skin tumor modifier of MSM 3) locus on chromosome 4 by crossing a resistant Japanese wild-derived inbred strain MSM/Ms with a susceptible strain FVB/N and subjected to the two-stage skin carcinogenesis protocol using DMBA/TPA. To narrow down Stmm3 region, we generated three congenic lines (p53+/+ or p53+/–) and subjected these mice to the two-stage skin carcinogenesis protocol using DMBA/TPA. As a result, we narrowed down Stmm3 region to 88-93 Mb on chromosome 4.These data suggested that Stmm3 locus, which maps near the Cdkn2a/p19Arf, was entirely p53-dependent. And there are nonsynonymous polymorphisms in p19Arf between FVB/N and MSM/Ms mice. Biological assays of p19Arf alleles from MSM/Ms and FVB/ N indicated that MSM_p19Arf stability was higher than FVB_p19Arf by TPA treatment. And MSM_p19Arf was more effective in inducing G1 arrest. These date suggested that p19Arf might be expected to be a candidate gene controlling the transition to large papillomas in MSM/Ms.

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Valproic acid induces vertebral malformation through global expression changes in Hox gene clusters ○Sho Tanimoto1, Masami Yaguchi1, Jyoji Mochida2, Koichiro Abe1 1 Department of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan, 2Department of Orthopaedic Surgery, Tokai University School of Medicine, Kanagawa, Japan

Environmental factors causes morphological changes in fetus, but their mechanisms including interacting genetic factors are almost unknown. To elucidate gene expression changes by environmental cues during embryogenesis, we employed valproic acid (VPA) induced vertebral malformation in mice as a model. VPA is a antiepileptic drug and also acts as teratogen which induces fetal vertebral malformation and as an inhibitor of histone deacetylase. VPA (500 mg/kg) was once administered intraperitoneally to ICR mice at the 9th day of pregnancy. All E18.5 embryos showed vertebral abnormality and anterior transformation. In addition, we also employed microarray and quantitative real-time PCR analyses using E10.5 embryos to know gene expression changes between VPA and control groups. Accordingly, we found differential gene expression in many of Homeobox genes. Interestingly, expression of the homeobox genes positioned in telomeric side of the cluster, which involved in thoracic, lumbar and sacral vertebrae formation, decreased dramatically, but the genes on centromeric side did not. These results demonstrated that VPA influences epigenetic global expression changes in Hox gene cluster. These may shed light on the complex interrelationships between genetic and environmental factors which leads to sporadic vertebral malformation.

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Novel ENU-induced mutant, KTA41, as a mouse model for adolescent idiopathic scoliosis ○Nobuho Sagawa1, Fuchs Helmut2, Sabrautzki Sibylle2, Hrabe de Angelis Martin2, Satoshi Ota3, Kiyoshi Ando4, Koichiro Abe1 1

Tokai University School of Medicine,Isehara, Japan, 2Helmholtz zentrum Muenchen, Institute of Experimental Genetics, Neuherberg, German, 3BioresourceCenter,Riken,Wako, Japan, 4Tokai University School of Medicine, Isehara, Japan

Scoliosis is clinically defined as lateral curvature and convolution of the spine. Scoliosis is not life-threatening disease but impairs locomotive and respirative functions in severe cases. Most of scoliosis patients are adolescent and idiopathic. The factors and mechanisms to cause vertebral abnomaly in scoliosis are almost unknown, and lack of animal models for scoliosis hampers developing new medical treatment. In this study, to establish a novel animal model for scoliosis, we focused on a novel ENU-induced recessive mutant strain, KTA41, derived from ENU mutagenesis screening. Because adult homozygous KTA41 mice show mild kinky tail and thoracic vertebral fusion, we performed detailed skeletal analyses in various developmental stages. Accordingly, hyperossification was detected from newborn KTA41 mice, and thereafter abnormal synostosis and curvature started around 3 months of age. From these findings, it is suggested that the skeletal abnormality and its progression in KTA41 mice are closed to adolescent idiopathic scoliosis. Thus we attempt to simulate how curvature occurs during vertebral growth in KTA41 mice as disease modeling. This might contribute to the diagnosis in early stage and prevention of the disease.

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The gene dosage effect of DNASE1L2 is associated with 16p13 deletion syndrome ○Yasuyo Kozawa, Kazuyuki Hirota, Hideaki Toki, Shigeharu Wakana, Masaru Tamura RIKEN BioResource Center

DNASE1 and DNASE1L2, both of which located on HSA16 16p13.3, belong to the DNASE1 family, and play pivotal roles in DNA degradation. It was reported that mutation of DNASE1 causes systemic lupus erythematosus, and that haploinsufficiency of DNASE1 is involved in 16p13.3 micro-deletion syndrome. However, function of DNASE1L2 and correlation of DNASE1L2 mutation and human disease are largely unknown. To disclose the function of DNASE1L2 gene, we analyzed morphological features of Dnase1l2 knockout (KO) homozygotes and heterozygous mice, mainly focused on bone morphology. We used wild-type, heterozygotes and homozygotes of KO mice with C57BL/6NTac background. We carried out dysmorphology, X-ray imaging and measurement of body weight analysis at 9, 14 and between 4 and 16 weeks old mice, respectively. The X-ray and dysmorphology analysis demonstrated that the KO homozygotes and the heterozygotes had digits and tail anomalies. These phenotypes especially in tail were observed more frequently and severer in the KO homozygotes. These results suggested that Dnase1l2 has a gene dosage effect. The measurement of body weight indicated a relation between abnormal digits and developmental delay. Both of KO heterozygotes and homozygotes had developmental delay and fusion of joints, which were observed in a patient affected with 16p13 deletion. These findings suggested that DNASE1L2 has critical roles during the bone development, and is involved in human 16p13 deletion syndrome.

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Exhaustive screening of outer hair cell-specific genes ○Kunie Matsuoka1, Shumpei P Yasuda1, Yuki Miyasaka1, Kenta Wada2, Hiroshi Shitara3, Midori Yamaguchi3, Choji Taya3, Yoshiaki Kikkawa1 1

Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 2Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Japan, 3Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science

In the cochlea, outer hair cells (OHCs) play an essential role in the amplification and frequency selection of sounds. We generated a mouse model OHC-TRECK by inducing selective depletion of OHCs by administration of diphtheria toxin (DT). We performed a differential expression analysis between DT-administered OHC-TRECK and wild-type mice using high-throughput mRNA sequence analysis (RNA-seq) to screen for novel, specifically expressed and functional genes involved in the development and maintenance of OHC. We administered DT to OHC-TRECK and WT mice at postnatal day 1 and at 4 weeks of age. Cochleae were prepared 7 days after DT administration and subjected to RNA-seq to detect OHC-specific genes that are important for development and hearing ability. We sequenced and analyzed 26-120 million paired 100-bp RNA-seq reads. Expression of the oncomodulin gene was remarkably decreased in DT-administered OHC-TRECK mice, confirming the results of a preliminary microarray analysis. Other genes that were significantly decreased in the cochleae of DT-administered OHC-TRECK mice are currently being analyzed to validate their expression patterns.

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Establishment of KFRS4/Kyo rats as a new model of atopic dermatitis ○Takashi Kuramoto1, Mayuko Yokoe1, Azusa Yuri1, Ai Nishitani1, Daisuke Tanaka1, Hiroshi Hiai2, Tadao Serikawa2,3 1

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Graduate School of Medicine, Kyoto University, Kyoto, Japan, 3Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan

Atopic dermatitis (AD) develops in patients with an individual or familial history of allergic diseases, and is characterized by chronic relapsing inflammation associated with IgE hyperproduction. Here, we report the establishment of the Kyoto Fancy Rat Stock 4 (KFRS4/Kyo) strain as a new rat model of AD. The KFRS4/Kyo strain was derived from hybrids of a fancy rat raised in the USA and a laboratory rat, PVG. Recently, we found that KFRS4/Kyo rats showed skin lesions which spontaneously appeared on neck and dorsal skin, accompanied with scratching behavior. These skin lesions, which were prominent in female, appeared from about 4 months of age and developed extensively from 6 to 8 months of age. Total IgE in plasma was markedly elevated from 4 months of age, correlating with clinical symptoms of dermatitis. Histological analysis of the skin lesions showed hyperplasia of the epidermis and infiltration of eosinophils into the dermis. These findings indicate that KFRS4/ Kyo rats show the dermatitis characterized by pruritus and elevation of plasma IgE. Thus, the KFRS4/Kyo rats can be used as a model of AD.

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RNA-seq analysis in microphthalmos rat, NAK/Nokh ○Saki Okubo1, Hironobu Uchiyama2, Shingo Ishihara3, Ryoichi Hashizume1,3, Yoshiaki Kikkawa4, Kenta Wada1,3,4 1

Graduate School of Bioindustry, Tokyo University of Agriculture, 2Graduate School of Bioindustry, Tokyo University of Agriculture, 3Department of Bioindustry, Tokyo University of Agriculture, 4Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science

Microphthalmia is one of the most profound congenital eye diseases which lead to completely loss of vision. We have been established the Nodai aphakia (NAK/Nokh) as promising rat model for human microphthalmia. Previously, we reported that phenotypic heterogeneities similar to human patients were observed in the progenies between NAK and several rat strains, and the causative mutation was localized in range of 20 Mb on chromosome 16. In this study, we performed mutation analysis and gene expression analysis in the embryonic eyes of NAK.We sequenced 8 lanes of cDNA library from the NAK, SD and BN, and average numbers of sequence data were 3.1, 4.6 and 3.7 Gb in NAK, SD and BN, respectively. The mutation analysis detected total 11,814 mutation sites between BN and NAK transcripts, and 28 mutations were found in genes which were located in the candidate region. On the other hand, gene expression analysis detected 100 genes which showed differential expression level between SD and NAK, and that contains up- (14) and down-regulated (86) genes. In addition, 25 of these down-regulated genes were categorized into the GO term, camera type eye development. Therefore, we speculated that phenotypes of NAK are caused by down regulation of essential genes for eye development.

