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Sample records for bovine nuclear transfer

  1. Bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  2. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kaňka, Jiří; Smith, Steven Dale; Soloy, Eva

    1999-01-01

    (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  3. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  4. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

    DEFF Research Database (Denmark)

    Booth, P J; VIUFF, D; THOMSEN, P D;

    2000-01-01

    comprised mainly triploid (8.2 6 10.3 [0–26.3]: SD [range]) and tetraploid (10.6 6 19.9 [0–54.9]) nuclei with other ploidy com- binations accounting for only 0.9 6 2.1 [0–2.1]% of deviant nuclei. The proportion of com- pletely normal nuclear transfer embryos was no less than those produced by in vitro...

  5. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen.

    Science.gov (United States)

    Liu, Jie; Westhusin, Mark; Long, Charles; Johnson, Gregory; Burghardt, Robert; Kraemer, Duane

    2010-12-01

    Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.

  6. Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J.; VIUFF, Dorte; Tan, Shijian;

    2002-01-01

    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona......-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential...... families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater...

  7. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde;

    2008-01-01

    the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy...... recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast...

  8. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique

    DEFF Research Database (Denmark)

    Booth, P J; Tan, S J; Reipurth, R

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods.......8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1 blastocysts per successfully fused and surviving reconstructed...... embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1 recipients pregnant at 5...

  9. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

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    F.D. Oliveira

    2015-12-01

    Full Text Available In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1 of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

  10. Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

    Science.gov (United States)

    Kim, Eun Young; Park, Min Jee; Park, Hyo Young; Noh, Eun Ji; Noh, Eun Hyung; Park, Kyoung Sik; Lee, Jun Beom; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2012-08-01

    In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

  11. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  12. In vitro development competence of bovine nuclear transfer embryos derived from Nanog-overexpressing fibroblast cells

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    Xi-bang Zheng, Yan Yun, Yong-ce Hu, Yong Li, Hua-yan Wang, Xiao-ling Ma, Jin-qiang Sui, An-min Lei and Zhong-ying Dou

    2014-04-01

    Full Text Available The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR, the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418. Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422 were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P<0.05 higher than in EGFP-Nanog expressing cells (82.14 vs 40.38 %, but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29% (P<0.05, indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.

  13. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts.

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    Wrenzycki, C; Wells, D; Herrmann, D; Miller, A; Oliver, J; Tervit, R; Niemann, H

    2001-07-01

    The successful production of embryos by nuclear transfer (NT) employing cultured somatic donor cells depends upon a variety of factors. The objective of the present study was to investigate the effects 1) of two different activation protocols, 2) the use of quiescent or nonquiescent donor cells (G(0) or G(1) of the cell cycle), and 3) passage number of donor cells on the relative abundance (RA) of eight specific mRNAs (DNA methyltransferase, DNMT; mammalian achaete-scute homologue, Mash2; glucose transporter-1, Glut-1; heat shock protein 70.1, Hsp; desmocollin II, Dc II; E-cadherin, E-cad; interferon tau, IF; insulin-like growth factor 2 receptor, Igf2r) in single blastocysts employing a semiquantitative reverse transcription-polymerase chain reaction assay. The results were compared with those for their in vitro (IVP)- and in vivo-generated noncloned counterparts. In experiment 1, employing either FBA (fusion before activation) or AFS (fusion and activation simultaneously) to generate NT blastocysts, Hsp mRNAs were not found in NT embryos from either protocol, whereas Hsp transcripts were detectable in IVP embryos. The relative abundance (RA) of IF transcripts was significantly increased in the AFS and IVP groups compared to the FBA treatment. In experiment 2, the use of either G(0) or G(1) donor cells to produce cloned embryos both significantly reduced the relative amount of DNMT transcripts and significantly increased the RA of Mash2 compared to the IVP embryos. In addition, IF transcript levels were significantly elevated in NT blastocysts employing G(1) donor cells for NT compared to IVP embryos and those generated using G(0) cells. In experiment 3, donor cells, either from passsage 5/6 or 8, were employed for NT. DNMT transcripts were significantly decreased, whereas Mash2 transcripts were significantly increased in both NT groups compared to their IVP counterparts. The amount of IF mRNA was significantly higher in P8-derived than in P5/6 and IVP embryos. In

  14. Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Booth, P J; Holm, P; Vajta, G;

    2001-01-01

    The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4......, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P ....7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P

  15. Gestaciones producidas con embriones bovinos clonados por transferencia nuclear Pregnancies produced by bovine embryos cloned by nuclear transfer

    OpenAIRE

    M A Martínez Díaz; R. Gatica; J E Correa; W Eyestone

    2007-01-01

    El presente estudio comunica la obtención de gestaciones de embriones clonados por transferencia nuclear de células somáticas en bovinos por primera vez en Chile. Ovocitos bovinos obtenidos de ovarios de matadero fueron madurados in vitro y enucleados por micromanipulación. Células donantes de núcleos fueron obtenidas de la oreja de una vaca adulta, cultivadas por 9-14 días y criopreservadas en nitrógeno líquido. Células somáticas confluentes fueron desagregadas e insertadas individualmente e...

  16. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    Science.gov (United States)

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  17. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...... the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed...... is completed toward the end of the 4th cell cycle. A substantial proportion of bovine embryos produced by nuclear transfer of embryonic or somatic cells to bovine ooplasts display aberrations in protein localization in one or more blastomers. This information is indicative of underlying aberrations in genomic...

  18. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...... is completed toward the end of the 4th cell cycle. A substantial proportion of bovine embryos produced by nuclear transfer of embryonic or somatic cells to bovine ooplasts display aberrations in protein localization in one or more blastomers. This information is indicative of underlying aberrations in genomic...

  19. Effect of culture time and gender of nuclei donor cells on bovine development produced by nuclear transfer

    OpenAIRE

    2010-01-01

    Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2) com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante se...

  20. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

  1. Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

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    Carolina Gonzales da Silva

    Full Text Available ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs that had not yet been tested for bovine embryo production by nuclear transfer (NT. Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+ were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%. Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

  2. Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors.

    Science.gov (United States)

    Gregg, K; Riddell, K P; Chen, S H; Galik, P K; Xiang, T; Guerra, T; Marley, M S; Polejaeva, I; Givens, M D

    2010-07-01

    The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

  3. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

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    Lee Rita SF

    2010-03-01

    Full Text Available Abstract Background Cloning of cattle by somatic cell nuclear transfer (SCNT is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI. Results Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1 showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. Conclusion These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a

  4. Nuclear donor cell lines considerably influence cloning efficiency and the incidence of large offspring syndrome in bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, J; Wang, Y; Su, J; Luo, Y; Quan, F; Zhang, Y

    2013-08-01

    Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.

  5. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  6. Nuclear transfer in rodents.

    Science.gov (United States)

    Mullins, Linda J; Wilmut, Ian; Mullins, John J

    2004-01-01

    Cloning is the asexual reproduction of an individual, such that the offspring have an essentially identical nuclear genome. Nuclear transfer and cloning have been achieved in a number of species, namely sheep, cows, goats, rabbits, cats and mice, but have been largely unsuccessful, so far, in dogs, primates and rats. Clearly, contributory factors which affect the outcome of successful cloning experiments are not universally applicable to all species. One theme common to all cloning experiments, however, is the overall inefficiency of the process, typically 0-4%. A number of factors contribute to nuclear transfer inefficiency, and we will review mouse cloning experiments, which address these problems, highlighting the importance of donor nucleus choice (somatic or ES cell, fetal or adult, quiescent or actively dividing). Finally, we will summarize the emerging principles which appear to govern nuclear reprogramming and production of clones, and will consider the application of nuclear transfer to the rat.

  7. Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies.

    Science.gov (United States)

    Ravelich, S R; Shelling, A N; Wells, D N; Peterson, A J; Lee, R S F; Ramachandran, A; Keelan, J A

    2006-01-01

    Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

  8. Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

    Science.gov (United States)

    Bressan, F F; Miranda, M S; Bajgelman, M C; Perecin, F; Mesquita, L G; Fantinato-Neto, P; Merighe, G F K; Strauss, B E; Meirelles, F V

    2013-04-01

    Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed

  9. Cloning in cattle: from embryo splitting to somatic nuclear transfer.

    Science.gov (United States)

    Heyman, Y; Vignon, X; Chesné, P; Le Bourhis, D; Marchal, J; Renard, J P

    1998-01-01

    The ability to obtain genetically identical offspring in cattle (clones) is useful for research and for potential applications to breeding schemes. Experimental possibilities for generating such animals have evolved considerably in the last two decades. Embryo splitting has become a relatively simple technique but is limited to twinning. Embryonic nuclear transfer has improved and is associated with sexing to generate sets of clones despite a great variability of results between parent embryos. The factors of progress are reviewed here. Recently, somatic cells used as a source of nuclei in bovine nuclear transfer has been demonstrated. Here we present the results of the developmental potential of nuclei from skin and muscle cells.

  10. Nuclear transfer to study the nuclear reprogramming of human stem cells.

    Science.gov (United States)

    Saito, Shigeo; Sawai, Ken; Murayama, Yoshinobu; Fukuda, Keiichi; Yokoyama, Kazunari

    2008-01-01

    Research of stem cells will enable us to understand the development and function of tissues and organs in mammals. The ability to induce regeneration of new tissues from embryonic stem (ES) cells derived from cloned blastocysts via nuclear transfer can be expected in the not-too-distant future. The fact that there is no way except nuclear cloning for the return of differentiated cells to undifferentiated cells remains an interesting problem to be solved. We describe protocols for the production of cloned calves from bovine ES cells to study nuclear reprogramming ability of stem cells. The frequency of term pregnancies for blastocysts from ES cells is higher than those of early pregnancies and maintained pregnancies after nuclear transfer with bovine somatic cells. We also describe protocols for gene introduction into bovine ES cells in vitro, particularly the human leukocyte antigens (HLA). Bovine ES cells provide a powerful tool for the generation of transgenic clonal offspring. This technique, when perfected for humans, may be critical for neural stem cell transplantation.

  11. Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear Effect of culture time and gender of nuclei donor cells on bovine development produced by nuclear transfer

    Directory of Open Access Journals (Sweden)

    Giovana Krempel Fonseca Merighe

    2010-10-01

    Full Text Available Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2 com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante sete dias, e transferidos para receptoras sincronizadas. As taxas de clivagem e desenvolvimento a blastocisto de embriões reconstruídos com células cultivadas por tempo maior foram inferiores às obtidas com os demais tempos de cultivo. Além disso, os blastocistos produzidos não resultaram no desenvolvimento de uma gestação a termo. Embora a taxa de clivagem em embriões fêmeas tenha sido maior, o número de embriões que atingiram o estádio de blastocisto foi maior nos embriões machos. No período gestacional, fêmeas apresentaram maior taxa de aborto entre 90 e 120 dias de gestação. Esses resultados indicam que células doadoras de núcleos cultivados por longos períodos dificultam a produção de blastocistos e aumentam as chances de perdas durante a gestação. Embriões clonados machos têm maior competência para se desenvolver a blastocisto e resultam em menor taxa de perda gestacional.The objective of this study was to evaluate the effects of culture time and sex of nuclei donor cells on embryo and fetal development after nuclear transfer. Thus, bovine oocytes were matured, enucleated and reconstructed with somatic cells from an adult animal. After fusion and chemical activation, the reconstituted zygotes were cultured in Charles Rosenkranz 2 (CR2 on a granular monolayer cell at 38.8ºC in a humidified atmosphere 5% CO2 in air for seven days, and transferred to synchronized receptors. Cleavage rates and development to

  12. Nuclear transfer in nonhuman primates.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Wolf, Don P

    2006-01-01

    The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman

  13. Epigenetic reprogramming in mammalian nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LI Shijie; DU Weihua; LI Ning

    2004-01-01

    Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embryonic ground state. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. This review summarizes the roles of various epigenetic mechanisms, including DNA methylation, histone acetylation, imprinting, X-chromosome inactivation, telomere maintenance and expressions of development-related genes on somatic nuclear transfer.

  14. Dry Transfer Systems for Used Nuclear Fuel

    Energy Technology Data Exchange (ETDEWEB)

    Brett W. Carlsen; Michaele BradyRaap

    2012-05-01

    The potential need for a dry transfer system (DTS) to enable retrieval of used nuclear fuel (UNF) for inspection or repackaging will increase as the duration and quantity of fuel in dry storage increases. This report explores the uses for a DTS, identifies associated general functional requirements, and reviews existing and proposed systems that currently perform dry fuel transfers. The focus of this paper is on the need for a DTS to enable transfer of bare fuel assemblies. Dry transfer systems for UNF canisters are currently available and in use for transferring loaded canisters between the drying station and storage and transportation casks.

  15. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  16. Cloned calves produced by nuclear transfer from cultured cumulus cells

    Institute of Scientific and Technical Information of China (English)

    AN; Xiaorong(安晓荣); GOU; Kemian(苟克勉); ZHU; Shien(朱士恩); GUAN; Hong(关宏); HOU; Jian(侯健); LIN; Aixing(林爱星); ZENG; Shenming(曾申明); TIAN; Jianhui(田见辉); CHEN; Yongfu(陈永福)

    2002-01-01

    Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

  17. The influence of interspecies somatic cell nuclear transfer on epigenetic enzymes transcription in early embryos

    Directory of Open Access Journals (Sweden)

    Martin Morovic

    2016-10-01

    Full Text Available One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a genes in early embryonic stages of interspecies (bovine, porcine nuclear transfer embryos (iSCNT by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly infl uenced by the ooplasmic environment.

  18. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...... (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures...

  19. Sheep: The First Large Animal Model in Nuclear Transfer Research

    OpenAIRE

    Loi, Pasqualino; Czernik, Marta; Zacchini, Federica; Iuso, Domenico; Scapolo, Pier Augusto; Ptak, Grazyna

    2013-01-01

    The scope of this article is not to provide an exhaustive review of nuclear transfer research, because many authoritative reviews exist on the biological issues related to somatic and embryonic cell nuclear transfer. We shall instead provide an overview on the work done specifically on sheep and the value of this work on the greater nuclear transfer landscape.

  20. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  1. Application of the zona-free manipulation technique to porcine somatic nuclear transfer

    DEFF Research Database (Denmark)

    Booth, P J; Tan, S J; Holm, P

    2001-01-01

    The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable...... 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem...

  2. Conjugative transfer of resistance determinants among human and bovine Streptococcus agalactiae.

    Science.gov (United States)

    Pinto, Tatiana Castro Abreu; Costa, Natália Silva; Corrêa, Ana Beatriz de Almeida; de Oliveira, Ivi Cristina Menezes; de Mattos, Marcos Correa; Rosado, Alexandre Soares; Benchetrit, Leslie Claude

    2014-01-01

    Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10(-7) and 9.10(-7), was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.

