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Publications (6)7.47 Total impact

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    ABSTRACT: Background: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. Method and results: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). Conclusions: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.
    Xenotransplantation 04/2013; 20(3). DOI:10.1111/xen.12031 · 2.84 Impact Factor
  • E Kim · J-S Kim · Y Lee · B-S Song · B-W Sim · S-U Kim · T Saitoh · H Yazawa · T Nunoya · K-T Chang ·
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    ABSTRACT: IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions.
    Reproduction in Domestic Animals 05/2012; 48(1). DOI:10.1111/j.1439-0531.2012.02037.x · 1.52 Impact Factor
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    ABSTRACT: Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background.
    Journal of Reproduction and Development 07/2008; 54(3):156-63. DOI:10.1262/jrd.19165 · 1.52 Impact Factor
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    ABSTRACT: The pigs have been a well-recognized experimental animal in biomedical research for many years, Minipigs particularly have gained in massive importance in biomedical research over the last few years. Pigs are increasingly being used as an alternative non-rodent animal species to the dog or monkey in toxicology because of the morphological and physiological similarities between porcine and human organs, especially the skin, cardiovascular system, gastrointestinal tract, and the urinary system. Accumulating data indicate that the minipig can be used for all routes of administration and is preferable to the dog or monkey in many cases. The advantages of the minipig compared to the domestic pig are its smaller size, even at full maturity, slower growth during studies, ease of handling, and controlled genotype as well as microbiologically obvious characteristics. Minipigs also have an advantage over traditional non-rodent animals because of increasing ethical concerns about the use of them in experiments. Reservoir of information from studies using minipigs is the keystone for the future diffusion of them as a good alternative to the non-rodent animals traditionally used in toxicology.
    Journal of Toxicologic Pathology 10/2007; 20(3):125-132. DOI:10.1293/tox.20.125 · 0.53 Impact Factor
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    ABSTRACT: Utility of Japanese White rabbit mutants with hypotrichosis (htr strain) was investigated in dermal toxicity studies. In the primary irritation study, the primary irritation indices (P.I.I.) by application of hexachlorophene and 3, 3′, 4′, 5-tetrachlorosalicylanilide (TCSA) were higher in the htr rabbits than in the control, haired rabbits. The P.I.I. by application of Tween 80 and sodium lauryl sulfate (SLS) were similar between the htr rabbits and the control rabbits. In the 16-day cumulative irritation study, irritation scores induced by SLS and sodium hydroxide were lower in the htr rabbits than in the control rabbits throughout the observation periods. However, approximately 30% of the dorsal skin of the control rabbits underwent the anagen phase of the hair cycle during the cumulative periods, resulting in difficulty to apply the chemicals and to estimate the skin reactions. In the phototoxicity study, irritation scores of the htr rabbits treated with 8-methoxypsoralen and TCSA were similar to those of the control rabbits. These results revealed advantages of the htr rabbits in the administration of test chemicals and the evaluation of skin reactions, suggesting the usefulness of htr rabbits for dermal toxicity studies.
    Journal of Toxicologic Pathology 01/2002; 15(3). DOI:10.1293/tox.15.153 · 0.53 Impact Factor
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    ABSTRACT: Rabit mutants with hypotrichosis were found in a colony of Japanese White strain (JW-NIBS) and descendants of the mutants have been successively produced. By genetic analyses, the hypotrichotic condition was inherited by a single autosomal recessive gene, symbolized as htr. The trunk of the htr rabbit did not grow hairs and was covered with a few, fine, soft and short hairs throughout life with the exception of wavy hairs around the nose and extremities. Histologic examination of the skin from htr rabbits revealed thickening of the epidermis, hyperplasia of the sebaceous gland, and dysplastic development of the large guard and small wool hairs. When immunostained with anti AE1 and AE3 antibodies, there were no significant differences in the keratin constituents of the skin between the htr rabbits and age-matched controls. While proliferating cell nuclear antigen (PCNA)-positive cells were numerous in the basal layer of epidermis, external root sheaths, and hair bulbs, apoptotic cells were also increased in the hair bulbs of htr rabbits. There were no significant differences in the number of T and B lymphocytes in the mesenteric lymph nodes between htr and control rabbits.
    Journal of Toxicologic Pathology 01/2000; 13(1):21-27. DOI:10.1293/tox.13.21 · 0.53 Impact Factor