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Epigenetic transcriptional regulation during early folliculogenesis ○Hitomi Suzuki1, Aleksandar Rajkovic2, Masami Kanai1 1 Dept. Experimental Animal Model for Human Disease, Tokyo Medical and Dental University, Tokyo, Japn, 2Dept. OBGYN-Reproductive science, University of Pittsburgh, PA, USA

Folliculogenesis in mammals is a complex process involving bi-directional communication between the oocyte and surrounding soma. In the mouse ovary, primordial follicles (PrdFs) that contain 15um oocyte surrounded by flatten soma are formed shortly after birth, and provide a source of future growing follicles such as primary follicle (PrmF) during the entire reproductive lifespan. PrdFs remain dormant for prolonged intervals. For retaining a long female reproductivity, it is essential to manage both ‘dormancy’ and ‘activation’ of PrdF and to prevent the consumption of all PrdF in a short period. We investigated PrdF specific factors and identified that the argininemethyltransferase Prmt5 was preferentially expressed in oocyte of PrdF. Kim et al. recently reported that Prmt5 was necessary for the silencing transposable elements via histone methylation, and Prmt5-null PGCs underwent apoptosis. However, Prmt5 function in postnatal oocyte is still unknown. We now analyze oocyte specific Prmt5 conditional knockout mouse and also investigate the binding co-factor of Prmt5. In the meeting, we would like to discuss about the phenotype of the mice and presumptive function of Prmt5 in folliculogenesis.

O-40

Replacement of the mouse management system (RIKEN LIMS) in the Japan mouse clinic (JMC) ○Kimio Kobayashi1, Tomohiro Suzuki1, Hideki Kaneda1, Ikuo Miura1, Ai Ozaki1, Daiki Usuda1, Kiyota Toguchi2, Tetsu Miyagi2, Shigeharu Wakana1, Hiroshi Masuya2 1

Japan Mouse Clinic: Technology and Development Team for Mouse Phenotype Analysis, RIKEN BRC, Tsukuba, Japan, 2Technology and Development Unit for Knowledge Base of Mouse phenotype, RIKEN BRC, Tsukuba, Japan

The Japan Mouse Clinic (JMC) has started for analyzing comprehensive and detailed phenotyping of a various mouse resources of domestic and aboard mouse researchers based on the standardized operating procedure of JMC in 2008. For the management of mouse resources, we have used the MUSDB (Mutagenesis Universal Support DataBase) which was developed in the RIKEN ENU Mutagenesis Program as the central database for the management of a lot of mouse mutant strains generated. In 2011, we joined the IMPC (International Mouse Phenotyping Consortium). Because it is strongly required that the flexibility for changes of the phenotyping platform and sustainability for future operations, we newly applied the web based database, RIKEN LIMS (Laboratory Information Management System). RIKEN LIMS is a modified version of WTSI Mouse Database developed by Sanger Institute based on OracleTM and JAVA (mainly JSP). It supports informational operations in mouse management and production, genotyping, phenotyping, and the reproduction technology. We have made a start the first trial of RIKEN LIMS for operations in mouse breeding. In this presentation, we will introduce functions, advantages and issues in the operation of RIKEN LIMS.

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Vacuum-drying-process-Free epoch-making autoclave sterilization by the soft-hydrothermal processing ○Miyamoto Toru1,3, Sadahiko Furuhata2, Noriyuki Kasai1, Nobuhiro Watanabe3, Motonari Hayashi3 1 2

Institute for Animal Experimentation, Tohoku University Graduate School of Meducine, Sendai, Japan, Shinshu Unversity Hospial, Matsumoto City, Japan, 3Maeda Seisakusho Co., Ltd., Nagano City, Japan

We have developed a groundbreaking sterilization method by means of the “Soft-hydrothermal process”, which has many advantages in terms of safety and cost-efficiency. This revolutionary technique makes it possible for sterilization to exclude vacuum-drying-process. In general, high temperature and high pressure steam sterilization which is so-called “Autoclaved sterilization”, is used to sterilize cages and cage bedding prior to do laboratory rodents experimentation. But there is a critical concern which should be considered to prevent developing bacteria, molds, spores and so forth caused by condensed water vapor and sterilization failure.We have suggested that the “Soft-hydrothermal Processing” is sufficient to assure the complete dehydration, and CI, BI and Bowie & Disk Test Pack results are judged as complete sterilization meeting “Guideline for Sterility Assurance in Healthcare Setting” by soft-hydrothermal processing for 121°C, 30 min and/or 134°C, 4 min in the 100% steam saturation ratio and in the flow system. These findings indicated that soft-hydrothermal processing, which was used in these conditions, has a strong effect of sterilization and might be applicable for dehydration, which does not requiring vacuum-drying-process, and retaining sterilization reliability and assurance.

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The effects on decontamination with the liquid- and vapor-phase of hydrogen peroxide ○Tohru Kimura1, Hiroyuki Yahata2, Hironobu Ichihara2 1

Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan, 2Santasalo & Steri-pro Solution Corporation, Kobe, Japan

Vaporized hydrogen peroxide (VHP) has an effect of a wide spectrum of disinfection on pathogenic microorganisms. However, the VHP-effects depend on the conditions of temperature and humidity. The purpose of this study was to investigate the effects on decontamination with the liquid- and vapor-phase of HP. After Geobacillus stearothermophilus (GS) samples were immersed in HP solution, the survivals of GS were chronologically determined. In addition, the laboratory room was exposed to VHP at low, moderate and high humidity. The effects on decontamination with VHP were determined by biological indicators (BI). Some GS samples survived after immersion in HP solution. The exposure test to VHP provided complete sterilized effects at humidity of not more than 75%. In contrast, a large number of positive BI samples were found at humidity above 80%. There was no damage of metallic materials following exposure to VHP. These results revealed that it took long process time to sterilized completely microorganisms with HP solution. We confirmed that VHP immediately sterilized microorganisms. At high humidity above 80%, however, VHP turned to the liquid phase, and then its effects of decontamination decreased. Hydroxyl radicals derived from HP caused degeneration of lipids and proteins in microorganisms, resulting in sterilization. Our results demonstrated that it was important to keep hydroxyl radical reaction in stable VHP.

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Consideration on bedding materials suitable for long-term breeding of mice ○Kohei Tomita1, Noboru Ogiso2, Satomi Takano2, Kazumichi Yamaguchi1,2, Kaori Muguruma2 1 KAC, Kyoto, Japan, 2Laboratory of Research Animal, National Center for Geriatrics and Gerontology, Aichi, Japan

Making an appropriate environment for animals is necessary to keep them for a long time. Our facility have kept aged animals for researches on gerontology and geriatric medicine. In the present study, we focused on bedding materials (BDs) and investigated their effects on the growth and the breeding environment of mice over long period. Fifty male and forty female mice (C57BL/6NCr Slc, twenty weeks-old at the start) were kept with five different kinds of BDs (three types of paper and two types of wood). To examine the health condition of mice, their body weight and food consumption were measured in addition to the observations of their external features. Significant differences in body weight were especially found in male mice over 68 weeks-old. Additionally loss or decoloration of hair were often observed in mice kept with particular BDs. Our results show that conditions of the animals might be changed greatly by their long-term breeding environment such as BDs.

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Sex differences in cynomolgus monkeys regarding affinity improvement training for animal technician ○Ai Nishimoto, Yuki Tachibana, Kaoru Takaura, Takehiro Ochi, Hironari Koyama Astellas Research Technologies Co., Ltd., Osaka, Japan

Objective: To characterize the effect of positive reinforcement training (PRT) on the affinity of cynomolgus monkeys for animal technician (AT), the effect of PRT focused on sex difference was investigated. Methods: Male and female cynomolgus monkeys aged 3 years were used. Following 28 days of PRT, monkeys accepted raisins and communicated with AT. Behavior was scored as follows: Score -1, fear to AT; Score 0, refusal to eat raisins in the presence of AT; Score 1, sampling of raisins in the presence of technician when served in cage; Score 2, acceptance of raisins from hand to hand via the upper part of the cage; Score 3, acceptance of raisins from hand to mouth via the upper part of the cage; Score 4, acceptance of raisins from hand to hand via the lower part of the cage; Score 5, acceptance of raisins from hand to mouth via the lower part of the cage; Score 6, acceptance of contact from AT. Results: Mean score in male monkeys increased from -0.4 to 3.4 following PRT. Similarly, the mean score of female monkeys increased from -0.8 to 3.0 following PRT. Before PRT, 82.6% male and 95.8% female monkeys refused raisins offered directly by AT (Score -1, 0, 1). After PRT, 26.1% male and 45.8% female monkeys refused raisins offered directly by AT. Conclusion: These results suggest that sex differences influence the success of PRT, with males being more likely to respond than females.