  3. Nuclear transfer technology in mammalian cloning.

    Science.gov (United States)

    Wolf, D P; Mitalipov, S; Norgren, R B

    2001-01-01

    The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

  4. 体细胞核移植技术生产转入溶菌酶基因(hLYZ)牛胚胎的研究%Human Lysozyme Gene (hLYZ) Transgenic Bovine Embryos Produced by Somatic Cell Nuclear Transfer

    Institute of Scientific and Technical Information of China (English)

    熊显荣; 李文哲; 王丽君; 王勇胜; 苏建民; 华松; 张涌

    2011-01-01

    The aim of this study was to investigate the effect of different sizes and treatments of donor cells on the in vitro development of bovine embryos after genetic modification and selection, so as to establish an effective system for production of transgenic bovine embryos.Bovine fetal fibroblasts were transfeected with the recombinant plasmid of mammary gland specific expression vector pEBH which containing human lysozyme (hLYZ) gene.Transgenic positive cells which were obtained through G418 selection were used as donor cells for production of transgenic cloned embryos.The result showed that genetic modification and screening of donor cells were harmful for the development of transgenic cloned embryos, the blastocyst rate decreased significantly;referred to internal diameter of injective needle, cells with the diameter of 15~20 μm were selected and used as nuclear transfer donor cells, the fusion rate and blastocyst rate of transgenic cloned embryos were significant higher than that of the other groups (P < 0.05); Compared with serum starvation and contact restrain groups, the cleavage rate and blastocyst rate of transgenic cloned embryos which were constructed with plant cells were significantly higher (P < 0.05); The expression of GFP was observed in each stage of in vitro development in all transgenic cloned embryos, but the expression levels seemed to be vary among individuals and even among different developmental stages of the same individual.We chose I 0 embroys randomly for PCR detection and found that all of the embroys were positive.Above all, we obtained hLYZ transgenic cloned embryos by somatic cell nuclear transfer technique, the reconstructed embryos can develop to blastocysts successfully; and when plant cells with diameter of 15~20 μm were used as donor cell, the developmental competence of somatic cell cloned embryos is improved significantly.%本研究主要探讨经基因修饰和筛选后转基因供体细胞的挑选及处理对牛转

  5. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

    DEFF Research Database (Denmark)

    Booth, P J; VIUFF, D; THOMSEN, P D;

    2000-01-01

    .2 (mean SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8 were potentially abnormal because in these, 1.1 1.4 5.3 7.5 26.3 30.4 and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors...

  6. Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; LI Rong; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Haiping; WANG Lili; FANG Changge; WAN Rong; LIU Ying

    2004-01-01

    The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

  7. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  8. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    Directory of Open Access Journals (Sweden)

    Julieta N Caballero

    Full Text Available Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  9. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    Science.gov (United States)

    Caballero, Julieta N; Frenette, Gilles; Belleannée, Clémence; Sullivan, Robert

    2013-01-01

    Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  10. Horizontal gene transfers link a human MRSA pathogen to contagious bovine mastitis bacteria.

    Directory of Open Access Journals (Sweden)

    Thomas Brody

    Full Text Available BACKGROUND: Acquisition of virulence factors and antibiotic resistance by many clinically important bacteria can be traced to horizontal gene transfer (HGT between related or evolutionarily distant microflora. Comparative genomic analysis has become an important tool for identifying HGT DNA in emerging pathogens. We have adapted the multi-genome alignment tool EvoPrinter to facilitate discovery of HGT DNA sequences within bacterial genomes and within their mobile genetic elements. PRINCIPAL FINDINGS: EvoPrinter analysis of 13 different Staphylococcus aureus genomes revealed that one of the human isolates, the hospital epidemic methicillin-resistant MRSA252 strain, uniquely shares multiple putative HGT DNA sequences with different causative agents of bovine mastitis that are not found in the other human S. aureus isolates. MRSA252 shares over 14 different DNA sequence blocks with the bovine mastitis ET3 S. aureus strain RF122, and many of the HGT DNAs encode virulence factors. EvoPrinter analysis of the MRSA252 chromosome also uncovered virulence-factor encoding HGT events with the genome of Listeria monocytogenes and a Staphylococcus saprophyticus associated plasmid. Both bacteria are also causal agents of contagious bovine mastitis. CONCLUSIONS: EvoPrinter analysis reveals that the human MRSA252 strain uniquely shares multiple DNA sequence blocks with different causative agents of bovine mastitis, suggesting that HGT events may be occurring between these pathogens. These findings have important implications with regard to animal husbandry practices that inadvertently enhance the contact of human and livestock bacterial pathogens.

  11. Two-staged nuclear transfer can enhance the developmental ability of goat-sheep interspecies nuclear transfer embryos in vitro.

    Science.gov (United States)

    Ma, Li-Bing; Cai, Lu; Li, Jia-Jia; Chen, Xiu-Li; Ji, Feng-Yu

    2011-02-01

    The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.

  12. Recent Progress of Somatic Cell Nuclear Transfer in Pigs

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaoming; Dou Zhongying

    2005-01-01

    Research in the field of somatic cell nuclear transfer (SCNT) and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low (<1% ). Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

  13. A new MV bus transfer scheme for nuclear power plants

    Directory of Open Access Journals (Sweden)

    Chang Choong-Koo

    2015-01-01

    Full Text Available Fast bus transfer method is the most popular and residual voltage transfer method that is used as a backup in medium voltage buses in general. The use of the advanced technology like open circuit voltage prediction and digital signal processing algorithms can improve the reliability of fast transfer scheme. However, according to the survey results of the recent operation records in nuclear power plants, there were many instances where the fast transfer scheme has failed. To assure bus transfer in any conditions and circumstances, uninterruptible bus transfer scheme utilizing the state of the art medium voltage UPS is discussed and elaborated.

  14. First-principles molecular dynamics study of proton transfer mechanism in bovine cytochrome c oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Katsumasa [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Boero, Mauro [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Tateno, Masaru [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Shiraishi, Kenji [CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Oshiyama, Atsushi [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan)

    2007-09-12

    Density functional based first-principles molecular dynamics calculations, performed on a model system extracted from the bovine cytochrome c oxidase, have been performed in an attempt to inspect the proton transfer mechanism across a peptide group. Our model system includes the specific Tyr440-Ser441 peptide group involved in a novel proton transfer path and shows that the Y440-S441 enol peptide group [-C(OH) = N-], which is a structural isomer of a keto form [-CO-NH-], is the product of the deprotonation of an imidic acid [-C(OH)-NH-] occurring in the vicinity of the deprotonated aspartic acid residue. For the subsequent enol-to-keto tautomerization, a direct H{sup +} transfer path in the Y440-S441 peptide group has been identified, in which the transition state takes a distorted four-membered ring structure.

  15. Electronic and Nuclear Factors in Charge and Excitation Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Piotr Piotrowiak

    2004-09-28

    We report the and/or state of several subprojects of our DOE sponsored research on Electronic and Nuclear Factors in Electron and Excitation Transfer: (1) Construction of an ultrafast Ti:sapphire amplifier. (2) Mediation of electronic interactions in host-guest molecules. (3) Theoretical models of electrolytes in weakly polar media. (4) Symmetry effects in intramolecular excitation transfer.

  16. Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos.

    Science.gov (United States)

    Popken, Jens; Brero, Alessandro; Koehler, Daniela; Schmid, Volker J; Strauss, Axel; Wuensch, Annegret; Guengoer, Tuna; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

    2014-01-01

    Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.

  17. Heat transfer and fluid flow in nuclear systems

    CERN Document Server

    Fenech, Henri

    1982-01-01

    Heat Transfer and Fluid in Flow Nuclear Systems discusses topics that bridge the gap between the fundamental principles and the designed practices. The book is comprised of six chapters that cover analysis of the predicting thermal-hydraulics performance of large nuclear reactors and associated heat-exchangers or steam generators of various nuclear systems. Chapter 1 tackles the general considerations on thermal design and performance requirements of nuclear reactor cores. The second chapter deals with pressurized subcooled light water systems, and the third chapter covers boiling water reacto

  18. Transfer factors for nuclear emergency preparedness

    Energy Technology Data Exchange (ETDEWEB)

    Kostiainen, E.; Haenninen, R. [Radiation and Nuclear Safety Authority (STUK) (Finland); Rosen, K.; Haak, E.; Eriksson, Aa. [Swedish Univ. of Agricultural Science (Sweden); Nielsen, S.P.; Keith-Roach, M. [Risoe National Lab. (Denmark); Salbu, B. [Agricultural Univ. of Norway (Norway)

    2002-12-01

    This report by the NKS/BOK-1.4 project subgroup describes transfer factors for radiocaesium and radiostrontium for the fallout year and the years after the fallout. The intention has been to collect information on tools to assess the order of magnitude of radioactive contamination of agricultural products in an emergency situation in Nordic environment. The report describes transfer paths from fallout to plant, from soil to plant and to animal products. The transfer factors of radionuclides (Sr, Cs, I) given in the report are intended to be used for making rough estimates of the contamination of agricultural products soon after the heaviness and composition of the deposition (Bq m{sup -2}) is known. (au)

  19. Factors that affect the reproductive efficiency of the recipient within a bovine embryo transfer program

    Directory of Open Access Journals (Sweden)

    Arturo Duica A.

    2007-12-01

    Full Text Available The embryo transfer is a biotechnological technique that allows increasing the descendant of animals with high genetic value. The positive results, represented in pregnancy after the application of this technique, are affected by some factors that are inherent to the donor, the embryo, the technique, and the recipients which receive a strange embryo in the uterus allowing pregnancy. This review describes some factors affecting the reproductive efficiency of the recipients of bovine embryos within a program of embryo transfer. Its important to evaluate the parameters in this kind of recipients, as race, age, physiological status, health status, weight, reproductive tract integrity and management, and also too monitoring the ovarian structures while the estrus synchronization, and within previous and posterior stages in embryo transfer procedure. Therefore an optimum follicular development will be determinant to corpus luteum formation which generates enough serum progesterone concentrations to offer a right uterine environment allowing the optimum embryo development. Controlling the factors that affect the efficiency of the embryo transfer, it will obtain an increasing of positive results represented in pregnancies and births of individuals come from animals with high genetic value.

  20. 1H-NMR and photochemically-induced dynamic nuclear polarization studies on bovine pancreatic phospholipase A2

    NARCIS (Netherlands)

    Egmond, M.R.; Slotboom, A.J.; Haas, G.H. de; Dijkstra, Klaas; Kaptein, R.

    1980-01-01

    Proton-NMR resonances of trytophan 3 and tyrosine 69 in bovine pancreatic phospholipase A2, its pro-enzyme and in Ala1-transaminated protein were assigned using photochemically-induced dynamic nuclear polarization (photo-CIDNP) as such or in combination with spin-echo measurements. In addition assig

  1. Somatic cell nuclear transfer in horses.

    Science.gov (United States)

    Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna

    2008-07-01

    The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.

  2. Human somatic cell nuclear transfer is alive and well.

    Science.gov (United States)

    Cibelli, Jose B

    2014-06-05

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well.

  3. Nuclear Waste Cross Site Transfer Pump Operational Resonance Resolution

    Energy Technology Data Exchange (ETDEWEB)

    HAUCK, F.M.

    1999-12-01

    Two single-volute, multi-stage centrifugal pumps are installed at a nuclear waste transfer station operated by the Department of Energy in Hanford, WA. The two parallel 100% pumps are Variable Frequency Drive operated and designed to transport waste etc.

  4. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  5. Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer em- bryos, the enucleated MⅡ oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse embryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (single nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zygote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were obtained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse embryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the development rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM MⅡ oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

  6. Electron-Nuclear Spin Transfer in Triple Quantum Dot Networks

    Science.gov (United States)

    Prada, Marta; Toonen, Ryan; Harrison, Paul

    2005-03-01

    We investigate the conductance spectra of coupled quantum dots to study systematically the nuclear spin relaxation of delta- and y-junction networks and observe spin blockade dependence on the electronic configurations. We derive the conductance using the Beenakker approach generalised to an array of quantum dots where we consider the nuclear spin transfer to electrons by hyperfine coupling. This allows us to predict the relevant memory effects on the different electronic states by studying the evolution of the single electron resonances in presence of nuclear spin relaxation. We find that the gradual depolarisation of the nuclear system is imprinted in the conductance spectra of the multidot system. Our calculations of the temporal evolution of the conductance resonance reveal that spin blockade can be lifted by hyperfine coupling.

  7. Electron nuclear spin transfer in quantum-dot networks

    Science.gov (United States)

    Prada, M.; Toonen, R. C.; Blick, R. H.; Harrison, P.

    2005-05-01

    We investigate the conductance spectra of coupled quantum dots to study systematically the nuclear spin relaxation of different geometries of a two-dimensional network of quantum dots and observe spin blockade dependence on the electronic configurations. We derive the conductance using the Beenakker approach generalized to an array of quantum dots where we consider the nuclear spin transfer to electrons by hyperfine coupling. This allows us to predict the relevant memory effects on the different electronic states by studying the evolution of the single electron resonances in the presence of nuclear spin relaxation. We find that the gradual depolarization of the nuclear system is imprinted in the conductance spectra of the multidot system. Our calculations of the temporal evolution of the conductance resonance reveal that spin blockade can be lifted by hyperfine coupling.

  8. The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

    Science.gov (United States)

    Rim, Chol Ho; Fu, Zhixin; Bao, Lei; Chen, Haide; Zhang, Dan; Luo, Qiong; Ri, Hak Chol; Huang, Hefeng; Luan, Zhidong; Zhang, Yan; Cui, Chun; Xiao, Lei; Jong, Ui Myong

    2013-12-01

    To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, pcloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

  9. Technological Transfer from Research Nuclear Reactors to New Generation Nuclear Power Reactors

    Science.gov (United States)

    Radulescu, Laura; Pavelescu, Margarit

    2010-01-01

    The goal of this paper is the analysis of the technological transfer role in the nuclear field, with particular emphasis on nuclear reactors domain. The presentation is sustained by historical arguments. In this frame, it is very important to start with the achievements of the first nuclear systems, for instant those with natural uranium as fuel and heavy water as moderator, following in time through the history until the New Generation Nuclear Power Reactors. Starting with 1940, the accelerated development of the industry has implied the increase of the global demand for energy. In this respect, the nuclear energy could play an important role, being essentially an unlimited source of energy. However, the nuclear option faces the challenges of increasingly demanding safety requirements, economic competitiveness and public acceptance. Worldwide, a significant amount of experience has been accumulated during development, licensing, construction, and operation of nuclear power reactors. The experience gained is a strong basis for further improvements. Actually, the nuclear programs of many countries are addressing the development of advanced reactors, which are intended to have better economics, higher reliability, improved safety, and proliferation-resistant characteristics in order to overcome the current concerns about nuclear power. Advanced reactors, now under development, may help to meet the demand for energy power of both developed and developing countries as well as for district heating, desalination and for process heat. The paper gives historical examples that illustrate the steps pursued from first research nuclear reactors to present advanced power reactors. Emphasis was laid upon the fact that the progress is due to the great discoveries of the nuclear scientists using the technological transfer.

