JFCIクラウン研究所・文献集
Rapid assignment of the swine major histocompatibility complex (SLA) class I and II genotypes in Clawn miniature swine using PCR-SSP and PCR-RFLP methods.
Xenotransplantation. 2005 Mar;12(2):121-6.
Ando A, Ota M, Sada M, Katsuyama Y, Goto R, Shigenari A, Kawata H, Anzai T, Iwanaga T, Miyoshi Y, Fujimura N, Inoko H.
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan.
BACKGROUND: Inbred miniature swine with defined novel SLA haplotypes will be useful in allo- and xeno-transplantation studies, which can be carried out representing variable combinations of SLA haplotypes. METHODS: In Clawn miniature swine, two haplotypes (c1 and c2) and one crossover haplotype (c3) have been assigned by nucleotide sequence determination of RT-PCR products of the three SLA classical class I genes and two SLA class II genes. To select SLA class I and II homozygotes in Clawn miniature swine individuals, we developed a rapid and simple SLA-class I- and II-DNA typing method by a combination of polymerase chain reaction-sequence specific primer (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. RESULTS: Seven allele specific primer pairs were designed for amplification of the second exons of three SLA class I genes, SLA-1, SLA-2, and SLA-3, and one SLA class II gene, DRB1. Furthermore, based on PCR-RFLP patterns in the SLA-DQB1 gene, two allelic variants were recognized in the second exon in the Clawn miniature swine. Three haplotypes, c1, c2 and c3, were simply identified by the combination of PCR-SSP and PCR-RFLP methods in 22 samples from five families. A single allele at each of the class I and II genes was also observed in seven samples as SLA class I and II homozygotes with either the c1 or c2 haplotype. CONCLUSIONS: The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.
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The miniature pig: a unique experimental model for Schistosoma japonicum infection.
Parasitol Int. 2004 Dec;53(4):293-9.
Watanabe K, Kikuchi M, Ohno A, Mohamed RT, Nara T, Ubalee R, Senba M, Iwasaki T, Chen H, Aoki Y, Hirayama K.
Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
As part of a search for good animal models for human schistosomiasis, two miniature pigs of the CLAWN strain (C-1, C-2) were inoculated percutaneously with 200 Schistosoma japonicum cercariae of the Chinese strain, and the subsequent infection was monitored parasitologically, pathologically and serologically. Egg excretion into feces began at 5 weeks post-infection (p.i.) and became pronounced from 8 weeks to 17-20 weeks p.i. The average number of eggs in 1 g feces of each pig at the peak period between 8 and 20 weeks were 288 and 277, respectively. C-1 and C-2 were killed and perfused at 27 and 47 weeks p.i. and adult worm numbers recovered were 35 and 15, respectively. C-2 had at least four pairs of viable mature worms but no detectable fecal eggs for a month before perfusion, suggesting that any produced eggs were not excreted into the feces during this period. Egg deposits associated with inflammatory reactions were observed by histological examination of the liver, spleen, pancreas, mesenteric lymph nodes, lung, and small intestine. This suggests that reduced fecal excretion of eggs into the feces did not correlate to reduced parasite numbers in the chronic phase of schistosomiasis. This is the first report showing the miniature pig to be a potential model for human S. japonicum infection.
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Molecular investigation of hepatitis E virus infection in domestic and miniature pigs used for medical experiments.
Xenotransplantation. 2004 Nov;11(6):503-10.
Tanaka H, Yoshino H, Kobayashi E, Takahashi M, Okamoto H.