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Constructing new surgical training program nourishing ethics view for primary residents ○Iki Yuko, Takuya Ito, Masahiko Kanehira, Katsuyoshi Kudoh, Masafumi Noda, Michiaki Unno Tohoku university hospital advanced medical training center (TAMTAC), Sendai, Japan

At Advanced Medical Training Center, survey is implemented about technical skills confidentiality, nontechnical experiences in OP, and ethical sense for maximizing educational effect of the program. Visual Analog Scale (VAS) is used just before and after training. Comparing to technical skills, ethics part couldn’t show currently significant difference. Yet, we can predicate the trend of primary residents ethical sense by comparison among averages in 0 to 10 cm line scale. “Should the ethics view of medical doctors be higher than the others?” is marked approximately 8. And the result of “Do you think the ethical study in professional school was sufficient as medical doctor?” shows about 3. Then, the answer of “Do you think the ethical view to animals is common to that to human beings?” was 8.5. Besides, a slightly less 8 was marked on “Do you think the ethical view to animals are raised by watching living animals?”. Certainly, the score of “Do you have counteracting feeling on the animal at sacrificing him?” was slightly above 6. So participants are surely stressed because of wet lab which living animals are sacrificed by them. However, watching in-life animals induce primary residents to sensitize the preciousness of animal life and it connects to rear the morality to human beings. So we suggest definitely the complement of the ethical educational shortage during professional school by this training program.

O-46

Thorough and expedited review of protocols in an academic institute using an a-synchronous IACUC software ○Rony Kalman Authority for Biological and Biomedical Models Hebrew University Jerusalem Israel

The protocol review process is a fundamental part of any animal related research. IACUCs (Institutional Animal Care and Use Committees) are required to review a large number of protocols under a time constraint. The review process combines administrative challenge with a need for a highly specialized professional input. IACUC members are required to go in depth into the protocol details. At the same time there is a need for a joint committee work where each member is exposed to the other members comments and inputs. The final result is the vote of all the committee members (CMs) following the input of the designated reviewers (DRs).The Hebrew University IACUC reviews over 250 applications annually. In order to make the review process both efficient and thorough, we have developed an in-house IACUC software. The PIs enter, write and send the application using a personal coded access (this serves as an electronic signature). The application covers all the aspects of the research (legal declaration, scientific abstract, collaborators, justification of species strain and number of animals, harm-benefit analysis, scoring tables, procedures, anesthesia analgesia and euthanasia per species, safety measures, ethical severity). Most fields have links to useful web-sites.Once the first draft is submitted and the DRs are appointed, the process is fully automated. An email is sent to all committee members with a specific indication of the DRs. Every CM can comment, see each other comments and give their vote using a built-in keyword search tool to explore past information. Only after all the DRs gave their vote, and the safety officer ( who is also a committee member) gives his input, the PI is automatically asked to submit a second version. The previous version remains unchanged for future audits. Reviewers automatically receive and act on the second version and so on up to the final approved version. The PI receives the final approval letter and can order animals and start the experiment.The approved version is ratified in the next IACUC meeting. This software has greatly facilitated the IACUC work expediting and streamlining the process adding background data and reporting capabilities to the IACUC.

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Evaluation of the medical training materials using the slaughtered experimental pigs ○Takumi Teratani1, Yukitoshi Makimura1, Noriko Hannda1, Naoya Kasahara2, Taizen Urahashi3, Tatsuya Takayama4, Syuji Hishikawa5, Eiji Kobayashi1, Satoshi Kunita1 1 Division of Development of Animal Resource, Jichi Medical University, Tochigi, Japan, 2Department of Surgery, Jichi Medical University, Tochigi, Japan, 3Department of Transplant Surgery, Jichi Medical University, Tochigi, Japan, 4Department of Urology, Jichi Medical University, Tochigi, Japan, 5Division for Medical Skill Training, Jichi Medical University, Tochigi, Japan

The medical training kit have been developed for facilitating the improvement of the surgical technique and its stability. However, concerning the organs with extremely complicated structures the training kits are still not available. The present study is to develop a reliable surgical training simulator by using the tissues and organs from slaughtered experimental pigs. First, the pig liver was removed and initially flushed by normal saline to wash out the blood. Second, the liver was perfused by model-preparing solutions (10% neutral buffered formalin and formalin substitute fixative solution) for 72 h with a perfusion system for graft fixation. Finally, the liver was rinsed by sterile water. Totally 4 kinds of livers, including frozen/thawed and fresh livers, were involved in the evaluation experiments. The following 4 examination items were used for the evaluations. Our method may provide a useful tool for preparing animal disease models by surgical treatment and for training of therapeutic techniques, especially at a preliminary stage before using a living animal.

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Developmental evaluation of cardiac function by fetal electrocardiograph in mouse ○Ito Takuya1, Yoshitaka Kimura1, Rika Sugibayashi1,2, Kiyoe Funamoto1, Yupeng Dong1, Miyuki Endo1, Keita Iida1 1

Advanced Interdisciplinary Biomedical Engineering Graduate School of Medicine, Tohoku University, Sendai, Japan, 2National Center for Child Health and Development, Tokyo, Japan

Mouse is useful for developmental study. The mainstream analysis are morphological and molecular biological techniques. However, biological monitoring by ultrasound is only method for evaluating fetal murine cardiac function. The method of electrical evaluation of myocardiac function of fetal mouse has not been established. Electrocardiograph is general cardiac function monitor after birth in human and mouse. We tried to introduced electrocardiograph to fetal mouse, in order to electrophysiological state evaluation extend until fetal period. From pregnancy 12.5 to 18.5 days, one or two fetuses were randomly chosen for fetal electrocardiogram (fECG) recording using two needle electrodes insertion into the uterine. Fetal heart rate calculated from the RR interval was 119.3 ± 24.2 (mean ± SEM) in 12.5 days. Fetal heart rate increases depend on pregnant day, it was 395.5 ± 17.5 in 18.5 days. These heart rate were consistent with the heart rate by Doppler obtained in literature. This result indicates that the fetul inslut caused by the needle electrode insertion into the uterine is mild same as caused by the Doppler method. The fECG can measure the fetal cardiac function from mid to late gestation. In addition, this method can be used to fetal monitor for fetal operation such as electroporation.

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Application of radiotelemetric method in animal experiments using MRI in cardiac function evaluation ○Makimura Yukitoshi1, Akinori Hirano2, Jun Fujita3, Shuji Hishikawa1, Satoshi Kunita1 1

Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan, Division of Cardiovascular Surgery, Keio University of Medicine, Tokyo, Japan, 3Department of Cardiology, Keio University of Medicine, Tokyo, Japan 2

Radiotelemetric sensors for in vivo assessment of blood pressure and heart rate are widely used in large animal models. MRI with implanted sensors is regarded as contraindicated as transmitter malfunction and injury of the animals may be caused. Moreover, artefacts are expected to compromise image evaluation. MRI with a radiotelemetric sensor (TA10CTA-D70, Data Sciences) was performed in amicrominipigs. Imaging artefacts were analysed at 1.5 T in vivo. The skin temperature and the core temperature were measured before and after MRI. The macroscopic observation of skin was performed. Electrocardiogram and 24 h heart rate were compared to verify the integrity of the telemetric sensor. Artefacts impeded the assessment of the right atirum in microminipigs, but not of the left ventricle. The temperature of skin and core were not changed by MRI. No burn injuries were observed after MRI. No changes of electrocardiogram and heart rate were found before and after MRI. The radiometric sensor turned off before MRI was automatically turned on after MRI. These results indicate that MRI with implanted radiotelemetric sensors is feasible and the tested sensor maintains functionality. Measures to prevent artefacts and excursion should be examined in future studies.

O-50

Analysis of body temperature rhythm in rats before and after weaning using radio telemetry ○Jun Wakai1, Kouki Kato2, Kiyoaki Katahira2, Miho Sekiguchi1,2 1

Laboratory Animal Research Center, Fukushima Medical University, Fukushima, Japan, 2Translational Research Center, Fukushima Medical University

[Background] It is thought that thermoregulation is incomplete in neonatal rats, but it is unclear about the details. In the present study, to reveal a detail of development of thermoregulation, we recorded body temperature and activity of rats from lactational period to postweaning period with radio telemetry system. [Materials and methods] Lactational CD(SD) rats (male, 14 days old, n=5) were used in this study. We implanted telemetric transmitter (TA10TA-F10, DSI) into abdominal cavity of the rats. Body temperature and activity were monitored every 10 minutes from 14 to 35 days old. Rats implanted the transmitter were weaned at 21 days old and housed singly. [Results and discussion]In light period, body temperatures of rats in lactational period, a week after weaning, two weeks after weaning, and three weeks showed the same value (37.2°C) in light period. Body temperatures in dark period of the rat in lactational period showed a similar value (37.3°C), but those of the rats in 1-3 weeks after weaning showed over 37.7°C. Light-dark rhythm of body temperature was not shown in rat in lactational period, but it was observed as soon as rats were weaned. Light-dark rhythm of activity was not shown in the lactational rat, but it was shown after weaning. According to these results, body temperatures in lactational rats may be maintained by swarming around their mother.

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Effect of lactic acid bacteria on obesity in mouse models ○Yasunori Yonejima1, Keiko Hisa1, Yuko Sakai2, Masakazu Kita2

1

Nitto Pharmaceutical Industries, Ltd, Kyoto, Japan, 2Laboratory Animal Center, Kyoto Prefectural University of Medicine, Kyoto, Japan

In modern societies, obesity has become a common problem because people conventionally eat low-carbohydrate and high-fat diets (HFD). An improved daily diet is important for preventing obesity; therefore, research on functional foods has proceeded. It has proved that functional fatty acids, fiber, and lactic acid bacteria have effects on obesity (Wang et al., 2006), (Ushida et al., 2011), (Kadooka et al., 2010); however, the mechanisms were not clear. On the other hand, it has been proved that intestinal microbiota have a role in the host’s metabolic homeostasis (Ridaura et al., 2013), so improvement of intestinal microbiota by probiotic bacteria may contribute to preventing obesity. We focused on the effects of a probiotic, Lactobacillus brevis NT, on lipid digestion and metabolism; these effects were evaluated using a pair-feeding approach in KK/Ta mice being fed a high-fat diet. Four-week-old male KK/Ta mice were divided into the following four groups: HFD feeding, HFD supplemented with chocolate feeding, HFD supplemented with chocolate and L. brevis NTM003 feeding, HFD supplemented with chocolate and L. brevis NT feeding. After five weeks of feeding, body weight, visceral fat weight, and liver weight were measured, and serum lipids were determined. The results show that dietary supplementation with chocolate and L. brevis significantly decreased fat in liver and serum lipids.