  10. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep

    OpenAIRE

    Evans, Matthew J.; Gurer, Cagan; Loike, John D.; Wilmut, Ian; Schnieke, Angelika E.; Schon, Eric A.

    1999-01-01

    Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals1-6 typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte7,8. Thus, ...

  11. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  12. Nuclear Effects in Neutrino Interactions at Low Momentum Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Miltenberger, Ethan Ryan [Univ. of Minnesota, Minneapolis, MN (United States)

    2015-05-01

    This is a study to identify predicted effects of the carbon nucleus environment on neutrino - nucleus interactions with low momentum transfer. A large sample of neutrino interaction data collected by the MINERvA experiment is analyzed to show the distribution of charged hadron energy in a region with low momentum transfer. These distributions reveal a major discrepancy between the data and a popular interaction model with only the simplest Fermi gas nuclear effects. Detailed analysis of systematic uncertainties due to energy scale and resolution can account for only a little of the discrepancy. Two additional nuclear model effects, a suppression/screening effect (RPA), and the addition of a meson exchange current process (MEC), are shown to improve the description of the data.

  13. Heat Transfer Modeling of Dry Spent Nuclear Fuel Storage Facilities

    Energy Technology Data Exchange (ETDEWEB)

    Lee, S.Y.

    1999-01-13

    The present work was undertaken to provide heat transfer model that accurately predicts the thermal performance of dry spent nuclear fuel storage facilities. One of the storage configurations being considered for DOE Aluminum-clad Spent Nuclear Fuel (Al-SNF), such as the Material and Testing Reactor (MTR) fuel, is in a dry storage facility. To support design studies of storage options a computational and experimental program has been conducted at the Savannah River Site (SRS). The main objective is to develop heat transfer models including natural convection effects internal to an interim dry storage canister and to geological codisposal Waste Package (WP). Calculated temperatures will be used to demonstrate engineering viability of a dry storage option in enclosed interim storage and geological repository WP and to assess the chemical and physical behaviors of the Al-SNF in the dry storage facilities. The current paper describes the modeling approaches and presents the computational results along with the experimental data.

  14. Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE).

    Science.gov (United States)

    Wrathall, A E; Brown, K F D; Sayers, A R; Wells, G A H; Simmons, M M; Farrelly, S S J; Bellerby, P; Squirrell, J; Spencer, Y I; Wells, M; Stack, M J; Bastiman, B; Pullar, D; Scatcherd, J; Heasman, L; Parker, J; Hannam, D A R; Helliwell, D W; Chree, A; Fraser, H

    2002-03-23

    Semen from 13 bulls, eight with clinical bovine spongiform encephalopathy (BSE), was used to artificially inseminate (AI) 167 cows with clinical BSE, and their resultant embryos were collected non-surgically seven days after AI. The viable and non-viable embryos with intact zonae pellucidae were washed 10 times (as recommended by the International Embryo Transfer Society) then frozen. Later, 587 of the viable embryos were transferred singly into 347 recipient heifers imported from New Zealand, and 266 live offspring were born of which 54.1 per cent had a BSE-positive sire and a BSE-positive dam. The recipients were monitored for clinical signs of BSE for seven years after the transfer, and the offspring were monitored for seven years after birth. Twenty-seven of the recipients and 20 offspring died while being monitored but none showed signs of BSE. Their brains, and the brains of the recipients and offspring killed after seven years, were examined for BSE by histopathology, PrP immunohistochemistry, and by electron microscopy for scrapie-associated fibrils. They were all negative. In addition, 1020 non-viable embryos were sonicated and injected intracerebrally into susceptible mice (20 embryos per mouse) which were monitored for up to 700 days, after which their brains were examined for spongiform lesions. They were all negative. It is concluded that embryos are unlikely to carry BSE infectivity even if they have been collected at the end-stage of the disease, when the risk of maternal transmission is believed to be highest.

  15. Expression of a transferred nuclear gene in a mitochondrial genome

    Directory of Open Access Journals (Sweden)

    Yichun Qiu

    2014-08-01

    Full Text Available Transfer of mitochondrial genes to the nucleus, and subsequent gain of regulatory elements for expression, is an ongoing evolutionary process in plants. Many examples have been characterized, which in some cases have revealed sources of mitochondrial targeting sequences and cis-regulatory elements. In contrast, there have been no reports of a nuclear gene that has undergone intracellular transfer to the mitochondrial genome and become expressed. Here we show that the orf164 gene in the mitochondrial genome of several Brassicaceae species, including Arabidopsis, is derived from the nuclear ARF17 gene that codes for an auxin responsive protein and is present across flowering plants. Orf164 corresponds to a portion of ARF17, and the nucleotide and amino acid sequences are 79% and 81% identical, respectively. Orf164 is transcribed in several organ types of Arabidopsis thaliana, as detected by RT-PCR. In addition, orf164 is transcribed in five other Brassicaceae within the tribes Camelineae, Erysimeae and Cardamineae, but the gene is not present in Brassica or Raphanus. This study shows that nuclear genes can be transferred to the mitochondrial genome and become expressed, providing a new perspective on the movement of genes between the genomes of subcellular compartments.

  16. Sperm Nuclear Transfer and Transgenic Production in the Fish Medaka

    Directory of Open Access Journals (Sweden)

    Tongming Liu, Ling Liu, Qiwei Wei, Yunhan Hong

    2011-01-01

    Full Text Available Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI is a powerful assisted reproductive technology (ART for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.

  17. Epigenetic reprogramming by somatic cell nuclear transfer in primates.

    Science.gov (United States)

    Sparman, Michelle; Dighe, Vikas; Sritanaudomchai, Hathaitip; Ma, Hong; Ramsey, Cathy; Pedersen, Darlene; Clepper, Lisa; Nighot, Prashant; Wolf, Don; Hennebold, Jon; Mitalipov, Shoukhrat

    2009-06-01

    We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

  18. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Séverine A Degrelle

    Full Text Available Somatic cell nuclear transfer (SCNT is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each; one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538, we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity and subsequent pregnancy loss. Finally

  19. 1H-NMR and photochemically-induced dynamic nuclear polarization studies on bovine pancreatic phospholipase A2.

    Science.gov (United States)

    Egmond, M R; Slotboom, A J; De Haas, G H; Dijkstra, K; Kaptein, R

    1980-06-26

    Proton-NMR resonances of trytophan 3 and tyrosine 69 in bovine pancreatic phospholipase A2, its pro-enzyme and in Ala1-transaminated protein were assigned using photochemically-induced dynamic nuclear polarization (photo-CIDNP) as such or in combination with spin-echo measurements. In addition assignments were made by suppression of cross-relaxation effects using short (0.1 s) high-power laser pulses.

  20. Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes.

    Science.gov (United States)

    White, K L; Bunch, T D; Mitalipov, S; Reed, W A

    1999-01-01

    Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation

  1. Nuclear transfer to prevent mitochondrial DNA disorders : revisiting the debate on reproductive cloning

    NARCIS (Netherlands)

    Bredenoord, A. L.; Dondorp, W.; Pennings, G.; De Wert, G.

    2011-01-01

    Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount

  2. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  3. Effect of Follicle Size and Follicle Stimulating Hormone Concentration on Nuclear Maturation of Bovine Oocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Uğur Şen

    2015-07-01

    Full Text Available The aim of the study was to investigate the effect of follicle size and follicle stimulating hormone (FSH concentration on nuclear maturation of bovine oocytes in vitro. Follicles on bovine ovary were classified into 3 groups according to the diameter; small (<3 mm, medium (3–8 mm and large (9–12 mm. Oocytes were aspirated from follicles with different size and matured in tissue culture medium (TCM–199 supplemented with 10% FCS and various concentrations of FSH (0.5, 1.0 or 10 and μg/ml for 22 hours filled with approximately 95% humidified and 5% CO2 in air at 38.5 °C. At the end of culture period, nuclear maturation (at metaphase II; MII of oocytes were determined by Bisbenzimide (Hoechst 33258 DNA staining under fluorescent microscope. In the present study, effect of follicle size on nuclear maturation of bovine oocytes were determined and the percentage of oocytes reached to M II stage was significantly lower in oocytes obtained small follicle than those of medium and large follicles. Supplementation of 10.0 μg/ml FSH into maturation media increased percentage of nuclear maturation compare to 0.5 and 1.0 μg/ml. Additionally, improving effect of high FSH concentration on nuclear maturation were more observed in oocytes obtained small follicles. The results of present study showed that oocytes from follicles with 3–8 mm diameters exhibited a more successful maturation, but oocytes obtained small follicles exhibited more maturation as a ratio under high FSH concentration.

  4. Heat transfer in nuclear fuels: Measurements of gap conductance

    Science.gov (United States)

    Cho, Chun Hyung

    Heat transfer in the fuel-clad gap in a nuclear reactor impacts the overall temperature distribution, stored energy and the mechanical properties of a nuclear fuel rod. Therefore, an accurate estimation of the gap conductance between the fuel and the clad is critically important for reactor design and operations. To obtain the requisite accuracy in the gap conductance estimation, it is important to understand the effects of the convective heat transfer coefficient, the gas composition, pressure and temperature, and so forth. The objectives of this study are to build a bench-scale experimental apparatus for the measurement of thermal gap conductances and to develop a better understanding of the differences that have been previously observed between such measured values and those predicted theoretically. This is accomplished by employing improved analyses of the experiments and improved theoretical models. Using laser heating of slightly separated stainless-steel plates, the gap conductance was measured using a technique that compares the theoretical and experimental time dependent temperatures at the back surface of the second plate. To consider the effects of surface temperature and gas pressure, the theoretical temperatures were calculated using a convective heat transfer coefficient that was dependent upon both the temperature and the gas pressure.

  5. Serial Nuclear Transfer of Goat-Rabbit Interspecies Reconstructed Embryos

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhi-guo; CHENG Li-zi; ZHANG Xiao-rong; LIU Ya; JING Ren-tao; WANG Cun-li; ZHAO Huan; LI Bin; CAO Chen-chong; LI Dong-wei

    2005-01-01

    The experiments of serial nuclear transfer were conducted between Boer goat and rabbit. The enucleated oocytes of rabbit were used as recipients while the blastomeres of goat morula was used as nuclear donor. The reconstructed embryos developing to morula were used as donor for serial cloning. As a result, two generations of reconstructed embryos were obtained, including 58 first generation reconstructed embryos and 14 second generation reconstructed embryos. The fusion rates were 79.5 and 70%, respectively. and there was no significant difference between them (P>0.05). The cleavage rates were 75.9 and 28.6% respectively with significant difference (P<0.01). No blastocyst was obtained from the second generation reconstructed embryos while 13.8% of first generation reconstructed embryos developed to blastocyst.

  6. Applications of somatic cell nuclear transfer in goats

    Directory of Open Access Journals (Sweden)

    Ton Yoisungnern

    2014-06-01

    Full Text Available A number of animals with genetically identical appearance can be produced by somatic cell nuclear transfer (SCNT. From current advancement of SCNT and molecular techniques, production of a transgenic animal becomes easier. Although cloning efficiency in goat is low, the ability to propagate genetically identical animals, with a gene or genes of interest, would be important for increasing productivity and ultimately the economic livelihood. In this paper, the potential applications and uses of SCNT technology like production of transgenic goat for production of quality milk and meat are discussed.

  7. Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Tian, J; Song, J; Li, H; Yang, D; Li, X; Ouyang, H; Lai, L

    2012-08-01

    Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.

  8. Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

    Science.gov (United States)

    Long, Charles R; Westhusin, Mark E; Golding, Michael C

    2014-02-01

    Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

  9. Birth of Beagle dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Hossein, Mohammad Shamim; Jeong, Yeon Woo; Park, Sun Woo; Kim, Joung Joo; Lee, Eugine; Ko, Kyeong Hee; Hyuk, Park; Hoon, Song Seung; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-09-01

    The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

  10. Producing primate embryonic stem cells by somatic cell nuclear transfer.

    Science.gov (United States)

    Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

    2007-11-22

    Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

  11. 78 FR 11904 - Zion Nuclear Power Station, Units 1 and 2; ZionSolutions, LLC; Consideration of Indirect Transfer

    Science.gov (United States)

    2013-02-20

    ... From the Federal Register Online via the Government Publishing Office NUCLEAR REGULATORY COMMISSION Zion Nuclear Power Station, Units 1 and 2; ZionSolutions, LLC; Consideration of Indirect Transfer AGENCY: Nuclear Regulatory Commission. ACTION: Request for license transfer; opportunity to...

  12. Propagation of elite rescue dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Oh, Hyun Ju; Choi, Jin; Kim, Min Jung; Kim, Geon A; Jo, Young Kwang; Choi, Yoo Bin; Lee, Byeong Chun

    2016-01-01

    The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.

  13. Dogs cloned from fetal fibroblasts by nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Jang, Goo; Kim, Min Kyu; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Dae Yong; Lee, Byeong Chun

    2009-10-01

    Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9-77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2-1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.

  14. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  15. Magnetization transfer contrast imaging in bovine and human cortical bone applying an ultrashort echo time sequence at 3 Tesla.

    Science.gov (United States)

    Springer, Fabian; Martirosian, Petros; Machann, Jürgen; Schwenzer, Nina F; Claussen, Claus D; Schick, Fritz

    2009-05-01

    Magnetization transfer (MT) contrast imaging reveals interactions between free water molecules and macromolecules in a variety of tissues. The introduction of ultrashort echo time (UTE) sequences to clinical whole-body MR scanners expands the possibility of MT imaging to tissues with extremely fast signal decay such as cortical bone. The aim of this study was to investigate the MT effect of bovine cortical bone in vitro on a 3 Tesla whole-body MR unit. A 3D-UTE sequence with a rectangular-shaped on-resonant excitation pulse and a Gaussian-shaped off-resonant saturation pulse for MT preparation was applied. The flip angle and off-resonance frequency of the MT pulse was systematically varied. Measurements on various samples of bovine cortical bone, agar gel, aqueous manganese chloride solutions, and solid polymeric materials (polyurethane) were performed, followed by preliminary applications on human tibial bone in vivo. Direct on-resonant saturation effects of the MT prepulses were calculated numerically by means of Bloch's equations. Corrected for direct saturation effects dry and fresh bovine cortical bone showed "true" MTR values of 0.26 and 0.21, respectively. In vivo data were obtained from three healthy subjects and showed MTR values of 0.30 +/- 0.08. In vivo studies into MT of cortical bone might have the potential to give new insights in musculoskeletal pathologies.

  16. Nuclear Transparency in Large Momentum Transfer Quasielastic Scattering

    Science.gov (United States)

    Mardor, I.; Durrant, S.; Aclander, J.; Alster, J.; Barton, D.; Bunce, G.; Carroll, A.; Christensen, N.; Courant, H.; Gushue, S.; Heppelmann, S.; Kosonovsky, E.; Mardor, Y.; Marshak, M.; Makdisi, Y.; Minor, E. D.; Navon, I.; Nicholson, H.; Piasetzky, E.; Roser, T.; Russell, J.; Sutton, C. S.; Tanaka, M.; White, C.; Wu, J.-Y.