Center for Experimental Medicine, Jichi Medical School, Tochigi 329-0498, Japan. htanaka@jichi.ac.jp
BACKGROUND: Hepatitis E virus (HEV) infection is highly prevalent among domestic pigs in Japan. It has been reported that pig handlers such as farmers and veterinarians are at increased risk of contracting HEV infection. Pigs are regarded as the most acceptable candidate animals for xenotransplantation and, recently, they are being used as experimental animals. METHODS: We investigated the prevalence of IgG class antibodies to swine HEV (anti-HEV) and HEV RNA among 152 2-month-old domestic pigs and 38 miniature pigs of 4 to 10 months of age that had been brought to our center for medical experiments from five swine farms (A-E) in Japan. Serum samples were tested for anti-HEV by in-house enzyme immunoassay, and for HEV RNA by reverse transcription-polymerase chain reaction using primers targeting the open reading frame 2 (ORF2) region. RESULTS: One percent (one of 84), 6% (one of 16), and 38% (20 of 52) of the domestic pigs from farms A, B and C, respectively, had detectable HEV RNA, and the 22 HEV isolates recovered from the viremic pigs were 89.8 to 100% identical to each other in the 412-nucleotide sequence of ORF2 and segregated into three clusters within genotype 3. Although one pig from farm A had detectable HEV RNA reproducibly, the HEV isolate recovered from this pig was up to 100% similar to those recovered from pigs from farm C, and the sera from all 84 pigs from farm A were negative for anti-HEV. These results suggested that farm A is free from HEV infection. As the viremic pig from farm A had been raised for 1 month in a barn at our center before serum sampling, it is most likely that the pig acquired HEV infection in the barn at our center where HEV-viremic pigs from farm C had been reared for several days approximately 3 months earlier. The 38 miniature pigs from farms D and E were negative for both anti-HEV and HEV RNA. In an attempt to further investigate the prevalence of HEV infection, pigs that were being raised in four swine farms (farms A, C, D, and E) were tested for anti-HEV. Although 96 (86%) of the 112 pigs from farm C were positive for anti-HEV, none of the 48 pigs in farm A and 138 miniature pigs in farms D and E was positive for anti-HEV. CONCLUSIONS: These results suggest that three of the five swine farms tested were free from HEV, and that periodic testing for anti-HEV and HEV RNA of pigs used as experimental animals and pigs raised in swine farms from which pigs are purchased, is useful for providing HEV-free pigs to researchers who are engaged in studies using pigs.
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The hemodynamic effects of acute myocardial ischemia and reperfusion in Clawn miniature pigs.
Exp Anim. 2003 Jul;52(4):335-8.
Kamimura R, Miura N, Suzuki S.
Division of Laboratory Animal Science, Research Center for Life Science Resources, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
Acute myocardial ischemia was induced by occluding the LAD in Clawn miniature pigs. Eight pigs (group 1) were subjected to 6 h ischemia and nine pigs (group 2) were subjected to 20 min ischemia, followed by reperfusion for 340 min. Three animals of the group 1 died due to ventricular fibrillation after occlusion and in group 2, four animals died due to the arrhythmia after reperfusion. Though the ischemic area of group 2 (15.6% of the ventricle) was narrower than that of group 1 (21.7%), the survival rate was lower. We supposed that ischemia-reperfusion injuries were strongly connected with the hemodynamics of group 2. Clawn miniature pigs are useful experimental animals for myocardial ischemic researches.
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Branching patterns in coronary artery and ischemic areas induced by coronary arterial occlusion in the CLAWN miniature pig.
Exp Anim. 1996 Apr;45(2):149-53.
Kamimura R, Suzuki S, Nozaki S, Sakamoto H, Maruno H, Kawaida H.
Institute of Laboratory Animal Sciences, Faculty of Medicine, Kagoshima University, Japan.
This study of 28 CLAWN miniature pigs (male 17, female 11, mean weight 29 kg) was undertaken to investigate the coronary arterial branching patterns and the ischemic area induced by surgical occlusion of the left anterior descending artery (LAD) and change in the ischemic area over time. These results were compared with those in dogs, which have frequently been used in myocardial ischemic research. Regarding the coronary arterial branching pattern, there were fewer ventricular branches from the right and left coronary arteries than in dogs. The septal branches arose from only the LAD and the posterior descending artery (PD). The largest septal artery branched from the LAD. There were two types of septal artery branching patterns. In approximately 80% of the CLAWN miniature pigs, the PD arose from the right coronary artery (Right dominance). The peculiarity of the coronary arterial branching pattern in the CLAWN miniature pigs was more similar to human beings than to dogs. The ischemic area induced by occlusion at three-fifths distal section of the LAD was 12.1% to 22.6% (mean 17.1%) of the left ventricle. The ischemic area in all animals that died of global left ventricular malfunction and hemodynamic instability after LAD occlusion was more than 25% of the left ventricle.
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ミニブタ心移植におけるMHCクラスII適合の重要性と免疫抑制剤短期投与の効果
移植, 39(5) : 542-548, 2004.
大津義徳1,3, 久 容輔1, 岩永健裕1, 亀澤康裕1, 矢澤 肇2, 天野 純3, 清水 章4, 佐田正晴1, 山田和彦1
1国立循環器病センター研究所再生医療部, 2日本生物科学研究所, 3信州大学心臓血管外科, 4日本医科大学第1病理
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Navigator-echo-based MR provides high-resolution images and precise volumetry of swine livers without breath holding or injection of contrast media.
Liver Transpl. 2006 Jan;12(1):72-7.
Kim ID, Azuma T, Ido A, Moriuchi A, Numata M, Teramukai S, Okamoto J, Tsutsumi S, Tanaka K, Tsubouchi H.
Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan. ildeok@kuhp.kyoto-u.ac.jp
The accurate calculation of hepatic volume by computed tomography (CT) or magnetic resonance (MR) is complicated by the need for breath holding and the injection of contrast media. These are often contraindicated in patients with liver failure, and we examined the ability of unenhanced 3-dimensional (3-D) navigator-echo-based MR (NE-MR) to accurately image livers and measure volumes without breath holding compared to unenhanced (plain) or gadolinium-diethylene triamine pentaacetic acid enhanced MR (Gd-MR) in miniature swine (n = 8). Without breath holding, diaphragm movement monitoring with NE-MR reduced motion artifacts in hepatic images compared with the other modalities. Without the injection of contrast media, the signal-to-noise ratios of the images obtained using NE-MR were significantly higher than those from plain MR; Gd-MR was superior to NE-MR, however (79.5 +/- 7.5 vs. 63.2 +/- 6.0 or 97.8 +/- 8.1, respectively; P < 0.01 for each). Overall, NE-MR produced improved high-resolution liver images. Consequently, liver volumes calculated based on NE-MR images were more highly correlated with actual liver weights compared to plain or Gd-MR in the whole livers (n = 8; r = 0.937 vs. 0.835 or 0.904, respectively). Also, NE-MR demonstrated significantly strong correlation between actual weights and volumetry-calculated volumes in regenerative livers 7 days after massive hepatectomy (n = 10, r = 0.989, P < 0.01). In conclusion, our results indicate that without breath holding or the injection of contrast media, 3-D NE-MR can provide both high-resolution liver images and precise hepatic volumes in patients with liver failure due to liver surgery (massive hepatectomy and living donor liver transplantation) or fulminant hepatic failure. Copyright 2005 AASLD
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Characteristics and gender differences concerning pulmonary hemodynamics in Clawn miniature pigs.
Exp Anim. 2007 Oct;56(5):375-8.
Kamimura R, Setoyama K, Yabuki A, Tottori J, Suzuki S.
Frontier Science Research Center, Kagoshima University, Japan.
The characteristics and gender differences of the pulmonary hemodynamic parameters of 16 Clawn miniature pigs were examined and the data were compared with reports concerning dogs and other pig species. The pulmonary systolic, diastolic and mean arterial blood pressures of the mini-pig were slightly higher than those of the dog, respectively, but both the right atrial pressure and pulmonary capillary wedge pressure were within the normal physiological ranges of the dog. Concerning gender differences in hemodynamic parameters of the mini-pig, the female values, except the right atrial pressure, were slightly higher than those of the male, but no significant differences were recognized. The present study results will help pulmonary researchers understand the differences between Clawn miniature pigs and dogs for accurate analysis of experimental results.
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Skin morphology of the Clawn miniature pig.
Exp Anim. 2007 Oct;56(5):369-73.
Yabuki A, Kamimura R, Setoyama K, Tottori J, Taniguchi K, Matsumoto M, Suzuki S.
Laboratory of Veterinary Anatomy, Department of Veterinary Medicine, Kagoshima University, Japan.
Skin morphology of the Clawn miniature pig (CMP) was investigated at the axilla, medial thigh, back and loin. The mean thickness of the epidermis (excluding the corneal layer), the mean number of layers of keratinocytes comprising the epidermis and the mean height of keratinocytes were assessed morphometrically. When observed under a light microscope, the skin of the CMP resembled human skin. Morphometrically, skin from the back and loin of the CMP most resembles human skin. Electron microscopic observations revealed sparse but typical Birbeck granules in the epidermal Langerhans cells of the CMP. The results of the present study indicate that CMP skin is potentially useful as a model for human skin.
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Establishment of cell lines that exhibit pluripotency from miniature swine periodontal ligaments.
Arch Oral Biol. 2007 Oct;52(10):1002-8. Epub 2007 Jun 1.
Ibi M, Ishisaki A, Yamamoto M, Wada S, Kozakai T, Nakashima A, Iida J, Takao S, Izumi Y, Yokoyama A, Tamura M.
Department of Oral Biochemistry and Molecular Biology, Graduate School of Dental Medicine, Hokkaido University, North 13, West 7, Sapporo 060-8586, Japan.
Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.
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TRAV gene usage in pig T-cell receptor alpha cDNA.
Immunogenetics. 2005 May;57(3-4):219-25. Epub 2005 Apr 5.
Yamamoto R, Uenishi H, Hatsuse H, Sato E, Awata T, Yasue H, Takagaki Y.
Genome Research Department, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, 305-8602, Japan.
Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.
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