O-52

Validation of DOHaD theory using mouse model (4): establishment of new maternal malnutrition model ○Tamio Furuse1, Kohda Takashi2, Kunio Miyake3, Takae Hirasawa4, Tomoko Kushida1, Ikuko Yamada1, Mishou Kashimura1, Hideki Kaneda1, Kimio Kobayashi1, Fumitoshi Ishino3, Takeo Kubota3, Shigeharu Wakana1 1

Japan mouse clinic, RIKEN BRC, Tsukuba, Ibaraki, Japan, 2Dept. of Epigenetics, Tokyo Med. & Dent. Univ., Tokyo, Japan, 1-5-45 Yushima, Bunkyoku, Tokyo, Japan, 3Dept. of Epigenetic Med., Univ. of Yamanashi, Shimokato, Chuo, Yamanashi 409-3898, Japan, 4Dept. of Bioscience., Teikyo University, Toyosatodai, Ustunomiya, Tochigi 320-8551, Japan

The developmental origins of health and disease paradigm (DOHaD) is a concept that fetal environmental factors affect the adult phenotypes. We are performing study that validates the DOHaD theory in the pathogenesis of psychiatric disorders and developmental disorders using mouse models. We provided following diets as experimental diets to pre-pregnant and pregnant females, AIN93G (control diet, CD), low-protein (LP), and low-protein diet that contains folate supplement (LP + FA). The offspring which were exposed to malnutrition in utero exhibited increased activity in the home cage, decreased contact to novel object, and decreased social investigation. The adult offspring of LP group and LP+FA group exhibited different pattern of mRNA expression and genomic methylation. In addition, we are developing new DOHaD model by using maternal mice which carry mutations in genes that relate to nutrient transport. In this meeting, we will report about the summary of past study and progress of the development of new model.

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The genetic analysis about acetylcholine sensitivity of mice ○Seiichi Tanaka1, Takeo Ichinose3, Mitsuru Matsuyama2, Yuko Takayashiki2, Hiroshi Nagashima2, Daisuke Torigoe4, Masami Morimatsu3, Takashi Agui3 1

Center for Experimental Animals, Fukuoka University, Fukuoka, Japan, 2Animal-care Co., Ltd.,Tokyo, Japan, 3School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 4Center for Animal Resources and Development, Kumsmoto University, Kumamoto, Japan

A mouse is the most popular in genetics research. The mouse models infecting pseudorabies virus latently suggested a possibility that acetylcholine (ACh) sensitivity varied. We objected to identify a regulatory gene by measuring ACh sensitivity difference and genetics analysis with C3H/HeJJcl (C3H) and BALB/CAJcl (BALB/C). We produced 68 (BALB/c × C3H)F2 progenies.We weaned at 3 weeks of age and gave acetylcholine chloride intraperitoneally at 5 weeks of age and observed having sensitivity or not. We cut a tail approximately 5mm for genotyping of the microsatellite about F2 and saved it at -20 degree celsius. LC 50 for ACh in F1 progenies was determined 3 × 10–2 M in males and 6 × 10–2 M in females using 34 F1 progenies. Next, sensitivity for ACh in 68 F2 progenies were evaluated and determined 18 F2 progenies showed susceptible phenotype, whereas 50 showed resistant phenotype, with approximately 1 to 3 ratio, suggesting that this phenotype was controlled by a single genetic locus. In the future, in regulation of ACh sensitivity without report by now, this research is hoped to contribute to investigate molecular mechanism in detail and make for genetic data accumulstion with a mouse of an experimental animal.

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Behavioral genetics of stress response associated with PACAP gene in mice ○Akira Tanave1,2, Aki Takahashi1,3, Tsuyoshi Koide1,4 1

Mouse Genomics Resource Laboratory, National Institute of Genetics, 2Transdisciplinary Research Integration Center, 3University of Tsukuba, 4The Graduate University for Advanced Studies

We have been conducted genetic mapping of anxiety-like behavior, and found PACAP gene as a candidate gene. Congenic mice that have PACAP gene derived from MSM/Ms strain showed altered open-field behaviors and high PACAP expression levels. PACAP is a peptidergic neurotransmitter that induces stress responses. It is known that PACAP is associated with the anxiety-like behaviors, but functional analysis of the PACAP gene is still not enough. Here we report further analyses using the congenic and transgenic mice that highly express the PACAP gene. We examined serum corticosterone level affected by restrained stress in the PACAP congenic mice. The congenic mice showed increased serum corticosterone levels similar with control mice immediately after the stress, but the increased serum corticosterone levels were significantly prolonged in the congenic mice compared to the control mice after 1-2 hour of the stress. These results suggest that the altered stress responses may be a cause of the altered open-field behaviors. In addition, we developed C57BL/6 transgenic mice with PACAP gene derived from MSM/Ms by using Tol2 transposon system. Now we are conducting behavioral analyses using the transgenic mice. Taken together, our results provide some evidences that indicate a relationship between the anxiety-like behavior and PACAP gene via a stress response pathway.

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Subordinate male mice in group housing show anxiety- and depression-like phenotypes ○Yasuyuki Horii1, Tatsuhiro Nagasawa2, Akira Tanave1, Aki Takahashi1,3, Kayoko Shimoi2, Tsuyoshi Koide1 1

Mouse Genomic Resource Laboratory, National Institute of Genetics, Shuzuoka, Japan, 2Laboratory of Biochemistry and Toxicology, University of Shizuoka, Shizuoka, Japan, 3Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, Ibaraki, Japan

Morbidity of anxiety and depression disorders is the highest among mental disorders. The previous studies suggested that developing risk is increased by social stress. In the laboratory, mice are socially stressed by social defeat stress test to establish animal model for the disease. In this test, subject male cohoused with apparently large and high aggressive strain male to give chronic social stress. By this treatment, the subject shows high plasma corticosterone and anxiety- and depression-like behaviors than control. It is questionable whether hierarchy which naturally ranked in group housing makes those differences. In the present study, we measured social hierarchy under group housing and compared the phenotypes which associated with stress-related mental disorders between boss and subordinate mice. Subordinate showed lower bodyweight, higher basal corticosterone and higher anxiety- and depression-like behaviors when compared to boss. The expression levels of hippocampal neurogenesis related genes were also significantly different between boss and subordinate. Currently, we are analyzing adult neurogenesis at hippocampus to see effects of the social stress on neural cells and will show the result at the meeting.

O-56

Immunohistochemical analyses of Timeless in the mouse brain development ○Yutaka Inaguma Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan

Timeless is an evolutionally conserved gene and its product is an essential component of the circadian rhythm regulation. Timeless is also implicated in cell cycle and embryonic development. In the present study, we developed a specific antibody against Timeless and examined the expression and localization of Timeless during mouse brain development. We used GST-fused mouse Timelss C-terminal fragment as an antigen and immunized rabbits. Specific antibody was purified by affinity column coupled with the antigen. In Western blotting, Timeless was detected throughout the developmental stage. In immunohistochemical analyses, Timeless was detected strongly in neurons in the ventricular zone/subventricular zone and moderately in cortical neurons during corticogenesis. In adult mouse brain, Timeless was observed moderately in cortical neurons. Timeless was enriched in the nucleus of cortical neurons from embryonic to early postnatal stages while it was distributed in the cytoplasm in the adult stage. Similar distribution change from nucleus to cytoplasm was observed in the hippocampal neurons between P0 and P30. In situ hybridization revealed that the tissue expression profile of Timeless-mRNA was similar to that of the protein. In differentiated primary cultured mouse hippocampal neurons, Timeless was detected in cell body, axon and dendrites. These results suggest that Timeless is expressed in neuronal tissues in a spatiotemporally regulated manner and involves in developmental stage-specific neuronal functions.

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Transcription factors that suppress Huntington’s disease and its molecular mechanism ○Naoki Hayashida Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan

In Huntington’s disease, glutamine repeats of huntingtin (Htt) are more than 150 in patients. The protein containing long glutamine tract is called as polyQ. PolyQ-Htt aggregates and acquires toxicity, and finally forms the inclusion bodies in cell nuclei. These bodies are formed in neurons, astrocytes, and microglia, and injure the neuronal functions. R6/2 model mice that express the Htt exon1 containing long CAG repeats have characteristic phenotypes. Inclusion bodies appear at very early stage in almost all tissues, and the longevity is dramatically short as less than 16 weeks. We maintained R6/2 by ovary transplantation for 10 years, CAG repeats decreased to 95-97 from 154. The longevity extended to more than 1 year. Thus, we named this line as R6/2(95-97) and analyzed. We established HSF1KO-R6/2(95-97) as HSF1 (heat shock factor 1) is required for heat shock protein induction. In addition, we established HSF2KO-R6/2(95-97) because HSF2 is highly expressed in CNS. Also, we established NFATc2KO-R6/2(95-97) because we found NFATc2 strongly suppresses polyQ aggregation in cellular model. Totally, all KO mice died at 14-40 weeks old even no WT-R6/2(95-97) died. In brain, polyQ-Htt began to aggregate at 8 weeks old in KO mice. Two weeks before their death, inclusion bodies were observed in nuclei, but not in other tissues at all. These results indicate that transcription factors HSFs and NFATc2 have important roles in suppression of Huntington’s disease.