    1998-12-01

    We measured simultaneously pp elastic and quasielastic \\(p,2p\\) scattering in hydrogen, deuterium, and carbon for momentum transfers of 4.8 to 6.2 \\(GeV/c\\)2 at incoming momenta of 5.9 and 7.5 GeV/c and center-of-mass scattering angles in the range θc.m. = 83.7°-90°. The nuclear transparency is defined as the ratio of the quasielastic cross section to the free pp cross section. At incoming momentum of 5.9 GeV/c, the transparency of carbon decreases by a factor of 2 from θc.m.~=85° to θc.m.~=89°. At the largest angle the transparency of carbon increases from 5.9 to 7.5 GeV/c by more than 50%. The transparency in deuterium does not depend on incoming momentum nor on θc.m..

  17. Cloning of ES cells and mice by nuclear transfer.

    Science.gov (United States)

    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  18. Recent advancements in cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  19. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    Science.gov (United States)

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

  20. Estimating transfer of bovine virus-diarrhoea virus in Danish cattle by use of register data

    DEFF Research Database (Denmark)

    Alban, L.; Stryhn, H.; Kjeldsen, A.M.;

    2001-01-01

    To study how routinely recorded data (also called "register data") might be used in disease monitoring on a regional or national level, a database for bovine virus-diarrhoea virus (BVDV) was made from existing databases, covering the period January 1995-November 1999. This paper includes a genera...

  1. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    Science.gov (United States)

    Niemann, H; Lucas-Hahn, A

    2012-08-01

    Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

  2. Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy

    NARCIS (Netherlands)

    Paridon, P.A. van; Visser, A.J.W.G.; Wirtz, K.W.A.

    1987-01-01

    The phosphatidylinositol transfer protein isolated from brain, liver, heart and platelets was found to be present in two subforms which could be distinguished on the basis of the isoelectric points. In this study we have demonstrated that the two subforms isolated from bovine brain are due to the pr

  3. Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering.

    Science.gov (United States)

    Dalgliesh, Ailsa J; Liu, Zhi Zhao; Griffiths, Leigh G

    2017-03-10

    Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are

  4. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  5. Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate.

    Science.gov (United States)

    Mitalipov, S M; White, K L; Farrar, V R; Morrey, J; Reed, W A

    1999-04-01

    The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72

  6. Identification of nuclear effects in neutrino-carbon interactions at low three-momentum transfer

    CERN Document Server

    Rodrigues, P A; Miltenberger, E; Aliaga, L; Altinok, O; Bercellie, A; Betancourt, M; Bodek, A; Bravar, A; Carneiro, M F; Chvojka, J; Devan, J; Eberly, B; Elkins, M; Felix, J; Fields, L; Fine, R; Gago, A M; Galindo, R; Gallagher, H; Ghosh, A; Golan, T; Gran, R; Harris, D A; Higuera, A; Hurtado, K; Kiveni, M; Kleykamp, J; Kordosky, M; Le, T; Leistico, J R; Lovlein, A; Maher, E; Manly, S; Mann, W A; Marshall, C M; Caicedo, D A Martinez; McFarland, K S; McGivern, C L; McGowan, A M; Messerly, B; Miller, J; Mislivec, A; Mousseau, J; Muhlbeier, T; Naples, D; Nelson, J K; Norrick, A; Nuruzzaman,; Patrick, C E; Perdue, G N; Ramirez, M A; Ransome, R D; Ray, H; Ren, L; Rimal, D; Ruterbories, D; Schmitz, D W; Salinas, C J Solano; Tice, B G; Valencia, E; Walton, T; Wolcott, J; Wospakrik, M; Zavala, G; Zhang, D

    2015-01-01

    Two different nuclear-medium effects are isolated using a low three-momentum transfer subsample of neutrino-carbon scattering data from the MINERvA neutrino experiment. The observed hadronic energy in charged-current $\

  7. From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?

    Science.gov (United States)

    Yamada, Mitsutoshi; Byrne, James; Egli, Dieter

    2015-10-01

    Nuclear transfer has seen a remarkable comeback in the past few years. Three groups have independently reported the derivation of stem cell lines by somatic cell nuclear transfer, from either adult, neonatal or fetal cells. Though the ability of human oocytes to reprogram somatic cells to stem cells had long been anticipated, success did not arrive on a straightforward path. Little was known about human oocyte biology, and nuclear transfer protocols developed in animals required key changes to become effective with human eggs. By overcoming these challenges, human nuclear transfer research has contributed to a greater understanding of oocyte biology, provided a point of reference for the comparison of induced pluripotent stem cells, and delivered a method for the generation of personalized stem cells with therapeutic potential.

  8. Epigenetic reprogramming by somatic cell nuclear transfer: questions and potential solutions.

    Science.gov (United States)

    Huili, Ji; Haosheng, Lu; Dengke, Pan

    2014-12-01

    Somatic cell nuclear transfer (SCNT) is a technology by which a highly differentiated somatic nucleus is transferred into an enucleated oocyte to generate a reconstructed embryo that subsequently develops to an offspring. However, to date, the efficiency of cloned animal is still low. The major reason is incomplete nuclear reprogramming of donor cells after nuclear transfer, which results in abnormal epigenetic modifications, including DNA methylation, histone acetylation, gene imprinting, X-chromosome inactivation, and telomere length. Most improvements have been made in somatic epigenetic reprogramming with small molecules and manipulating expression of specific genes. It is expected that SCNT will soon have broad applications in both basic research and practical production. In this review, we summarize the recent progress in epigenetic reprogramming by somatic cell nuclear transfer; in particular, we focus on strategies for rescuing the epigenetic errors occurring during SCNT.

  9. Molten Chloride Salts for Heat Transfer in Nuclear Systems

    Science.gov (United States)

    Ambrosek, James Wallace

    2011-12-01

    A forced convection loop was designed and constructed to examine the thermal-hydraulic performance of molten KCl-MgCl2 (68-32 at %) salt for use in nuclear co-generation facilities. As part of this research, methods for prediction of the thermo-physical properties of salt mixtures for selection of the coolant salt were studied. In addition, corrosion studies of 10 different alloys were exposed to the KCl-MgCl2 to determine a suitable construction material for the loop. Using experimental data found in literature for unary and binary salt systems, models were found, or developed to extrapolate the available experimental data to unstudied salt systems. These property models were then used to investigate the thermo-physical properties of the LINO3-NaNO3-KNO 3-Ca(NO3), system used in solar energy applications. Using these models, the density, viscosity, adiabatic compressibility, thermal conductivity, heat capacity, and melting temperatures of higher order systems can be approximated. These models may be applied to other molten salt systems. Coupons of 10 different alloys were exposed to the chloride salt for 100 hours at 850°C was undertaken to help determine with which alloy to construct the loop. Of the alloys exposed, Haynes 230 had the least amount of weight loss per area. Nickel and Hastelloy N performed best based on maximum depth of attack. Inconel 625 and 718 had a nearly uniform depletion of Cr from the surface of the sample. All other alloys tested had depletion of Cr along the grain boundaries. The Nb in Inconel 625 and 718 changed the way the Cr is depleted in these alloys. Grain-boundary engineering (GBE) of Incoloy 800H improved the corrosion resistance (weight loss and maximum depth of attack) by nearly 50% as compared to the as-received Incoloy 800H sample. A high temperature pump, thermal flow meter, and pressure differential device was designed, constructed and tested for use in the loop, The heat transfer of the molten chloride salt was found to

  10. Innovations in the supply chain and construction engineering of nuclear-based heat transfer components

    Energy Technology Data Exchange (ETDEWEB)

    Perales, A. [Equipos Nucleares, S.A. - Ensa, Jose Ortega y Gasset, 28006 Madrid (Spain); Woolf, G. [Tecnicas Reunidas - TR, Arapiles, 28014 Madrid (Spain)

    2010-07-01

    Equipos Nucleares S.A. (Ensa) and Tecnicas Reunidas S.A. (TR), both long-established Spanish companies, have brought together innovative approaches for the supply of heat transfer solution packages by combining their respective experiences in heat exchanger design (TR), manufacturing (Ensa), and nuclear materials procurement (Ensa and TR), thereby founding a new potent European component supplier for nuclear power plants with over 50 years of experience in the global nuclear market. The combined strategy of the Ensa-TR association which addresses the problems currently faced by nuclear component suppliers is described herein. (authors)

  11. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    Science.gov (United States)

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7.

  12. Förster resonance energy transfer between pyrene and bovine serum albumin: Effect of the hydrophobic pockets of cyclodextrins

    Science.gov (United States)

    Maity, Arnab; Mukherjee, Puspal; Das, Tarasankar; Ghosh, Prasun; Purkayastha, Pradipta

    The phenomenon of Förster resonance energy transfer (FRET) between pyrene and bovine serum albumin (BSA) protein in presence of cyclodextrins (CDs) is explored in the present work. CDs provide hydrophobic environment and thus the aromatic molecules get encapsulated in them depending on the relative size and space. In this work we revealed that along with pyrene monomer, the side chains of amino acids in BSA can get trapped partly in the hydrophobic cavities of CDs if space permits. While being encapsulated by β-CD as pyrene monomer, it can interact with the BSA tryptophan moiety exposed toward the aqueous environment to form a dimer through π-π interaction. This, in turn, affects the energy transfer process by reducing the efficiency. On the other hand, pyrene excimer gets encapsulated in a γ-CD molecule due to availability of enough space. The excimer shows a new band at a higher wavelength. This further reduces FRET efficiency due to scarcity of acceptor for the tryptophan moieties in BSA.

  13. Heat Transfer Phenomena in Supercritical Water Nuclear Reactors

    Energy Technology Data Exchange (ETDEWEB)

    Mark H. Anderson; MichaelL. Corradini; Riccardo Bonazza; Jeremy R. Licht

    2007-10-03

    A supercritical water heat transfer facility has been built at the University of Wisconsin to study heat transfer in ancircular and square annular flow channel. A series of integral heat transfer measurements has been carried out over a wide range of heat flux, mas velocity and bulk water temperatures at a pressure of 25 MPa. The circular annular test section geometry is a 1.07 cm diameter heater rod within a 4.29 diameter flow channel.

  14. ARMS CONTROL: U.S. Efforts to Control the Transfer of Nuclear-Capable Missile Technology

    Science.gov (United States)

    2015-12-11

    risk of diversion is an important part of its analysis, Commerce focuses its reviews on certain countries that are developing nuclear-capable missiles...ogy to countries developing nuclear-capable missile systems. Our work was conducted in the United States at the Departments of State, Commerce , and...the MTCR in halting the transfer of MTCR-related systems and technology to countries developing nuclear-capable missile systems. The objectives, scope

  15. Effect of synchronization of donor cells in early G1-phase using shake-off method on developmental potential of somatic cell nuclear transfer embryos in cattle.

    Science.gov (United States)

    Goto, Yuji; Hirayama, Muneyuki; Takeda, Kazuya; Tukamoto, Nobuyuki; Sakata, Osamu; Kaeriyama, Hiroshi; Geshi, Masaya

    2013-08-01

    In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0-phase (G0-SCNT group) or early G1-phase (eG1-SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0-phase and eG1-phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake-off method). The fusion rate in the G0-SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1-SCNT groups (39.2 ± 1.9%) (P cells in eG1-phase using the shake-off method improved the overall production efficiency of the clone offspring per transferred embryo.

  16. Nuclear transfer to prevent mitochondrial DNA disorders: revisiting the debate on reproductive cloning.

    Science.gov (United States)

    Bredenoord, A L; Dondorp, W; Pennings, G; De Wert, G

    2011-02-01

    Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders.

  17. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  18. Thermoacoustic sensor for nuclear fuel temperaturemonitoring and heat transfer enhancement

    Energy Technology Data Exchange (ETDEWEB)

    James A. Smith; Dale K. Kotter; Randall A. Alli; Steven L. Garrett

    2013-05-01

    A new acoustical sensing system for the nuclear power industry has been developed at The Pennsylvania State University in collaboration with Idaho National Laboratories. This sensor uses the high temperatures of nuclear fuel to convert a nuclear fuel rod into a standing-wave thermoacoustic engine. When a standing wave is generated, the sound wave within the fuel rod will be propagated, by acoustic radiation, through the cooling fluid within the reactor or spent fuel pool and can be monitored a remote location external to the reactor. The frequency of the sound can be correlated to an effective temperature of either the fuel or the surrounding coolant. We will present results for a thermoacoustic resonator built into a Nitonic-60 (stainless steel) fuel rod that requires only one passive component and no heat exchangers.

  19. Effect of freeze-thaw cycles on load transfer between the biomineral and collagen phases in bovine dentin

    Energy Technology Data Exchange (ETDEWEB)

    Deymier-Black, A.C., E-mail: AlixDeymier2010@u.northwestern.edu [Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208 (United States); Almer, J.D., E-mail: almer@aps.anl.gov [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Haeffner, D.R., E-mail: haeffner@aps.anl.gov [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 (United States); Dunand, D.C., E-mail: dunand@northwestern.edu [Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208 (United States)

    2011-10-10

    Stabilization of biological materials by freezing is widespread in the fields of medicine and biomaterials research and yet, in the case of hard biomaterials such as dentin, there is not a good understanding of how such treatments might affect the mechanical properties. The freezing and thawing may have a number of different effects on dentin including formation of cracks in the microstructure and denaturation of the collagen. Using high-energy synchrotron X-ray diffraction, the apparent moduli of bovine dentin samples were measured before and after various numbers of freeze-thaw cycles. It was determined that repeated freezing and thawing has no measurable effect on the hydroxyapatite or fibrillar apparent moduli up to 10 cycles. This confirms that the use of low temperature storage for stabilization of dentin is reasonable in cases where stiffness is a property of importance. Highlights: {yields} Studied the effect of freezing on the load transfer of HAP and fibrils in dentin. {yields} X-ray scattering measured HAP and fibril apparent moduli vs. freezing cycles. {yields} Apparent moduli did not vary significantly between 0 and 10 freeze thaw cycles. {yields} Residual strains imply no widespread cracking due to volumetric expansion of water. {yields} Dentin can be freeze-thawed with no significant effects on elastic properties.

  20. Fluorescent copper(II complexes: The electron transfer mechanism, interaction with bovine serum albumin (BSA and antibacterial activity

    Directory of Open Access Journals (Sweden)

    Madhumita Hazra

    2017-01-01

    Full Text Available Dinuclear copper(II complexes with formula [Cu2(L2(N32] (1 and [Cu2(L2(NCS2] (2 HL = (1-[(3-methyl-pyridine-2-ylimino-methyl]-naphthalen-2-ol were synthesized by controlling the molar ratio of Cu(OAC2·6H2O, HL, sodium azide (1 and ammonium thiocyanate (2. The end on bridges appear exclusively in azide and thiocyanate to copper complexes. The electron transfer mechanism of copper(II complexes is examined by cyclic voltammetry indicating copper(II complexes are Cu(II/Cu(I couple. The interactions of copper(II complexes towards bovine serum albumin (BSA were examined with the help of absorption and fluorescence spectroscopic tools. We report a superficial solution-based route for the synthesis of micro crystals of copper complexes with BSA. The antibacterial activity of the Schiff base and its copper complexes were investigated by the agar disc diffusion method against some species of pathogenic bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and Bacillus cereus. It has been observed that the antibacterial activity of all complexes is higher than the ligand.