O-58

Stress-reactive rats has shorter life spans than does stress nonreactive rats ○Ryo Ohta, Fumiaki Kumagai, Hideki Marumo, Kenji Usumi, Yoshiaki Saito, Makiko Kuwagata Hatano Research Institute, Food and Drug Safety Center

Although the Hatano high- and low-avoidance rats had been selectively bred for good vs. poor avoidance learning, high-avoidance rats are known to more reactive to stress than low-avoidance rats. In this study, highand low-avoidance females were compared onset of menopause by observing estrous cycles from 8 to 11 months of age. Furthermore, these females were allowed to live their natural life until 24 months of age in order to compare survival days. At eight months of age, 2 of 35 females in high-avoidance line and 20 of 35 females in low-avoidance line had abnormal estrous cycles. At 11 months of age, 22 of 35 females in high-avoidance line and all females in low-avoidance line had abnormal estrous cycles. Median life-span of high-avoidance females (673 days) was shorter than that of low-avoidance females (733 days). The incident of pituitary neoplasia was higher in high-avoidance females and that of mammary neoplasia was higher in low-avoidance females. These results suggest that low-avoidance females had accelerated ovarian aging than high-avoidance females. However, high-avoidance (stress-reactive) females had shorter life spans than did low-avoidance (stress non-reactive) females, had developed pituitary neoplasia which was causal factors in their accelerated mortality.

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The influence of life span in calorie restricted rat ○Yoshihiro Noda1, Naoko Maekawa1,2, Daijyu Muto1,2, Yui Miyazawa1,2, Yoko Takahashi1,2, Tamao Endo1 1

Animal Facility, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2KAC CO., LTD., Tokyo, Japan

Aged animals breeding are important in gerontological research. As for fostering of an aged animal breeding throughout the life from the juvenile age period, long-term stable environment is required, but temperaturehumidity management and the microbe control are not easy by facilities administration. In Tokyo Metropolitan Institute of Gerontology, since 1979, mice (C57BL/6NCrSlc, B6D2F1/Crlj, C57BL/6J), and rats (Wistar, F344/ DuCrlCrlj) have been brought up into aged animals. In rats, average lifespan of the Wistar were 810 days (male), 927 days (female) and those of Fischer were 859 days (male), 946 days (female). In this study, we investigated weight gain and longevity by aged rat. Moreover, fertility rate, the number of pups, and weaning rate which were examined by Wistar. Furthermore, it discussed weight gain and life-span in caloric restriction of Fischer rat.

O-60

Analysis of mammalian egg activation mechanism by quantitative live imaging system ○Kaori Nozawa1,2, Yuhkoh Satouh1, Kazuo Yamagata1, Masahito Ikawa1,2 1

Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 2Graduate School of Medicine, Osaka University, Osaka, Japan

In mouse case, ovulated oocytes are arrested at the metaphase stage of the second meiotic division (MII) and resumption of cell cycle is triggered by the sperm factors after sperm-egg fusion. This resumption is referred to as egg activation. During the egg activation, cortical granule exocytosis, extrusion of the second polar body and pronuclear formation are also triggered by Ca2+ oscillation, a series of intracellular Ca2+ elevations. It is suggested that the organized and sufficient pattern of the Ca2+ oscillation is required not only for activation but also for embryonic development. Thus, we have constructed low-invasive live imaging system to detect Ca 2+ and other subsequent events in qualitative and quantitative manner.As a result of the combination of Ca 2+ indicator with long-wavelength fluorescence and spinning-disc confocal microscopy, we succeeded to detect Ca 2+ oscillation and following developmental process to blastocyst of the same embryos and to sire pups after transplantation of the observed embryos. We also analyzed the relationship between Ca2+ oscillation and the cortical reaction using LCA-lectin and succeeded to visualize how the pattern of Ca2+ oscillation influences the cortical reaction.We expect these systems will be used to analyze events during egg activation responsible for the quality of embryos and developmental processes in detail.

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Abnormal sperm morphology and male subfertility in mice ○Hideo Gotoh Agrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan

Abnormal sperm morphology and male subfertility in miceHideo GotohAgrogenomics Research Center, National Institute of Agrobiological Sciences, Tsukuba, JapanB10.M strain shows both high frequencies of abnormalities in sperm morphology and subfertility. The abnormal sperm morphology has been found to be genetic. QTL analysis and gene mapping analysis indicated two recessive responsible loci. Sperm head morphology 1 (Shm1) locus was located on chromosome 1, and Shm2 locus was located on chromosome 4. In this study, F2 males (n = 40) produced between B10.M and C3H strains were mated with C3H females, and fertility rates of individual males were observed. Fertility rates of four pregnant females were observed for each male. After the fertility tests, genotype of Shm1 and Shm2 loci of the male mice were determined, and sperm morphology was also observed. Fertility rates of males distributed from normal fertility to subfertility, and about 25% of the males showed subfertility. The results indicated that the subfertility of B10.M males is a genetic phenotype. However, it was also found that the male subfertility was not linked either Shm1 or Shm2 locus, and that the male subfertility showed no correlation to the frequencies of abnormal sperm morphology. Further genetic analysis is expected to determine genes responsible for male subfertility of B10.M strain.

O-62

Breeding optimization of highly immunodeficient mice: NOD/SCID/ JAK3null ○Maki Sakaguchi1, Nobuyuki Mikoda1, Satoshi Nakamura1, Kouki Matsuda2, Sinichiro Hattori2, Seiji Okada2 1

Kyudo Co., Ltd, Saga, Japan, 2Division of Hematopoiesis,Center for AIDS Resarch, Kumamoto University, Kumamoto, Japan

Introduction:NOD/SCID/JAK3null(NOJ) is a highly immunodeficient strain prepared by crossbreeding an NOD/SCID mouse with a JAK-3 deficient mouse. The NOJ display poor breeding efficiency, which makes it difficult to obtain many pups. Here we investigate the effective production of pups using in-vitro fertilization and embryo transfer techniques.Methods:We prepared the NOJ 2-cell embryo via in-vitro fertilization, and then transferred the embryo into the oviduct of the pseudopregnant ICR strain mice. Thereafter we carried out a blood test, a biochemical test and an organ weight test to analyze the physiology of the pups. We also compared the two breeding methods by transplanting hematopoietic stem cells derived from a human umbilical cord to the liver of the newborns.Results:In natural-bred mice, the average litter size and the weaning rate were low (5.3 pups and 70.1% respectively). On the other hand, in embryo-transferred mice the average litter size was 8.5 pups. Moreover, the weaning rate of embryo-transferred mice significantly increased (95.6%, compared to 70.1% for natural-bred mice). In addition, no significant physiological difference was found between natural-bred and embryo-transferred mice, and we were able to confirm the development of the human hematopoietic stem cells/ immune system containing human T-cells in pups produced via both the embryo transfer method and natural breeding.

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Retrotransposon alteration in rat liver by long term treatment with DDT ○Makio Takeda, Ryoichi Ohtsuka, Satoru Yamaguchi, Nobuaki Nakashima, Takanori Harada The Institute of Environmental Toxicology, Ibaraki, Japan

Long interspersed element-1 (Line-1) is a kind of retrotransposons and has autonomous retrotransposition activity. In mammals, Line-1 has similarity to telomerase, and sirtuin participates in protection of telomere. Therefore we hypothesize that there are some kind of cross-talk between sirtuin and Line-1. It is well known that long term treatment of liver drug metabolize enzyme inducer such as DDT and phenobarbital induces hepatocarcinogenesis in rodents, and we revealed that CYP2B1 induction modified sirtuin activity leading to alteration in glucometabolism in the rat liver. As details of the mechanism are still unclear, we performed mechanistic analysis using rat liver samples in 2-year carcinogenesis study with DDT in order to clarify carcinogenetic mechanism as well as extrapolation to human. After 104 weeks of treatment, genome Line-1 copies of high dose group were significantly reduced compare to control. Furthermore, the copies of controls were significantly reduced in time dependent manner. Sequence data suggest that human genome LINE-1 is more highly methylated compared to rat genome LINE-1. Furthermore, the average human genome is estimated to contain 80-100 retrotransposition-competent. By comparison, the rodent genome is estimated to contain about 3000 complete Line-1s. In conclusion, it is suggested that rat genome may depend more highly on Line-1 than human genome and the reduction of copies may be rodent-specific carcinogenetic factor.

O-64

Activation of Wnt/β-catenin signaling disrupts homeostasis of gastric epithelial cells in mice ○Akihiro Hirata1, Yasuhiro Yamada2, Hiroyuki Tomita3, Tetsuya Tsukamoto4, Akira Hara3 1

Division of Animal Experiment, Life Science Research Center, Gifu University, Gifu, Japan, 2Center for iPS cell Research and Application (CiRA), Kyoto University, Kyoto, Japan, 3Department of Tumor Pathology, Gifu University Graduate School of Medicine, Gifu, Japan, 4Department of Diagnostic Pathology, School of Medicine, Fujita Health University, Aichi, Japan

The Wnt/β-catenin signaling is frequently activated in gastric cancers but it remains poorly understood how Wnt activation contributes to the gastric carcinogenesis. In the present study, we first investigated the effect of Wnt activation in gastric epithelial cells using doxycycline-inducible β-catenin mice. When we fed adult mice doxycycline for 5 days, cytoplasmic and nuclear β-catenin accumulation were observed in both fundic and pyloric glands of the stomach. The number of Ki-67-positive proliferating cells significantly increased in the isthmus and gastric pits of both fundic and pyloric glands in doxycycline-treated mice. In addition, the significantly decreased mucus production was observed in the gastric pits of β-catenin-induced stomach, demonstrating a suppression of cellular differentiation toward surface mucous cells following β-catenin induction. qRT-PCR demonstrated that β-catenin induction led to the up-regulations of canonical Wnt target genes and gastric and intestinal epithelial stem cell markers. Our data indicate that Wnt activation maintains gastric epithelial cells in an undifferentiated and proliferative state.

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Combination therapy of β2-adrenoceptor and glucocorticoid for sepsis-induced renal injury ○Makoto Miyagawa1, Kumiko Kurosaki2, Akio Nakamura2 1 Central Experimental Animal Center,Teikyo University,Tokyo, Japan, 2Department of pediatrics, Teikyo University School of Medicine, Tokyo, Japan

Sepsis and its sequelae are still a major cause of morbidity. Recently, we reported that functional β2adrenoceptor (β2-AR) activation via β2-AR gene delivery, during sepsis, would enhance glucocorticoid antiinflammatory action in the kidney,resulting in kidney protection. The current study was undertaken using the CLP rat model to explore whether the combination therapy of β2-AR gene delivery and glucocorticoid might be able to prevent the progressive renal damage associated with sepsis. Rats was subjected to ligation of the cecum from the cecal tip followed by an intraperitoneal injection of adenoviral transgenes containing the human β2AR or β-galgene. Furthermore, glucocorticoid were given to some rats. 8,24,48 hours following CLP, kidneys were collected to perform histological evaluation in the kidneys. All group rats were survived at 8 h following the onset of sepsis. By contrast, the survival rate for the rats was approximately 53-67% at 24 h following the onset of sepsis. On the other hand, renal histological findings in the CLP rat model showed renal cellular infiltrates and glomerular inflammation. However, these pathological findings were improved in the kidneys of the CLP+β2AR+Dex rats.Crosstalk betweenβ 2-AR and glucocrticoid may produce additive or even synergistic anti-inflammatory effects, resulting in kidney protection against sepsis-induced renalinjury.

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Mesenchymal progenitor-like gene expression of pleomorphic rhabdomyosarcoma generating cell lines ○Noboru Suzuki1, Hiroko Ito2, Hiromitsu Saito1 1

Department of Animal Functional Genomics, Functional Genomics Institute, Mie University Life Science Research Center, 2Department of Bioresources, Mie University

Pleomorphic rhabdomyosarcomas (pRMSs) arise predominantly in the skeletal musculature of adults. Here we report that a cell line was established in culture from the primary tumor induced in the intramuscular tissue of a conditional K-rasG12V animal model and designated RMS3. RMS3 cells were fibroblastic and could grow exponentially in DMEM containing 10% fetal calf serum. RMS3 cells gave rise to pRMSs with characteristic bizarre giant cells, positive for desmin and alpha-sarcomeric actin and colonized in the lung by orbital vein injection. RT-PCR showed that the tumor generating RMS3 cells were positive for tissue-resident fibro-adipogenic progenitor (FAP)/ mesenchymal stem cell (MSC) markers; Sca-1, CD34, PDGFRalpha, Adam12 and Tcf4, but negative for satellite cell markers; Pax7 and Myf5, suggesting the cellular origin of RMS3 cells is not satellite cells but FAPs/MSCs. We found that mRNA levels of IL-6 of RMS3 cells in culture significantly enhanced by subcutaneous implantation with no change of K-rasG12V-mRNA levels. And we could recapture both the enhancement of mRNA levels of IL-6 and its secretion in a 3-D culture system. Further, we demonstrated that blazein which is a steroid isolated from Agaricus blazei suppressed the IL-6 enhancement in dose-dependent manner. Thus, we provide a novel system which is useful for our better understanding and developing drugs for pRMSs.

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K-rasG12V-mediated lung tumor models identified five new QTL modifying events post-K-ras mutation ○Hiromitsu Saito, Ryouya Yamaguchi, Noboru Suzuki Department of Animal Genomics Functional Genomics Institute, Mie University Life Science Research Center, Mie, Japan

The process of oncogenic K-ras-mediated lung adenocarcinogenesis can be dissected into two parts: pre- and post-K-ras mutation. Adoption of transgenic lines containing a flox-K-rasG12V transgene eliminates the use of chemical carcinogens and enables us to study directly crucial events post-K-ras mutation without considering the cellular events involved with oncogenic K-ras mutation, e.g., distribution and metabolism of chemical carcinogens and DNA repair by host factors. We generated two mouse strains C57BL/6J-RyR2tm1Nobs and A/ J-RyR2tm1Nobsin which K-rasG12V can be transcribed from the cytomegalovirus early enhancer/chicken beta actin promoter in virtually any tissue. Upon K-rasG12V induction in lung epithelial cells by an adenovirus expressing the Cre recombinase, the number of tumors in the B6-RyR2tm1Nobs/+ mouse line was 12.5 times that in the A/ J-RyR2tm1Nobs/+ mouse line. QTL analysis revealed that new 5 modifier loci, D3Mit19, D3Mit45, D6Mit188, D9Mit191 and D11Mit20, were involved in the differential susceptibility between the two lines. In addition, we found that differential expression of the wild-type K-ras gene, which was genetically turn out to be antioncogenic activity on K-rasG12V, could not account for the different susceptibility in our two K-rasG12Vmediated lung tumor models. Thus, we provide a genetic system that enables us to explore new downstream modifiers post-K-ras mutation.

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Kras mutation develop oncogenic activity with HPV E6/E7 ○Katsuyoshi Kumagai1, Masakatsu Takanashi2, Shin-ichiro Ohno2, Hirotaka Nishi3, Katsuko Sudo1, Masahiko Kuroda2 1

Animal Research Center, University-related Facilities, Tokyo Medical University, Tokyo, Japan, Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan, 3Department of Obatetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

2

Human papilloma viruse (HPV) are the primary causal agents responsible for development of cervical cancer, and expression of two viral agents E6 and E7 is considered to develop of cervical cancer. However, a disease develops in 0.15% of infected women by HPV in spite of approximately 90-100% of women infected with HPV during their lives. KRAS was identified as an oncogene in 1982 and is found to be mutanted gene have been observed in the KRAS oncogene. Activating KRAS mutation were found in human cancers including 14% of cervical cancers. Moreover, Kras mutation promotes metastatic liver tumorigenesis that is significantly accelerated by inactivation of p53 in mice. Thus, Kras mutation were considered to develop canceration with E6/ E7. Recently, we have demonstrated that transduction of Kras mutation with E6/E7 is sufficient for development of canceration in epithelium cells. Here we show that transduction of Kras mutation with E6/E7 expression causes increase not only number of colonies but also formation of colonies. Furthermore, The survival rate of E6/E7 expression female mice were lower than wild type mice. These result suggest that Kras mutation could be develop not only oncogenic activity in epithelium cells with E6/E7 but also canceration of female mice.

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The role of parathyroid hormone in mouse skin tumorigenesis ○Kazuhiro Okumura1, Megumi Saito1, Eriko Isogai1, Ikuo Miura2, Shigeharu Wakana2, Midori Shimanuki3, Koji Shitara3, Choji Taya3, Ryo Kominami4, Yuichi Wakabayashi1 1

Division of Experimental Animal Research, Chiba Cancer Center Research Institute, Chiba, Chiba, Japan, Technology and Development Team for Mouse Phenotype Analysis, Riken Bioresource Center, Tsukuba, Ibaraki, Japan, 3Laboratory for Transgenic Technology, the Animal Research Division, Tokyo Metropolitan Institute Medical Science, Setagaya, Tokyo, Japan, 4Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan 2

Using a forward genetics approach to map several loci in a mouse skin cancer model, we previously identified strong genetic loci Skin tumor modifier of MSM 1 (Stmm1) on chromosome 7 with a large number of [(FVB/ N x MSM/Ms) x FVB/N] F1 backcross mice. The parathyroid hormone (Pth) gene was found in the vicinity of Stmm1. Here, we report a genetic polymorphism located in Pth gene, which produces Val/Met pro-Pth variants. Skin carcinogenesis experiments with MSM-BAC transgenic mouse lines (FVB-PthMet Tg) showed PthMet (MSM/ Ms) alleles conferred strong resistance to tumors. In addition, we investigated the amount of serum intact-Pth of FVB-PthMet Tg and FVB-PthVal Tg mice. As a result, the concentration of serum intact-Pth was more than two times higher in PthMet-Tg than in PthVal-Tg mice. These results suggested that Pth might be a good candidate gene for Stmm1 and the amount of serum Pth could be a marker of skin cancer risk.

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The role of zinc transporter ZIP14 in the regulation of osteoclast functions ○Tatsuya Furuichi1, Syun Sasaki1, Satoki Ichimura1, Shintaro Hojyo2,3, Toshiyuki Fukada2,4 1

Lab. of Laboratoy Animal Science and Medicine, Co-Department of Veterinary Medicine, Iwate University, RIKEN Center for Integrative Medical Sciences, 3Deutsches Rheuma-Forschungszentrum Berlin, 4Division of Pathology, School of Dentistry, Showa University 2

Bone is constantly renewed by the process of bone remodeling. In this process, osteoclasts resorb old bone whereas osteoblasts deposit new bone. Zinc (Zn) concentration is relatively high in bone tissues and Zn deficiency causes bone loss. However, the mechanisms regulating bone mass by Zn have not been clarified. Zn homeostasis is controlled by over twenty members of zinc transporter family. The Zn transporter ZIP14 is a cell surface-localized Zn transporter that contributes to the Zn influx into cells. In this study, we examined the bone phenotypes of Zip14-KO mice to reveal the role of ZIP14 in bone homeostasis. X-ray and p-QCT analyses showed the bone mass was significantly decreased in Zip14-KO mice compared to wild-type mice. Bone morphometric analysis showed osteoblast number and bone formation rate were not significantly changed between the two genotypes, indicating the osteoblast function in Zip14-KO mice is normal. On the contrary, percent of eroded surface/bone surface was significantly increased in the mutant indicating the osteoclast function is activated in Zip14-KO mice. In situ hybridization analysis showed Zip14 mRNA was expressed in osteoclasts . These results strongly suggest the ZIP14-mediated Zn influx negatively regulates osteoclast function.