  1. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.

    Science.gov (United States)

    D'Amours, Olivier; Frenette, Gilles; Bordeleau, Louis-Jean; Allard, Nancy; Leclerc, Pierre; Blondin, Patrick; Sullivan, Robert

    2012-10-01

    Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

  2. An improved heat transfer configuration for a solid-core nuclear thermal rocket engine

    Science.gov (United States)

    Clark, John S.; Walton, James T.; Mcguire, Melissa L.

    1992-01-01

    Interrupted flow, impingement cooling, and axial power distribution are employed to enhance the heat-transfer configuration of a solid-core nuclear thermal rocket engine. Impingement cooling is introduced to increase the local heat-transfer coefficients between the reactor material and the coolants. Increased fuel loading is used at the inlet end of the reactor to enhance heat-transfer capability where the temperature differences are the greatest. A thermal-hydraulics computer program for an unfueled NERVA reactor core is employed to analyze the proposed configuration with attention given to uniform fuel loading, number of channels through the impingement wafers, fuel-element length, mass-flow rate, and wafer gap. The impingement wafer concept (IWC) is shown to have heat-transfer characteristics that are better than those of the NERVA-derived reactor at 2500 K. The IWC concept is argued to be an effective heat-transfer configuration for solid-core nuclear thermal rocket engines.

  3. Fatal attraction: Explaining Russia's sensitive nuclear transfers to Iran

    Science.gov (United States)

    Kuchinsky, Leah R.

    This paper explores Russia's sensitive nuclear assistance to Iran in an effort to determine why a supplier state might proliferate against its own apparent security interests. The goal is to help readers understand the supply-side dynamics of nuclear proliferation. Through careful reconstruction of the historical narrative, using open source data, this study tests the plausibility of a "fatalistic calculus" explanation, identified by Stephen Sestanovich as a possible driver for Russia's behavior. According to the hypothesis, Russia has cooperated with Iran as a way both to stay in the good graces of a neighbor that is suspected of developing nuclear weapons and to win short-term influence and profits. The paper also examines the role of other factors advanced in the existing supply-side literature, such as economic motives identified by physicist and nonproliferation scholar David Albright. The findings show that bureaucratic, economic and fatalistic factors have each played a role in motivating Russia's cooperation with Iran, with their relative importance shifting over time. Fatalism begets a strategy of Russian "minimaxing," in the lexicon of Russia scholar Robert Freedman, wherein Russia attempts to minimize damage to its relationship with the U.S. while maximizing influence in Iran via nuclear cooperation. Fatalism, as actualized by minimaxing, best explains Russia's behavior after former Russian president Vladmir Putin came to power, when the bureaucratic and economic arguments become less cogent.

  4. Modeling transient heat transfer in nuclear waste repositories.

    Science.gov (United States)

    Yang, Shaw-Yang; Yeh, Hund-Der

    2009-09-30

    The heat of high-level nuclear waste may be generated and released from a canister at final disposal sites. The waste heat may affect the engineering properties of waste canisters, buffers, and backfill material in the emplacement tunnel and the host rock. This study addresses the problem of the heat generated from the waste canister and analyzes the heat distribution between the buffer and the host rock, which is considered as a radial two-layer heat flux problem. A conceptual model is first constructed for the heat conduction in a nuclear waste repository and then mathematical equations are formulated for modeling heat flow distribution at repository sites. The Laplace transforms are employed to develop a solution for the temperature distributions in the buffer and the host rock in the Laplace domain, which is numerically inverted to the time-domain solution using the modified Crump method. The transient temperature distributions for both the single- and multi-borehole cases are simulated in the hypothetical geological repositories of nuclear waste. The results show that the temperature distributions in the thermal field are significantly affected by the decay heat of the waste canister, the thermal properties of the buffer and the host rock, the disposal spacing, and the thickness of the host rock at a nuclear waste repository.

  5. Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

    Institute of Scientific and Technical Information of China (English)

    曾溢滔; 张美兰; 陈美珏; 周霞娣; 黄英; 任兆瑞; 黄淑帧; 胡明信; 吴学清; 高建明; 张斌; 徐慧如

    1994-01-01

    This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

  6. Functional full-term placentas formed from parthenogenetic embryos using serial nuclear transfer.

    Science.gov (United States)

    Hikichi, Takafusa; Ohta, Hiroshi; Wakayama, Sayaka; Wakayama, Teruhiko

    2010-09-01

    Mammalian parthenogenetic embryos invariably die in mid-gestation from imprinted gene defects and placental hypoplasia. Based on chimera experiments, trophoblastic proliferation is supposed to be inhibited in the absence of a male genome. Here, we show that parthenogenetic mouse embryonic cell nuclei can be reprogrammed by serial rounds of nuclear transfer without using any genetic modification. The durations of survival in uteri of cloned foetuses derived from green fluorescent protein (GFP)-labelled parthenogenetic cell nuclei were extended with repeated nuclear transfers. After five repeats, live cloned foetuses were obtained up to day 14.5 of gestation; however, they did not survive longer even when we repeated nuclear transfer up to nine times. All foetuses showed intestinal herniation and possessed well-expanded large placentas. When embryonic stem (ES) cells derived from fertilised embryos were aggregated with the cloned embryos, full-term offspring with large placentas were obtained from the chimeric embryos. Those placentas were derived from parthenogenetic cell nuclei, judging from GFP expression. The patterns of imprinted gene expression and methylation status were similar to their parthenogenetic origin, except for Peg10, which showed the same level as in the normal placenta. These results suggest that there is a limitation for foetal development in the ability to reprogramme imprinted genes by repeated rounds of nuclear transfer. However, the placentas of parthenogenetic embryos can escape epigenetic regulation when developed using nuclear transfer techniques and can support foetal development to full gestation.

  7. MODELAGEM BIOECONÔMICA DA TRANSFERÊNCIA DE EMBRIÕES EM BOVINOS BIOECONOMIC MODEL IN BOVINE EMBRYO TRANSFER

    Directory of Open Access Journals (Sweden)

    Renato Travassos Beltrame

    2010-04-01

    Full Text Available

    O objetivo deste trabalho foi desenvolver um modelo matemático orientado a eventos de simulação, para auxiliar tomadas de decisão relativas à transferência de embriões em bovinos, considerando-se as dinâmicas de dois componentes da transferência de embriões: receptoras e embriões. Na simulação, não se avaliaram respostas individuais de doadoras a coletas consecutivas e eventos correspondentes na transferência de embriões. Simulou-se o mesmo protocolo para superovulação a todas as doadoras. Receptoras foram sincronizadas simulando-se o uso de prostaglandina. O número de embriões viáveis produzido por doadora e sua variabilidade tiveram como base um processo aleatório de simulação de Monte Carlo, que pressupôs uma distribuição exponencial negativa de densidade de probabilidade. Custos e receitas foram inseridos no modelo por meio de um cenário-base para calcular indicadores econômicos de rentabilidade. A análise sugeriu a impraticabilidade da atividade, se realizada diante do cenário proposto (VPL – R$: 57.596,69. A partir do cenário proposto, o custo médio estimado foi de R$ 1.178,19, e de R$ 980,03, para se obter uma prenhez a partir de uma situação otimizada, sugerida pelo modelo (5/100; 5/190.

    PALAVRAS-CHAVES: Otimização, receptoras, simulação, transferência de embriões, viabilidade econômica.

    A simulation model related to embryo transfer programs in bovine was carried out through a mathematical model directed to events, considering the dynamic of two resources: recipients and embryos. Individual answers of donors to consecutive collections and corresponding events in embryo transfer were not evaluated. The same protocol for superovulation was simulated for all the donor collections, using similar doses of hormones and drugs for all the animals. Recipients were synchronized using prostaglandin. Meantime, the number of viable embryos produced by donor and its variability were based at

  8. Characteristics of pregnancies and offspring following transfer of bovine in vivo embryos assessed by nanorespirometry

    DEFF Research Database (Denmark)

    Lopes, Ana Sofia; Madsen, S E; Greve, Torben;

    2008-01-01

    It has been speculated whether the metabolism of the pre-implantation embryo may be reflected on the pregnancy and characteristics of the newborn animal. The present study investigated whether respiration rates of individual embryos were correlated with gestation length, type of parturition, birth......, III), stage of development, and diameter and were subsequently transferred individually (n = 43) to synchronized recipients. Gestation length of the recipients (n = 22) was calculated and the type of parturition (no assistance, light traction, heavy traction, or caesarean section) recorded. Sex......, weight, and condition of the calves at birth (weak, normal, or very active) were also assessed. Results were evaluated by chi-square analysis and using a linear mixed model. The pregnancy rate was 60% (26/43), and the respiration rates of individual embryos influenced gestation length as well...

  9. Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer.

    Science.gov (United States)

    Salilew-Wondim, Dessie; Hölker, Michael; Rings, Franca; Ghanem, Nasser; Ulas-Cinar, Mehmet; Peippo, Jaana; Tholen, Ernst; Looft, Christian; Schellander, Karl; Tesfaye, Dawit

    2010-07-07

    Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30-40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

  10. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    Science.gov (United States)

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  11. Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

    Science.gov (United States)

    Zakhartchenko, V; Flisikowska, T; Li, S; Richter, T; Wieland, H; Durkovic, M; Rottmann, O; Kessler, B; Gungor, T; Brem, G; Kind, A; Wolf, E; Schnieke, A

    2011-02-01

    The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

  12. Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    JI Jingjuan; GUO Tonghang; TONG Xianhong; LUO Lihua; ZHOU Guixiang; FU Yingyun; LIU Yusheng

    2007-01-01

    Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P<0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.

  13. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...... stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high...

  14. Somatic cell nuclear transfer in Oregon: expanding the pluripotent space and informing research ethics.

    Science.gov (United States)

    Lomax, Geoffrey P; DeWitt, Natalie D

    2013-12-01

    In May, Oregon Health and Science University (OHSU) announced the successful derivation, by the Mitalipov laboratory, of embryonic stem cells by somatic cell nuclear transfer. This experiment was recognized as a "formidable technical feat" and potentially a key step toward developing cell-based therapies. The OHSU report is also an example of how a scientific breakthrough can inform research ethics. This article suggests ways that nuclear transfer embryonic stem cell lines may contribute to research ethics by adding rigor to studies addressing pressing research questions important to the development of cell-based therapies.

  15. Passive heat-transfer means for nuclear reactors. [LMFBR

    Science.gov (United States)

    Burelbach, J.P.

    1982-06-10

    An improved passive cooling arrangement is disclosed for maintaining adjacent or related components of a nuclear reactor within specified temperature differences. Specifically, heat pipes are operatively interposed between the components, with the vaporizing section of the heat pipe proximate the hot component operable to cool it and the primary condensing section of the heat pipe proximate the other and cooler component operable to heat it. Each heat pipe further has a secondary condensing section that is located outwardly beyond the reactor confinement and in a secondary heat sink, such as air ambient the containment, that is cooler than the other reactor component. By having many such heat pipes, an emergency passive cooling system is defined that is operative without electrical power.

  16. Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

    Science.gov (United States)

    Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

    2013-10-01

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.

  17. Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos.

    Science.gov (United States)

    Bevacqua, R J; Canel, N G; Hiriart, M I; Sipowicz, P; Rozenblum, G T; Vitullo, A; Radrizzani, M; Fernandez Martin, R; Salamone, D F

    2013-07-15

    Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

  18. Real-time electron dynamics simulation of two-electron transfer reactions induced by nuclear motion

    Science.gov (United States)

    Suzuki, Yasumitsu; Yamashita, Koichi

    2012-04-01

    Real-time electron dynamics of two-electron transfer reactions induced by nuclear motion is calculated by three methods: the numerically exact propagation method, the time-dependent Hartree (TDH) method and the Ehrenfest method. We find that, as long as the nuclei move as localized wave packets, the TDH and Ehrenfest methods can reproduce the exact electron dynamics of a simple charge transfer reaction model containing two electrons qualitatively well, even when nonadiabatic transitions between adiabatic states occur. In particular, both methods can reproduce the cases where a complete two-electron transfer reaction occurs and those where it does not occur.

  19. Tie Tube Heat Transfer Modeling for Bimodal Nuclear Thermal Rockets

    Science.gov (United States)

    Clough, Joshua A.; Starkey, Ryan P.; Lewis, Mark J.; Lavelle, Thomas M.

    2007-01-01

    Bimodal nuclear thermal rocket systems have been shown to reduce the weight and cost of space vehicles to Mars and beyond by utilizing the reactor for power generation in the relatively long duration between burns in an interplanetary trajectory. No information, however, is available regarding engine and reactor-level operation of such bimodal systems. The purpose of this project is to generate engine and reactor models with sufficient fidelity and flexibility to accurately study the component-level effects of operating a propulsion-designed reactor at power generation levels. Previous development of a 1-D reactor and tie tube model found that ignoring heat generation inside of the tie tube leads to under-prediction of the temperature change and over-prediction of pressure change across the tie tube. This paper will present the development and results of a tie tube model that has been extended to account for heat generation, specifically in the moderator layer. This model is based on a 1-D distribution of power in the fuel elements and tie tubes, as a precursor to an eventual neutron-driven reactor model.

  20. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    孙永华; 陈尚萍; 汪亚平; 朱作言

    2000-01-01

    The transcriptional onset ot hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

  1. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blas-tula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

  2. High-fidelity transfer and storage of photon states in a single nuclear spin

    Science.gov (United States)

    Yang, Sen; Wang, Ya; Rao, D. D. Bhaktavatsala; Hien Tran, Thai; Momenzadeh, Ali S.; Markham, M.; Twitchen, D. J.; Wang, Ping; Yang, Wen; Stöhr, Rainer; Neumann, Philipp; Kosaka, Hideo; Wrachtrup, Jörg

    2016-08-01

    Long-distance quantum communication requires photons and quantum nodes that comprise qubits for interaction with light and good memory capabilities, as well as processing qubits for the storage and manipulation of photons. Owing to the unavoidable photon losses, robust quantum communication over lossy transmission channels requires quantum repeater networks. A necessary and highly demanding prerequisite for these networks is the existence of quantum memories with long coherence times to reliably store the incident photon states. Here we demonstrate the high-fidelity (˜98%) coherent transfer of a photon polarization state to a single solid-state nuclear spin that has a coherence time of over 10 s. The storage process is achieved by coherently transferring the polarization state of a photon to an entangled electron-nuclear spin state of a nitrogen-vacancy centre in diamond. The nuclear spin-based optical quantum memory demonstrated here paves the way towards an absorption-based quantum repeater network.