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PLAC1 regulates placental size and layer formation ○Masanaga Muto1, Yoshitaka Fujihara2, Masahito Ikawa1,2 1

Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan, 2Research Institute for Microbial Diseases, Suita, Osaka 565-0871, Japan

The placenta is a feto-maternal interface and is required for nutrient supply and gas exchange in mammalian reproductive systems. The investigation of the function of placental genes is important to understand pregnancy related diseases. Previously, the X-linked Plac1 (placental specific protein 1) gene was reported to be involved in trophoblast cell invasion and migration. In the present study, we generated Plac1 knockout (KO) mice and the biological functions of PLAC1 were investigated during mouse placental formation. To evaluate the fertility of Plac1 KO mice, we checked the number of pups produced from natural mating. Plac1 KO females mated with WT males showed smaller litter sizes and intrauterine growth restriction. The placental weight of Plac1 KO mice was drastically increased during mid-gestation and reached sizes around 2-fold larger than WT placentas. Plac1 KO placentas showed an expanded junctional zone that migrated and encroached into the labyrinth layer. Further, Plac1 KO placentas had morphological abnormalities in the labyrinth layer. We next transduced blastocysts with lentiviral vectors and over-expressed the Plac1 gene in a placenta specific manner. The morphology of blood vessels in the labyrinth layer and the birth rate of Plac1 KO mice were improved. Our findings suggest that the level of PLAC1 gene expression regulates placental size and function of the labyrinth trophoblast cells.

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Jmjd3, but not Utx, deficiency leads to homeotic transformation in mice ○Chie Naruse1,2, Shibata Shinwa2, Abe Kanae2, Kawaguchi Takayuki2, Sugihara Kazushi1,2, Ikawa Masahito3, Asano Masahide1,2 1

Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan, 3Animal Resource Center for Infectious Diseases Research Institute for Microbial Diseases, Osaka University, Suita, Japan

Jmjd3 and Utx are known as specific demethylases of lysine 27 on histone H3 (H3K27). Previous reports indicate that Jmjd3 is essential for differentiation of macrophages and epidermal cells. Although Jmjd3-/- mice are reported to be postnatal lethal, the cause of death and function of Jmjd3 during development is still unclear. We produced Jmjd3 homozygous (Jmjd3-/-) mutant mice and Utx hemizygous (Utx-/y) mutant mice to analyze the functions of Kdm6 family members in mice. Jmjd3-/- embryos, but not Utx-/y embryos, showed anterior homeotic transformation of vertebrae and ribs, and mRNA levels of Hox genes were reduced at their expression initiation stages (E8.5-9.5). Chromatin immunoprecipitation analyses suggested Jmjd3 was accumulated at the Hox gene locus before their transcription started, and Jmjd3 was released from the locus after transcription was started. Although H3K27me3 at the locus was decreased and H3K4me3 was increased in company with increase of Hox transcription level in wild-type embryos, H3K27me3 was remained at the locus in Jmjd3-/- embryos.

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Expression of truncated PITX3 in the developing lens leads to microphthalmia and aphakia in mice ○Kenta Wada1,2, Yoshibumi Matsushima3, Tomoki Tada1, Yasuhiro Yoshizawa1, Sayaka Hasegawa1, Midori Shimanuki2, Kei Watanabe2, Yoshiaki Kikkawa2 1 Tokyo University of Agriculture, 2Tokyo Metropolitan Institute of Medical Science, 3Research Institute for Clinical Oncology, Saitama Cancer Center

Paired-like homeodomain factor 3 (PITX3) has a critical role in lens development. Recently, we isolated spontaneously microphthalmia and aphakia (miak) mouse and identified a nonsense mutation (148Tyr>X) in the Pitx3. We investigated expression pattern of truncated PITX3 protein and of its downstream genes, in miak mice. During lens development, immunofluorescence of PITX3 was observed in lens vesicle and epithelium at E11.5 and E12.5, respectively. Although the localization of PITX3 was similar in the wild-type, the immunofluorescence was more abundant in miak/+ and miak/miak mice. Similar to that protein level, miak/+ and miak/miak showed higher expression of Pitx3 transcripts than wild-type. Furthermore, the expression levels of FOXE3 and PROX1 proteins showed obvious reduction in miak/miak mice, compared with wild-type and miak/+. To confirm binding activity of truncated PITX3 protein to Foxe3 enhancer and Mip promoter containing bicoid elements, the EMSA were performed. We observed the specific EMSA complexes in wild-type and miak mice, and obvious enhanced reactivity was shown in miak mice. Thus, we suggested that OAR domain has a role for self-regulator in PITX3, and that it is essential for normal lens development via transcriptional regulation of target genes coupled with homeodomain.

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Expression analysis of leucine-rich repeat containing 30 in the inner ear ○Yuki Miyasaka1,2, Shumpei Yasuda1, Yuta Seki1, Kunie Matsuoka1, Hiroshi Hibino2, Ryo Kominami2, Yoshiaki Kikkawa1,2 1

Tokyo Metropolitan Institute of Medical Science, 2Niigata University

We previously reported that age-related hearing loss (AHL) in C57BL/6J (B6J) mice could be prevented by introducing an ahl3 allele from MSM/Ms (MSM) mice. We identified a strong candidate ahl3 gene, leucinerich repeat containing 30 (Lrrc30), which is expressed in the stereocilia of the hair cells in the inner ear. To investigate the functions of LRRC30 during the aging process, we analyzed its mRNA and protein expression. First, we conducted an RT-PCR assay with RNA preparations from cochleae isolated at several developmental and maturational stages. Lrrc30 expression was first detectable in the mouse cochlea at P6, became markedly elevated during the course of development, and was expressed at high levels during the maturational stages. Moreover, we showed that the mean expression levels of Lrrc30 were 4 to 8-fold higher in MSM mice than in B6J mice. Next, we investigated the localization of LRRC30 protein in stereocilia using stimulated emission depletion microscopy. In mature stereocilia, we found that LRRC30 was localized in the stereocilia links. We also showed that LRRC30 was partially co-localized with cadherin 23 (CDH23) at the links. It is known that the AHL in B6J mice can be caused by a reduction in Cdh23 expression due to the ahl mutation. Therefore, we suggest that the increased levels of LRRC30 in MSM-derived ahl3 allele confer resistance to AHL by compensating for CDH23 down-regulation due to the ahl mutation.

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Species specificity of the inheritance modes of cataract phenotypes caused Mip mutations ○Yoshiaki Kikkawa1, Kei Watanabe1, Yuta Seki1, Shumpei Yasuda1, Hiroaki Taketsuru2, Takehito Kaneko2, Tomoji Mashimo2, Takashi Kuramoto2, Kenta Wada1,3, Hiroshi Shitara1 1

Tokyo Metropolitan Institute of Medical Science, 2Kyoto University, 3Tokyo University of Agriculture

Major intrinsic polypeptide gene (MIP) is a representative causative gene for congenital cataract in humans. We previously reported that the null mutation (MipL121TfsX7) resulted in congenital cataract in rats and that it was inherited in a recessive fashion. However, patients (MIPR113X) in a human family carrying a similar mutation within MipL121TfsX7 of rat developed cataract in the dominant mode. To confirm the difference between inheritance modes of cataract phenotypes between human and rat whether the recessive inheritance of cataract is specific phenomenon in rodent, we produced Mip mutant mice carrying similar mutations in MIPL121TfsX7 of rat and MIPR113X of human by using CRISPR/Cas9-mediated genome editing. We established two mutant mice carrying MipA111EfsX3 and MipY105SfsX4 mutations and analyzed the phenotypes. The both heterozygous mutant mice showed cataract, which was milder than that in homozygous mutant mice, suggesting that the Mip mutant alleles functions as semi-dominantly in mice. However, the expression patterns of the Mip transcripts were identical between mice and rats. The findings of our study suggest that there is a difference in phenotypes and inheritance modes of cataract between humans/mice and rats.

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Insufficient levels of SPINK induces impaired autophagy resulting to chronic pancreatitis ○Masaki Ohmuraya, Kimi Araki, Ken-ichi Yamamura Institute of Resource Development and Analysis, Kumamoto University

Background & Aims: Mutations in serine protease inhibitor Kazal type 1 (SPINK1) are associated with human chronic pancreatitis (CP). Genetic deletion of Spink3, mouse homolog of SPINK1, causes postnatal lethality precluding mechanistic investigations into the effects of SPINK deficiency. Here we have developed Spink3SPINK1/- transgenic mice (SPINK1-in) in which one ablated Spink3 allele is replaced by knockedin SPINK1. This partial restoration of SPINK function rescues mice from lethality; but SPINK1-in mice progressively develop spontaneous CP. Methods: We used Cre-Lox technology to generate Spink3SPINK1/mice and their controls, Spink3SPINK1/+. Pancreas damage and pancreatitis responses were analyzed.Results: In contrast to Spink3SPINK1/+ and Spink3SPINK1/SPINK1, SPINK1-in mice within 4 weeks developed pancreas damage the earliest manifestations of which were impaired autophagy and increased trypsin activity. This resulted in accumulation of large vacuoles, macrophage-type inflammation, intralobular fibrosis with activated stellate cells, and acinar cell death, i.e., apoptosis. Pancreas damage (i.e., vacuolization) was not prevented by Atg5 deletion, indicating the involvement of non-canonical autophagy. Conclusions: SPINK1-in mice represent a novel, clinically relevant, genetic model of human CP, revealing the mechanisms whereby SPINK insufficiency causes CP. The results identify new targets of SPINK regulating autophagic and lysosomal pathways in pancreas.

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Hypertension-responsiveness of the mouse renin gene in tsukuba hypertensive mouse ○Aki Ushiki1, Akiyoshi Fukamizu2,3, Keiji Tanimoto2,3 1

Grad. School of Life and Environmental Sci., Univ. of Tsukuba, Ibaraki, Japan, 2Faculty of Life and Environmental Sci., Univ. of Tsukuba, Ibaraki, Japan, 3TARA Center, Univ. of Tsukuba, Ibaraki, Japan

The renin-angiotensin system is essential for blood pressure homeostasis, in which renin (REN) catalyzes cleavage of its unique substrate, angiotensinogen (AGT), to produce vasopressor peptide angiotensin II. Because increased REN activity causes blood pressure elevation, its expression is normally suppressed in the hypertensive state. However, a mechanism of how REN gene expression is regulated by blood pressure remains to be determined. We hypothesized that REN gene has cis DNA sequences that is responsible for hypertensionmediated suppression of its transcription. To test if this hypothetical element locates proximal or distal to REN gene body, we generated transgenic mouse (TgM) lines carrying either small or large mouse REN gene fragment. Then, each transgene (Tg) was introduced into Tsukuba Hypertensive Mice (THM) by cross-mating and its hypertension-responsiveness was tested. A large Tg with a deleted mouse distal enhancer (mdE) element was also analyzed in the same way. Based on our results, mechanism of hypertension-responsiveness of REN genes would be discussed.

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Genome mutations of persistently infected Hepacivirus which is related to hepatitis-C in primates ○Atsunori Higashino1,2,3,4, Saori Suzuki1, Ken-ichi Mori2, Hirotaka Ode3, Kazuhiro Matsoka3, Yuko Katakai4, Sachi Okabayashi4, Noboru Maki2, Yasumasa Iwatani3, Wataru Sugiura3, Hrofumi Akari1 1

Primate Research Institute, Kyoto University, Inuyama, Japan, 2Advanced Life Science Institute, Wako, Japan, 3National Hospital Organization, Nagoya Medical Center, Nagoya, Japan, 4Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan

Our laboratory has reported that two common marmosets infected with GBV-B derived from a molecular clone pGBB developed long-term persistent infection among four common marmosets. GBV-B infection produces a chronic and progressive hepatitis C-like disease in marmosets. However, it is still unclear how virus mutations relate to disease progression in hepatitis C. In this study, we analyzed the viral genome sequence between acute clearance and persistent infection. We were able to analyze GBV-B adaptive or escape mutations for persistent infection. Many mutations were located in NS5A, NS5B regions on the GBV-B, which might cause long-term persistent infection or liver fibrosis. It is known that NS5A involve IFN resistance by inhibiting ISG expression pathway. NS5A may have had diversity in order to inhibit anti-virus factors. Since it has reported that NS5B is RNA depend RNA polymerase in HCV, mutations observed in this region seem to increase the efficiency of replication of the viral RNA. Our studies revealed that the mutations on GBV-B derived from infectious clone.

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Characterization of prediabetic state in a novel model of obese type 2 diabetes: the ZFDM rat ○Norihide Yokoi1, Takuro Yamaguchi1, Gheni Ghupurjan1, Masayuki Beppu1, Shihomi Hidaka1, Ayako Kawabata1, Yoshikazu Hoshino2, Masayuki Hoshino2, Susumu Seino1 1

Division of Molecular and Metabolic Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, 2Hoshino Laboratory Animals, Inc., Ibaraki, Japan

We recently established a novel animal model of obese type 2 diabetes, the Zucker fatty diabetes mellitus (ZFDM) rat strain harboring the fatty mutation (fa) in the leptin receptor gene. Here we performed a phenotypic characterization of prediabetic state in the ZFDM strain. At 7 weeks of age, fa/fa male rats exhibited mild glucose intolerance and severe insulin resistance. In the isolated pancreatic islets, the insulin secretory responses to both glucose stimulation and the incretin GLP-1 were retained. At 11 weeks of age, fa/fa male rats exhibited marked glucose intolerance accompanied by impaired insulin secretion. In the pancreatic islets, the insulin secretory response to glucose stimulation was maintained but the response to the incretin was diminished. In nondiabetic Zucker fatty (ZF) rats, the insulin secretory responses to both glucose stimulation and the incretin in the pancreatic islets were similar to those of ZFDM rats. A combination of severe insulin resistance, impaired insulin secretion, and diminished insulin secretory response to incretin may cause the development of diabetes in the ZFDM strain.

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Identification of diabetogenic genes associated with obesity using new animal models of rats(1) ○Daiki Sasaki1, Jun Koto1, Risa Watadani1, Yu Seto2, Ayaka Tanida2, Kohei Honda2, Kozo Matsumoto1,2 1

The Faculty of Life Science,Kyoto Sangyo University, Kyoto, Japan, 2The Department of Laboratory Animal Science, Kyoto Sangyo University

Type 2 diabetes is brought on by sedentary life style, rich nutrition and obesity. However, little is known how obesity induce the onset of type 2 diabetes. To elucidate these mechanisms, it is necessary to identify diabetogenic gene associated with obesity. We have created the new diabetic animal models that can search diabetogenic gene under condition of obesity. 14 loci were identified in OLETF rat. We used single congenic introgressed Nidd2 QTL region into F344 (F.O-Nidd2/of), and single obese congenic introgressed Lepr-/- locus into F344 (F.ZF-Lepr). To obtain double congenic rats (F.ZF-Lepr&Nidd2/of), both lines were crossed and selected homozygotes fo Nidd2 and Lepr-/- locus. In intraperitoneal insulin tolerance test (i.p.ITT), both normal F344 and F.O-Nidd2/of rat never showed insulin resistant, while the ipITT showed the isulin resistance in the F.ZF-Lepr&Nidd2/of rats. These results indicate that Nidd2/of QTL region includes an insulin resistant gene with obesity. We found the candidate gene for the onset of type 2 diabetes with obesity.

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Identification of diabetogenic genes associated with obesity using new animal models of rats (2) ○Jun Koto1, Daiki Sasaki1, Risa Watadani1, Yu Seto2, Ayaka Tanida2, Kohei Honda2, Kozo Matsumoto1 1

Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan, 2Kyoto Sangyo University

Recently, it is well understood that obesity is a major risk factor for the onset of type 2 diabetes. Genetic predisposition is also major risk factors as well, and the interactions between these factors are likely to be important in the etiology of this disease. However little is known how obesity induces the onset of type 2 diabetes. Therefore, identification of causative genes for diabetes associated with obesity is necessary for elucidation of the mechanism of the onset of type 2 diabetes. There are 14 diabetogenic loci are identified by QTL analysis of OLETF rats. But it is not clear which locus is associated with obesity. In this study, we used sigle congenic introgressed Nidd6 QTL region into F344, and single obese congenic introgressed Lepr-/locus into F344. To obtain double congenic rats, both lines were crossed and selected homozygotes for Nidd6 and Lepr-/- locus. Double congenic rats named as F.ZF-lepr&Nidd6/of rats displayed significantly higher blood glucose levels than those of obese control rats (F.ZF-lepr). This result indicates that the Nidd6 region is responsible for the onset of type 2 diabetes with obesity. Nidd6 region contains 60 genes. Therefore, we investigated genes expression of Nidd6 region in liver of these rats using Real Time PCR. We finally found three candidate genes for the onset of type 2 diabetes associated with obesity.

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Initiation and progression of coronary atherosclerosis in WHHLMI rabbits ○Tomonari Koike1, Ying Yu1, Atsushi Yamashita2, Ryosuke Nagasaka1, Satoshi Yamada1, Yujiro Asada2, Masashi Shiomi1,3 1

Institute for Experimental Animals, Kobe University Graduate School of Medicine, Kobe, Japan, Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, 3Division of Comparative Pathophysiology, Kobe University Graduate School of Medicine, Kobe, Japan 2

Objectives: Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits shows severe coronary atherosclerosis spontaneously. However, the detailed process of the coronary atherogenesis is still unclear. Methods: Coronary arteries from 136 WHHLMI rabbits were evaluated for lesion components, lesion thickness and cross-sectional narrowing (CSN). Results: Early lesions showed mainly intimal thickening (IT) with smooth muscle cells at the bifurcation of arteries. Fibrous plaques were mainly observed in 30~50% CSN. Layered plaques consisting of fibromuscular and lipids/foam cell layers were increased with plaque growth. Pre-fibroatheroma (Pre-FA), which has foam cell accumulation instead of lipid core, were decreased, and FA were increased with further progression of plaques, respectively. Thin-capped FA were frequent in >90% CSN. Conclusions: This study suggest that the development of coronary lesions are as follows; 1) the initiation is IT, 2) macrophage infiltration and increase in fibromuscular components promote lesions; 3) these lesions transfer to FA and thin-capped FA over time. The observation suggests that WHHLMI rabbits become a powerful tool for the investigation of coronary atherogenesis.

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