  3. Perspectives of heat transfer enhancement in nuclear reactors toward nanofluids applications

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, Marcelo S.; Cabral, Eduardo L.L.; Sabundjian, Gaiane, E-mail: msrocha@ipen.br, E-mail: elcabral@ipen.br, E-mail: gdjian@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); and others

    2013-07-01

    Nanofluids are colloidal suspensions of nanoparticles in a base fluid with interesting physical properties and large potential for heat transfer enhancement in thermal systems among other applications. There are an increasing number of nanofluids investigations concerning many aspects of synthesis and fabrication technologies, physical properties, and special applications. Results demonstrate that physical properties like high thermal conductivities and high critical heat flux (CHF) of some nanofluids classifies them as potential working fluids for high heat flux transportation in special systems, including thermal management of microelectronic devices (MEMS) and nuclear reactors. Understanding the importance of such investigations for the knowledge development of nuclear engineering a new research is being conducted at the Nuclear Engineering Center (CEN) of the Nuclear and Energy Research Institute (IPEN/CNEN-SP) to analyze the application potentiality of some nanofluids in nuclear systems for heat transfer enhancement under ionizing radiation influence. In this work a revision of theoretical and experimental studies of nanofluids is performed and its potentiality for using in future generations of nuclear reactors is highlighted showing the status of the research at present. (author)

  4. 牛胚胎移植技术的产业化研究%Study of Industrilization of Bovine Embryo Transferring

    Institute of Scientific and Technical Information of China (English)

    冯建忠; 史远刚; 张秀陶; 梁小军; 薛伟; 扬奇; 孟华; 李毓华

    2001-01-01

    Embryo transfer and relative technology was explicated, including setting up of nucleus herd of cattle by importing frozen embryo, choice of donor, superovalution, cryopreservation of embryo and embryo splitting. This study make a base for the industrilization of bovine embryo transferring.%本文探讨了引进国外优质奶、肉牛冷冻胚胎建立核心群,以及供体牛选择、超数排卵、胚胎冷冻保存和分割等相关技术,为牛胚胎移植产业化奠定基础。

  5. Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

    Science.gov (United States)

    Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun

    2016-10-01

    Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.

  6. Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer

    Directory of Open Access Journals (Sweden)

    Herrick Jason R

    2009-12-01

    Full Text Available Abstract Background Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer. Methods First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats. Results All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7% and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%; incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220 were

  7. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

    Institute of Scientific and Technical Information of China (English)

    YING CHEN; QING ZHANG YANG; DA YUAN CHEN; MIN KANG WANG; JIN SONG LI; SHAO LIANG HUANG; XIANG YIN KONG; YAO ZHOU SHI; ZHI QIANG WANG; JIA HUI XIA; ZHI GAO LONG; ZHI XU HE; ZHI GANG XUE; WEN XIANG DING; HUI ZHEN SHENG; AILIAN LIU; KAI WANG; WEN WEI MAO; JIAN XIN CHU; YONG LU; ZHENG FU FANG; YING TANG SHI

    2003-01-01

    To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

  8. Development of interspecies nuclear transfer embryos reconstructed with argali (Ovis ammon) somatic cells and sheep ooplasm.

    Science.gov (United States)

    Pan, Xiaoyan; Zhang, Yanli; Guo, Zhiqin; Wang, Feng

    2014-02-01

    Interspecies nuclear transfer has already achieved success in several species, which shows great potential in recovery and conservation of endangered animals. The study was conducted to establish an efficient system for in vitro argali (Ovis ammon)-sheep embryo reconstruction via interspecies somatic cell nuclear transfer (iSCNT). The competence of domestic sheep cytoplasts to reprogram the adult argali fibroblast nuclei was evaluated, and the effects of enucleation methods and donor cell passage and cell state on the in vitro development of argali-sheep cloned embryos were also examined. Sheep oocytes could support argali and sheep fibroblast cell nuclei transfer and develop to blastocysts in vitro. Oocytes matured for 21–23 h and enucleated by chemically assisted enucleation (CAE) had a higher enucleation rate than blind enucleation (BE), but the development rate of iSCNTembryos was the same (P>0.05). Moreover, passage numbers of fibroblast cells cell cycle stages did not affect the development rate of iSCNT reconstructed embryos. Thus sheep cytoplasm successfully supports argali nucleus development to blastocyst stage after optimising the nuclear transfer procedure, which indicates that iSCNT can be used to conserve endangered argali in the near future.

  9. Nuclear Transfer of Embryonic Cell Nuclei to Non-enucleated Eggs in Zebrafish, Danio rerio

    Directory of Open Access Journals (Sweden)

    Manabu Hattori, Hisashi Hashimoto, Ekaterina Bubenshchikova, Yuko Wakamatsu

    2011-01-01

    Full Text Available We previously established a novel method for nuclear transfer in medaka (Oryzias latipes using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio, we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8% operated eggs developed normally and reached the adult stage. Twenty-eight (15.2% of these transplants showed wild-type phenotype and the remaining three (1.6% were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4% were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism. Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5 as well as the phenotype, the wild-type body color, to their F1 and F2 progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.

  10. Technical support to the Nuclear Regulatory Commission for the boiling water reactor blowdown heat transfer program

    Energy Technology Data Exchange (ETDEWEB)

    Rice, R.E.

    1976-09-01

    Results are presented of studies conducted by Aerojet Nuclear Company (ANC) in FY 1975 to support the Nuclear Regulatory Commission (NRC) on the boiling water reactor blowdown heat transfer (BWR-BDHT) program. The support provided by ANC is that of an independent assessor of the program to ensure that the data obtained are adequate for verification of analytical models used for predicting reactor response to a postulated loss-of-coolant accident. The support included reviews of program plans, objectives, measurements, and actual data. Additional activity included analysis of experimental system performance and evaluation of the RELAP4 computer code as applied to the experiments.

  11. Epigenetic memory in somatic cell nuclear transfer and induced pluripotency: evidence and implications.

    Science.gov (United States)

    Firas, Jaber; Liu, Xiaodong; Polo, Jose M

    2014-07-01

    Six decades ago, seminal work conducted by John Gurdon on genome conservation resulted in major advancements towards nuclear reprogramming technologies such as somatic cell nuclear transfer (SCNT), cell fusion and transcription factor mediated reprogramming. This revolutionized our views regarding cell fate conversion and development. These technologies also shed light on the role of the epigenome in cellular identity, and how the memory of the cell of origin affects the reprogrammed cell. This review will discuss recent work on epigenetic memory retained in pluripotent cells derived by SCNT and transcription factor mediated reprogramming, and the challenges attached to it.

  12. Effect of high-pressure processing of bovine colostrum on immunoglobulin G concentration, pathogens, viscosity, and transfer of passive immunity to calves.

    Science.gov (United States)

    Foster, Derek M; Poulsen, Keith P; Sylvester, Hannah J; Jacob, Megan E; Casulli, Kaitlyn E; Farkas, Brian E

    2016-11-01

    This study aimed to determine the effects of high-pressure processing on the immunoglobulin concentration, microbial load, viscosity, and transfer of passive immunity to calves when applied to bovine colostrum as an alternative to thermal pasteurization. A pilot study using Staphylococcus aureus was conducted to determine which pressure-time treatments are most appropriate for use with bovine colostrum, with the goals of maximizing bacterial inactivation while minimizing IgG content and viscosity changes. Following the pilot study, an inoculation study was conducted in which first-milking colostrum samples from Holstein-Friesian cows were inoculated with known concentrations of various bacteria or viruses and pressure processed at either 300 MPa for up to 60min or at 400MPa for up to 30min. The recovery of total native aerobic bacteria, Escherichia coli, Salmonella enterica ssp. enterica serovar Dublin, Mycobacterium avium ssp. paratuberculosis, bovine herpesvirus type 1, and feline calicivirus were determined after processing. Colostrum IgG content was measured before and after pressure processing. Shear stress and viscosity for each treatment was determined over shear rates encompassing those found during calf feeding and at normal bovine body temperature (37.8°C). Following a calf trial, serum IgG concentration was measured in 14 calves fed 4 L of colostrum pressure processed at 400MPa for 15min. In the pilot study, S. aureus was effectively reduced with pressure treatment at 300 and 400MPa (0, 5, 10, 15, 30, and 45min), with 2 treatments at 400MPa (30, 45min) determined to be inappropriate for use with bovine colostrum due to viscosity and IgG changes. High-pressure processing at 300MPa (30, 45, and 60min) and 400MPa (10, 15, and 20min) was shown to effectively reduce total native aerobic bacteria, E. coli, Salmonella Dublin, bovine herpesvirus type 1, and feline calicivirus populations in bovine colostrum, but no decrease occurred in Mycobacterium avium ssp

  13. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  14. Neutron Transfer Reactions: Surrogates for Neutron Capture for Basic and Applied Nuclear Science

    Science.gov (United States)

    Cizewski, J. A.; Jones, K. L.; Kozub, R. L.; Pain, S. D.; Peters, W. A.; Adekola, A.; Allen, J.; Bardayan, D. W.; Becker, J. A.; Blackmon, J. C.; Chae, K. Y.; Chipps, K. A.; Erikson, L.; Gaddis, A.; Harlin, C.; Hatarik, R.; Howard, J.; Jandel, M.; Johnson, M. S.; Kapler, R.; Krolas, W.; Liang, F.; Livesay, R. J.; Ma, Z.; Matei, C.; Matthews, C.; Moazen, B.; Nesaraja, C. D.; O'Malley, P.; Patterson, N.; Paulauskas, S. V.; Pelham, T.; Pittman, S. T.; Radford, D.; Rogers, J.; Schmitt, K.; Shapira, D.; Shriner, J. F.; Sissom, D. J.; Smith, M. S.; Swan, T.; Thomas, J. S.; Vieira, D. J.; Wilhelmy, J. B.; Wilson, G. L.

    2009-03-01

    Neutron capture reactions on unstable nuclei are important for both basic and applied nuclear science. A program has been developed at the Holifield Radioactive Ion Beam Facility at Oak Ridge National Laboratory to study single-neutron transfer (d,p) reactions with rare isotope beams to provide information on neutron-induced reactions on unstable nuclei. Results from (d,p) studies on 130,132Sn, 134Te and 75As are discussed.

  15. Neutron transfer reactions: Surrogates for neutron capture for basic and applied nuclear science

    Energy Technology Data Exchange (ETDEWEB)

    Cizewski, J. A. [Rutgers University; Jones, K. L. [University of Tennessee; Kozub, R. L. [Tennessee Technological University; Pain, Steven D [ORNL; Peters, W. A. [Rutgers University; Adekola, Aderemi S [ORNL; Allen, J. [Rutgers University; Bardayan, Daniel W [ORNL; Becker, J. [Lawrence Livermore National Laboratory (LLNL); Blackmon, Jeff C [ORNL; Chae, K. Y. [University of Tennessee; Chipps, K. [Colorado School of Mines, Golden; Erikson, Luke [Colorado School of Mines, Golden; Gaddis, A. L. [Furman University; Harlin, Christopher W [ORNL; Hatarik, Robert [Rutgers University; Howard, Joshua A [ORNL; Jandel, M. [Los Alamos National Laboratory (LANL); Johnson, Micah [ORNL; Kapler, R. [University of Tennessee; Krolas, W. [University of Warsaw; Liang, J Felix [ORNL; Livesay, Jake [ORNL; Ma, Zhanwen [ORNL; Matei, Catalin [Oak Ridge Associated Universities (ORAU); Matthews, C. [Rutgers University; Moazen, Brian [University of Tennessee; Nesaraja, Caroline D [ORNL; O' Malley, Patrick [Rutgers University; Patterson, N. P. [University of Surrey, UK; Paulauskas, Stanley [University of Tennessee; Pelham, T. [University of Surrey, UK; Pittman, S. T. [University of Tennessee, Knoxville (UTK); Radford, David C [ORNL; Rogers, J. [Tennessee Technological University; Schmitt, Kyle [University of Tennessee; Shapira, Dan [ORNL; ShrinerJr., J. F. [Tennessee Technological University; Sissom, D. J. [Tennessee Technological University; Smith, Michael Scott [ORNL; Swan, T. P. [University of Surrey, UK; Thomas, J. S. [Rutgers University; Vieira, D. J. [Los Alamos National Laboratory (LANL); Wilhelmy, J. B. [Los Alamos National Laboratory (LANL); Wilson, Gemma L [ORNL

    2009-04-01

    Neutron capture reactions on unstable nuclei are important for both basic and applied nuclear science. A program has been developed at the Holifield Radioactive Ion Beam Facility at Oak Ridge National Laboratory to study single-neutron transfer (d,p) reactions with rare isotope beams to provide information on neutron-induced reactions on unstable nuclei. Results from (d,p) studies on {sup 130,132}Sn, {sup 134}Te and {sup 75}As are discussed.

  16. Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

    OpenAIRE

    Domenico Iuso; Marta Czernik; Fiorella Di Egidio; Silvestre Sampino; Federica Zacchini; Michal Bochenek; Zdzislaw Smorag; Modlinski, Jacek A.; Grazyna Ptak; Pasqualino Loi

    2013-01-01

    The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA da...

  17. Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  18. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

    OpenAIRE

    Engelhardt John F; Li Ziyi

    2003-01-01

    Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not...

  19. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  20. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  1. Heat transfer analysis of fuel assemblies in a heterogeneous gas core nuclear rocket

    Science.gov (United States)

    Watanabe, Yoichi; Appelbaum, Jacob; Diaz, Nils; Maya, Isaac

    1991-01-01

    Heat transfer problems of a heterogeneous gaseous core nuclear rocket were studied. The reactor core consists of 1.5-m long hexagonal fuel assemblies filled with pressurized uranium tetrafluoride (UF4) gas. The fuel gas temperature ranges from 3500 to 7000 K at a nominal operating condition of 40 atm. Each fuel assembly has seven coolant tubes, through which hydrogen propellant flows. The propellant temperature is not constrained by the fuel temperature but by the maximum temperature of the graphite coolant tube. For a core achieving a fission power density of 1000 MW/cu m, the propellant core exit temperature can be as high as 3200 K. The physical size of a 1250 MW gaseous core nuclear rocket is comparable with that of a NERVA-type solid core nuclear rocket. The engine can deliver a specific impulse of 1020 seconds and a thrust of 330 kN.

  2. Modeling of the heat transfer performance of plate-type dispersion nuclear fuel elements

    Science.gov (United States)

    Ding, Shurong; Huo, Yongzhong; Yan, XiaoQing

    2009-08-01

    Considering the mutual actions between fuel particles and the metal matrix, the three-dimensional finite element models are developed to simulate the heat transfer behaviors of dispersion nuclear fuel plates. The research results indicate that the temperatures of the fuel plate might rise more distinctly with considering the particle swelling and the degraded surface heat transfer coefficients with increasing burnup; the local heating phenomenon within the particles appears when their thermal conductivities are too low. With rise of the surface heat transfer coefficients, the temperatures within the fuel plate decrease; the temperatures of the fuel plate are sensitive to the variations of the heat transfer coefficients whose values are lower, but their effects are weakened and slight when the heat transfer coefficients increase and reach a certain extent. Increasing the heat generation rate leads to elevating the internal temperatures. The temperatures and the maximum temperature differences within the plate increase along with the particle volume fractions. The surface thermal flux goes up along with particle volume fractions and heat generation rates, but the effects of surface heat transfer coefficients are not evident.

  3. PRESTO polarization transfer to quadrupolar nuclei: implications for dynamic nuclear polarization.

    Science.gov (United States)

    Perras, Frédéric A; Kobayashi, Takeshi; Pruski, Marek

    2015-09-21

    We show both experimentally and numerically on a series of model systems that in experiments involving transfer of magnetization from (1)H to the quadrupolar nuclei under magic-angle-spinning (MAS), the PRESTO technique consistently outperforms traditionally used cross polarization (CP), affording more quantitative intensities, improved lineshapes, better overall sensitivity, and straightforward optimization. This advantage derives from the fact that PRESTO circumvents the convoluted and uncooperative spin dynamics during the CP transfer under MAS, by replacing the spin-locking of quadrupolar nuclei with a single central transition selective 90° pulse and using a symmetry-based recoupling sequence in the (1)H channel. This is of particular importance in the context of dynamic nuclear polarization (DNP) NMR of quadrupolar nuclei, where the efficient transfer of enhanced (1)H polarization is desired to obtain the highest sensitivity.

  4. Nonideal rotations in nuclear magnetic resonance: Estimation of coherence transfer leakage

    Energy Technology Data Exchange (ETDEWEB)

    Jerschow, Alexej [Materials Sciences Division, 11-D62, Lawrence Berkeley Laboratory, Berkeley, California 94720 (United States); Chemistry Department, D62 Hildebrand, University of California at Berkeley, Berkeley, California 94720 (United States)

    2000-07-15

    When spherical tensors are rotated by certain angles, coherence transfer selection rules may apply. For example, a {pi} rotation cleanly inverts the coherence order. A {pi}/2 rotation of a T{sub 0}{sup 1} tensor creates only T{sub {+-}}{sub 1}{sup 1} tensors. In this work estimations are given for the coherence transfer leakage under the action of rotations with small errors in the rotation angle or axis. Although the theory is stated with particular applications to NMR (nuclear magnetic resonance) in mind it is equally applicable wherever nonideal rotations of spherical tensors are considered (e.g., quantum computing and relaxation theory). In NMR it is useful for the estimation of coherence transfer leakage, especially in pulse sequences with many n{pi} pulses. The results are also applicable to spinors and half-integer representations of the rotation group. (c) 2000 American Institute of Physics.

  5. Preliminary assessment of somatic cell nuclear transfer in the dromedary (Camelus dromedarius).

    Science.gov (United States)

    Khatir, H; Anouassi, A

    2008-12-01

    Somatic cloning may enable the maintenance/expansion of the population of camels with the highest potential for milk production or the best racing performances. However, there have been no reports of embryonic or somatic nuclear transfer in camels. The aim of this study was to produce dromedary embryos by nuclear transfer using in vitro matured oocytes and two somatic cells from two sources (adult fibroblasts or granulosa cells). A total of 58 adult females were superstimulated by a single dose of eCG (3500 IU). Ten days later, their ovaries were collected postmortem. Cumulus-oocytes-complexes (COCs) were aspirated from stimulated follicles and were matured in vitro for 30 h. Fibroblasts (from live adult male) and granulosa cells (from slaughtered adult females) were used as donor karyoplasts and injected into mature enucleated dromedary oocytes. The cleavage rate was significantly higher (Pdromedary embryos from the two sources of donor cells (fibroblasts; n=5 vs. granulosa cells; n=7) was examined by transferring them to synchronized recipients. Two females (fibroblasts: 1/5; 20%, granulosa cells: 1/7; 14%) were confirmed pregnant by ultrasonography at 15 and 25 days following transfer. Later, the pregnancies were followed by pregnancy empirical-symptoms. These two pregnancies were lost between 25 and 60 days following transfer, respectively. In conclusion, the present study shows for the first time that the development of dromedary NT embryos derived from either adult fibroblasts or granulosa cells can occur in vitro and the transfer of these cloned embryos to recipients can result in pregnancies.

  6. PENICILLIN-STREPTOMYCIN IN THE CULTURE MEDIUM DURING IN VITRO MATURATION (IVM OF BOVINE OOCYTES AFFECTS NUCLEAR MATURATION AND SUBSEQUENT EMBRYO DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    SHIRAZI A

    2001-01-01

    Full Text Available Introduction: Standard concentrations of antibiotics in culture media are thought to have no detectable toxic effects on the cultured cells. However, since antibiotics are biologically active substances, the possibility that they interfere to some extent with cellular processes occurring in the cultured cells can not always be totally excluded. This study, therefore, was conducted to assess whether the presence of penicllin-streptomycin (pen-strep during in vitro maturation (IVM of bovine cumulus oocyte complexes (COCs affect nuclear and cytoplasmic maturation and subsequent embryo development. Materials and Methods: Bovine COCs were matured at 39oC in a humidified atmosphere with 5 % CO2 in air for 24 h in: 1- culture medium M 199 supplemented with 10 % FCS (Fetal calf serum, 0.05 IU/ml rhFSH (recombinant human FSH and 100 units penicillin and 100 ?g streptomycin/ ml. 2- culture medium M 199 without FCS and rhFSH in the presence of pen-strep. Cultures without antibiotics served as control. Six series of experiments, each consisted of at least 3 replicates, were performed. Results: In vitro maturation in the presence of pen-strep in culture medium supplemented with FCS and rhFSH significantly (P<0.05 increased the percentage of MII oocytes, however, when the COCs were divided, on the basis of appearance of the cumulus investment, into bright and dark groups, this effect was less obvious in both types of COCs, 76% vs 72% in bright COCs (P= 0.149 or 83% vs 80% in dark COCs (P=0.296 in treated and control groups respectively. The percentage of oocytes with type III of cortical granules (CGs distribution was not affected in the presence of pen-strep. The COCs expansion after IVM was not affected by the presence of antibiotics in culture medium. The subsequent embryo development of IVM/IVF produced ova, which were exposed to pen-strep during IVM, was significantly (P<0.05 decreased with respect to blastocyst formation by day 9. In vitro maturation in

  7. Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

    Science.gov (United States)

    Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

    2011-09-15

    The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.

  8. Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors.

    Directory of Open Access Journals (Sweden)

    Yan Jiang

    Full Text Available Interspecies somatic cell nuclear transfer (iSCNT involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%; and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%. Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38% compared to embryos generated from non-supplemented oocytes (P<0.01. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They

  9. Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse.

    Science.gov (United States)

    Markoulaki, Styliani; Meissner, Alexander; Jaenisch, Rudolf

    2008-06-01

    Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct--through genetic manipulations--degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body--characteristics that are essential for transplantation therapy or any other in vivo application.

  10. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  11. Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Hwang, In-Sul; Kwon, Dae-Jin; Oh, Keun Bong; Ock, Sun-A; Chung, Hak-Jae; Cho, In-Cheol; Lee, Jeong-Woong; Im, Gi-Sun; Hwang, Seongsoo

    2015-06-01

    The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.

  12. Birth of viable female dogs produced by somatic cell nuclear transfer.

    Science.gov (United States)

    Jang, G; Kim, M K; Oh, H J; Hossein, M S; Fibrianto, Y H; Hong, S G; Park, J E; Kim, J J; Kim, H J; Kang, S K; Kim, D Y; Lee, B C

    2007-03-15

    Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6-22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.

  13. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

    Science.gov (United States)

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A; Lambrecht, Marlies A; Koehler, Peter; Delcour, Jan A

    2015-07-20

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

  14. Targeting cellular memory to reprogram the epigenome, restore potential, and improve somatic cell nuclear transfer.

    Science.gov (United States)

    Eilertsen, K J; Power, R A; Harkins, L L; Misica, P

    2007-03-01

    Successful cloning by somatic cell nuclear transfer (SCNT) is thought to require reprogramming of a somatic nucleus to a state of restored totipotentiality [Dean, W., Santos, F., Reik, W., 2003. Epigenetic programming in early mammalian development and following somatic cell nuclear transfer. Semin. Cell. Dev. Biol. 14, 93-100; Jouneau, A., Renard, J.P., 2003. Reprogramming in nuclear transfer. Curr. Opin. Genet. Dev. 13, 486-491; ]. Though SCNT-induced reprogramming is reminiscent of the reprogramming that occurs after fertilization, reprogramming a differentiated nucleus to an embryonic state is delayed and incomplete in comparison (for review, see ). This is likely due to the existence of an epigenetic-based cellular memory, or program, that serves to regulate global patterns of gene expression, and is the basis of a genome defense mechanism that silences viruses and transposons. The mechanisms of this memory include CpG methylation and modification of histones. Recent evidence by Feng et al. [Feng, Y.-Q., Desprat, R., Fu, H., Olivier, E., Lin, C.M., Lobell, A., Gowda, S.N., Aladjem, M.I., Bouhasira, E.E., 2006. DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLOS Genet. 2, 0461-0470], using a transgenic experimental system, indicates that these marks may be acquired in more than one order and thus, silent heterochromatic structure can be initiated by either methylation of CpG dinucleotides or by histone modifications. In this system, however, CpG methylation appears to differ from histone modifications because it bestows a persistent epigenetic, or cellular, memory. In other words, CpG methylation can independently confer cellular memory, whereas histone modifications appear to be limited in this capacity. Therefore, in the context of genomic reprogramming induced by SCNT, efficient demethylation is likely a key (if not the only) rate-limiting step to improving the efficiency and outcomes of SCNT cloning. This review discusses the

  15. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    Science.gov (United States)

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  16. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek;

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  17. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin;

    2011-01-01

    in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls......To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...

  18. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    Science.gov (United States)

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

  19. Conservation of the Sapsaree (Canis familiaris), a Korean Natural Monument, using somatic cell nuclear transfer.

    Science.gov (United States)

    Jang, Goo; Hong, SoGun; Kang, JungTaek; Park, JungEun; Oh, HyunJu; Park, ChanKyu; Ha, JiHong; Kim, DaeYong; Kim, MinKyu; Lee, ByeongChun

    2009-09-01

    A recent emerging technology, somatic cell nuclear transfer (SCNT), has been considered for conserving threatened or endangered species. Sapsaree is a native breed in Korea and has been designated as a Natural Monument. The aim of this study was to produce a Sapsaree by SCNT for breed conservation. Donor fibroblasts from a 9-year-old male Sapsaree were placed into the perivitelline spaces of enucleated in vivo matured oocytes and fused electrically. A total of 309 cloned embryos were transferred into the oviducts of 15 naturally synchronized recipients. Two recipients were diagnosed as pregnant, and each delivered one cloned puppy, both of which weighed 530 g. Overall, this study demonstrated that an endangered canine breed can be conserved by SCNT.

  20. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  1. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    2014-11-01

    Full Text Available Cloned pigs generated by somatic cell nuclear transfer (SCNT show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP. q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

  2. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Engelhardt John F

    2003-11-01

    Full Text Available Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.

  3. Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

    2008-06-10

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

  4. Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis).

    Science.gov (United States)

    Golla, K; Selokar, N L; Saini, M; Chauhan, M S; Manik, R S; Palta, P; Singla, S K

    2012-10-01

    Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.

  5. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  6. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  7. Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish

    Directory of Open Access Journals (Sweden)

    Mahe Sophie

    2010-06-01

    Full Text Available Abstract Background Nuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (Carassius auratus, the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation. Results Trout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL allowed the same hatching rates as the non injected controls (84.8% ± 23. In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage. Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage. Conclusions The best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min

  8. Application of Direct Assessment Approaches and Methodologies to Cathodically Protected Nuclear Waste Transfer Lines

    Energy Technology Data Exchange (ETDEWEB)

    Dahl, Megan M. [ARES Corporation, Richland, WA (United States); Pikas, Joseph [Schiff Associates, Sugar Land TX (United States); Edgemon, Glenn L. [ARES Corporation, Richland, WA (United States); Philo, Sarah [ARES Corporation, Richland, WA (United States)

    2013-01-22

    The U.S. Department of Energy's (DOE) Hanford Site is responsible for the safe storage, retrieval, treatment, and disposal of approximately 54 million gallons (204 million liters) of radioactive waste generated since the site's inception in 1943. Today, the major structures involved in waste management at Hanford include 149 carbon steel single-shell tanks, 28 carbon-steel double-shell tanks, plus a network of buried metallic transfer lines and ancillary systems (pits, vaults, catch tanks, etc.) required to store, retrieve, and transfer waste within the tank farm system. Many of the waste management systems at Hanford are still in use today. In response to uncertainties regarding the structural integrity of these systems,' an independent, comprehensive integrity assessment of the Hanford Site piping system was performed. It was found that regulators do not require the cathodically protected pipelines located within the Hanford Site to be assessed by External Corrosion Direct Assessment (ECDA) or any other method used to ensure integrity. However, a case study is presented discussing the application of the direct assessment process on pipelines in such a nuclear environment. Assessment methodology and assessment results are contained herein. An approach is described for the monitoring, integration of outside data, and analysis of this information in order to identify whether coating deterioration accompanied by external corrosion is a threat for these waste transfer lines.

  9. Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows.

    Science.gov (United States)

    Moreira, F; Badinga, L; Burnley, C; Thatcher, W W

    2002-03-01

    Previous studies indicated that the use of bovine somatotropin (bST) in concurrence with a timed artificial insemination (TAI) protocol increased pregnancy rates. However, the mechanisms for such a bST effect on fertility were not clear. Objectives of this study were to determine the effects of bST on fertilization and early embryonic development after cows received a superovulation treatment, test whether embryos recovered from bST-treated cows were more likely to survive after transfer to recipients, and evaluate whether treatment of recipient cows with bST affects pregnancy rates. Lactating (n = 8) and nonlactating (n = 4) Holstein donor cows were superovulated, inseminated at detected estrus and assigned to a nontreated control group or to a treatment group receiving a single injection of bST (500 mg, sc) at insemination. Embryos were nonsurgically flushed 7 days after AI and frozen in ethylene glycol for direct transfer. Embryos derived from bST-treated (bST-embryos) or control (control-embryos) donors were transferred to lactating Holstein recipient cows that received either bST treatment 1 day after estrus (500 mg, sc; bST-recipients) or were untreated controls (control-recipients). Thus, there were four treatment groups: control-embryos/control-recipients (n = 43), bST-embryos/control-recipients (n = 41), control-embryos/bST-recipients (n = 37), and bST-embryos/bST-recipients (n = 60). Pregnancy was determined by palpation per rectum 33-43 days after embryo transfer. Unfertilized ova per flush was less for bST than for control (1.0 +/- 0.9 56.4%; P 0.4 +/- 0.7; P cows with bST increased pregnancy rates as compared to control-recipients that received a control-embryo. However, there was no additive effect when bST-recipients received a bST-embryo. Administration of bST at AI decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both

  10. Heat Transfer Enhancement By Three-Dimensional Surface Roughness Technique In Nuclear Fuel Rod Bundles

    Science.gov (United States)

    Najeeb, Umair

    This thesis experimentally investigates the enhancement of single-phase heat transfer, frictional loss and pressure drop characteristics in a Single Heater Element Loop Tester (SHELT). The heater element simulates a single fuel rod for Pressurized Nuclear reactor. In this experimental investigation, the effect of the outer surface roughness of a simulated nuclear rod bundle was studied. The outer surface of a simulated fuel rod was created with a three-dimensional (Diamond-shaped blocks) surface roughness. The angle of corrugation for each diamond was 45 degrees. The length of each side of a diamond block is 1 mm. The depth of each diamond block was 0.3 mm. The pitch of the pattern was 1.614 mm. The simulated fuel rod had an outside diameter of 9.5 mm and wall thickness of 1.5 mm and was placed in a test-section made of 38.1 mm inner diameter, wall thickness 6.35 mm aluminum pipe. The Simulated fuel rod was made of Nickel 200 and Inconel 625 materials. The fuel rod was connected to 10 KW DC power supply. The Inconel 625 material of the rod with an electrical resistance of 32.3 kO was used to generate heat inside the test-section. The heat energy dissipated from the Inconel tube due to the flow of electrical current flows into the working fluid across the rod at constant heat flux conditions. The DI water was employed as working fluid for this experimental investigation. The temperature and pressure readings for both smooth and rough regions of the fuel rod were recorded and compared later to find enhancement in heat transfer coefficient and increment in the pressure drops. Tests were conducted for Reynold's Numbers ranging from 10e4 to 10e5. Enhancement in heat transfer coefficient at all Re was recorded. The maximum heat transfer co-efficient enhancement recorded was 86% at Re = 4.18e5. It was also observed that the pressure drop and friction factor increased by 14.7% due to the increased surface roughness.

  11. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Domenico Iuso

    Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  12. Modeling Electronic-Nuclear Interactions for Excitation Energy Transfer Processes in Light-Harvesting Complexes.

    Science.gov (United States)

    Lee, Mi Kyung; Coker, David F

    2016-08-18

    An accurate approach for computing intermolecular and intrachromophore contributions to spectral densities to describe the electronic-nuclear interactions relevant for modeling excitation energy transfer processes in light harvesting systems is presented. The approach is based on molecular dynamics (MD) calculations of classical correlation functions of long-range contributions to excitation energy fluctuations and a separate harmonic analysis and single-point gradient quantum calculations for electron-intrachromophore vibrational couplings. A simple model is also presented that enables detailed analysis of the shortcomings of standard MD-based excitation energy fluctuation correlation function approaches. The method introduced here avoids these problems, and its reliability is demonstrated in accurate predictions for bacteriochlorophyll molecules in the Fenna-Matthews-Olson pigment-protein complex, where excellent agreement with experimental spectral densities is found. This efficient approach can provide instantaneous spectral densities for treating the influence of fluctuations in environmental dissipation on fast electronic relaxation.

  13. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  14. Somatic cell nuclear transfer and transgenesis in large animals: current and future insights.

    Science.gov (United States)

    Galli, C; Lagutina, I; Perota, A; Colleoni, S; Duchi, R; Lucchini, F; Lazzari, G

    2012-06-01

    Somatic cell nuclear transfer (SCNT) was first developed in livestock for the purpose of accelerating the widespread use of superior genotypes. Although many problems still exist now after fifteen years of research owing to the limited understanding of genome reprogramming, SCNT has provided a powerful tool to make copies of selected individuals in different species, to study genome pluripotency and differentiation, opening new avenues of research in regenerative medicine and representing the main route for making transgenic livestock. Besides well-established methods to deliver transgenes, recent development in enzymatic engineering to edit the genome provides more precise and reproducible tools to target-specific genomic loci especially for producing knockout animals. The interest in generating transgenic livestock lies in the agricultural and biomedical areas and it is, in most cases, at the stage of research and development, with few exceptions that are making the way into practical applications.

  15. Prolonged interval between fusion and activation impairs embryonic development by inducing chromosome scattering and nuclear aneuploidy in pig somatic cell nuclear transfer.

    Science.gov (United States)

    You, Jinyoung; Song, Kilyoung; Lee, Eunsong

    2010-01-01

    The aim of the present study was to examine the effect of various intervals between electrofusion and activation (FA interval) on the nuclear remodelling and development of somatic cell nuclear transfer (SCNT) embryos in pigs. Reconstructed oocytes were activated at 0 (simultaneous fusion and activation; SFA), 1, 2 and 3 h (delayed activation) after electrofusion; these groups were designated as DA1, DA2 and DA3, respectively. When oocyte nuclear status was examined at 0.5, 1, 2 and 3 h after electrofusion, the incidence of chromosome scattering was increased (P or=3) pseudopronuclei (PPN) (0.0% of SFA; 5.3% of DA1; 21.7% of DA2; and 33.5% of DA3). The development of SCNT embryos to the blastocyst stage was decreased (P nuclear aneuploidy.

  16. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    Directory of Open Access Journals (Sweden)

    Richter Anne

    2012-11-01

    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

  17. Transfer-induced fission in inverse kinematics: Impact on experimental and evaluated nuclear data bases

    Science.gov (United States)

    Farget, F.; Caamaño, M.; Ramos, D.; Rodrıguez-Tajes, C.; Schmidt, K.-H.; Audouin, L.; Benlliure, J.; Casarejos, E.; Clément, E.; Cortina, D.; Delaune, O.; Derkx, X.; Dijon, A.; Doré, D.; Fernández-Domınguez, B.; Gaudefroy, L.; Golabek, C.; Heinz, A.; Jurado, B.; Lemasson, A.; Paradela, C.; Roger, T.; Salsac, M. D.; Schmitt, C.

    2015-12-01

    Inverse kinematics is a new tool to study nuclear fission. Its main advantage is the possibility to measure with an unmatched resolution the atomic number of fission fragments, leading to new observables in the properties of fission-fragment distributions. In addition to the resolution improvement, the study of fission based on nuclear collisions in inverse kinematics beneficiates from a larger view with respect to the neutron-induced fission, as in a single experiment the number of fissioning systems and the excitation energy range are widden. With the use of spectrometers, mass and kinetic-energy distributions may now be investigated as a function of the proton and neutron number sharing. The production of fissioning nuclei in transfer reactions allows studying the isotopic yields of fission fragments as a function of the excitation energy. The higher excitation energy resulting in the fusion reaction leading to the compound nucleus 250Cf at an excitation energy of 45MeV is also presented. With the use of inverse kinematics, the charge polarisation of fragments at scission is now revealed with high precision, and it is shown that it cannot be neglected, even at higher excitation energies. In addition, the kinematical properties of the fragments inform on the deformation configuration at scission.

  18. Water-to-Wildlife Transfer of Radionuclides in Freshwater Ecosystems around the Gyeongju Nuclear Site

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Ho; Lim, Kwang-Muk; Jun, In; Kim, Byeong-Ho; Keum, Dong-Kwon [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-10-15

    The IAEA and ICRP have recognized that not only humans but also wildlife needs to be protected from the impact of ionizing radiations. In many advanced countries, it is legally required to evaluate the radiological impact to wildlife. Therefore, it can be expected that the wildlife dose assessment will also soon become a legal requirement in Korea. One of the key parameters in evaluating radiation doses to wildlife is the concentration ratio (CR), which is used for quantifying radionuclide transfer from an environmental medium such as soil and water to an organism. CR values can vary greatly with environmental conditions and wildlife species. Accordingly, it is important for a reliable dose assessment that site-specific CR data be used. In this study, CR values of various radionuclides were measured for several freshwater wildlife species living around the Gyeongju nuclear site. CR values of a total of 20 elements were determined for three fish species and three plant species living in freshwater ecosystems around the Gyeongju nuclear site. The CR values showed considerable variations with the elements and with wildlife species. For the establishment of a reliable input data file of K-BIOTA, a Korean wildlife dose assessment model, data on CR values needs to be increased to cover a wider range of domestic wildlife.

  19. Transfer-induced fission in inverse kinematics: Impact on experimental and evaluated nuclear data bases

    Energy Technology Data Exchange (ETDEWEB)

    Farget, F.; Schmidt, K.H.; Clement, E.; Delaune, O.; Derkx, X.; Dijon, A.; Golabek, C.; Lemasson, A.; Roger, T.; Schmitt, C. [CEA/DSM-CNRS/IN2P3, GANIL, Caen (France); Caamano, M.; Ramos, D.; Benlliure, J.; Cortina, D.; Fernandez-Dominguez, B.; Paradela, C. [Universidade de Santiago de Compostela, Santiago de Compostela (Spain); Rodriguez-Tajes, C. [CEA/DSM-CNRS/IN2P3, GANIL, Caen (France); Universidade de Santiago de Compostela, Santiago de Compostela (Spain); Audouin, L. [Universite Paris-Sud 11, CNRS/IN2P3, Institut de Physique Nucleaire, Orsay (France); Casarejos, E. [Universidade de Vigo, Vigo (Spain); Dore, D.; Salsac, M.D. [Centre de Saclay, CEA, Irfu, Gif-sur-Yvette (France); Gaudefroy, L. [CEA DAM Ile-de-France, BP 12, Bruyeres-le-Chatel (France); Heinz, A. [Chalmers Tekniska Hoegskola, Fundamental Fysik, Goeteborg (Sweden); Jurado, B. [Universite Bordeaux, CENBG, UMR 5797 CNRS/IN2P3, Gradignan (France)

    2015-12-15

    Inverse kinematics is a new tool to study nuclear fission. Its main advantage is the possibility to measure with an unmatched resolution the atomic number of fission fragments, leading to new observables in the properties of fission-fragment distributions. In addition to the resolution improvement, the study of fission based on nuclear collisions in inverse kinematics beneficiates from a larger view with respect to the neutron-induced fission, as in a single experiment the number of fissioning systems and the excitation energy range are widden. With the use of spectrometers, mass and kinetic-energy distributions may now be investigated as a function of the proton and neutron number sharing. The production of fissioning nuclei in transfer reactions allows studying the isotopic yields of fission fragments as a function of the excitation energy. The higher excitation energy resulting in the fusion reaction leading to the compound nucleus {sup 250}Cf at an excitation energy of 45MeV is also presented. With the use of inverse kinematics, the charge polarisation of fragments at scission is now revealed with high precision, and it is shown that it cannot be neglected, even at higher excitation energies. In addition, the kinematical properties of the fragments inform on the deformation configuration at scission. (orig.)

  20. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of); Shim, Hosup [Department of Physiology, Dankook University School of Medicine, Cheonan 330 714 (Korea, Republic of); Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June [Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Suwon (Korea, Republic of); Kim, Jin-Hoi [Department of Animal Biotechnology, Konkuk University, Seoul 143 701 (Korea, Republic of); Lee, Jeong Woong, E-mail: jwlee@kribb.re.kr [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  1. Knowledge transfer in Swedish Nuclear Power Plants in connection with retirements; Kompetensoeverfoering paa svenska kaernkraftverk i samband med pensionsavgaangar

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, Annika; Ohlsson, Kjell; Roos, Anna

    2007-12-13

    This report displays how the Swedish nuclear power plants Forsmark, Oskarshamn and Ringhals work with knowledge management. The report also consists of a literature review of appropriate ways to extract tacit knowledge as well as methods to transfer competence. The report is made up of a smaller number of interviews at the nuclear power plants in combination with a questionnaire distributed to a larger number of people at the plants. The results of the interview study is that only one of the Swedish nuclear power plants have a programme to transfer knowledge from older staff to newer. This is, however, not a programme for everyone. Another plant has a programme for knowledge building, but only for their specialists. At both plants, which lack a programme, the interviewees request more structure in knowledge transfer; even though they feel the current way of transferring knowledge with mentors works well. Besides more structure, interviewees present a wish to have more time for knowledge transfer as well as the opportunity to recruit more than needed. Recruiting more than needed is however not very simple due to multiple causes such as nominal sizing departments and a difficulty of recruiting people to work far from larger cities. The way things are now, many feel too under-staffed and under a lot of time pressure daily to also have time for knowledge transfer besides their normal work.

  2. Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed embryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.

  3. Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise.

    Science.gov (United States)

    Fletcher, C J; Roberts, C T; Hartwich, K M; Walker, S K; McMillen, I C

    2007-01-01

    The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with approximately 5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT die in utero. Placentomes from SCNT pregnancies (n = 9) and age matched, naturally mated controls (n = 20) were collected at two gestational age ranges (105-134 days and 135-154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105-134 days: control 55.0 +/- 4.2, clone 44.7 +/- 8.0 and 135-154 days: control 72.2 +/- 5.1, clone 36.6 +/- 5.1; P clone 18.6 +/- 2.8 g and 135-154 days: control 6.6 +/- 0.6 g, clone 7.0 +/- 2.0 g; P cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones.

  4. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  5. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  6. Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

    Science.gov (United States)

    Lee, W-J; Lee, J-H; Jeon, R-H; Jang, S-J; Lee, S-C; Park, J-S; Lee, S-L; King, W-A; Rho, G-J

    2017-02-13

    Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

  7. Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer.

    Science.gov (United States)

    Shimatsu, Yoshiki; Horii, Wataru; Nunoya, Tetsuo; Iwata, Akira; Fan, Jianglin; Ozawa, Masayuki

    2016-01-01

    Most cases of ischemic heart disease and stroke occur as a result of atherosclerosis. The purpose of this study was to produce a new Nippon Institute for Biological Science (NIBS) miniature pig model by somatic cell nuclear transfer (SCNT) for studying atherosclerosis. The human apolipoprotein(a) (apo(a)) genes were transfected into kidney epithelial cells derived from a male and a female piglet. Male cells were used as donors initially, and 275 embryos were transferred to surrogates. Three offspring were delivered, and the production efficiency was 1.1% (3/275). Serial female cells were injected into 937 enucleated oocytes. Eight offspring were delivered (production efficiency: 0.9%) from surrogates. One male and 2 female transgenic miniature pigs matured well. Lipoprotein(a) was found in the male and one of the female transgenic animals. These results demonstrate successful production of human apo(a) transgenic NIBS miniature pigs by SCNT. Our goal is to establish a human apo(a) transgenic NIBS miniature pig colony for studying atherosclerosis.

  8. Current status and applications of somatic cell nuclear transfer in dogs.

    Science.gov (United States)

    Jang, Goo; Kim, Min Kyu; Lee, Byeong Chun

    2010-11-01

    Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications.

  9. Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

    Science.gov (United States)

    Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

    2013-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (Pcloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

  10. Radiocesium Transfer in Forest Insect Communities after the Fukushima Dai-ichi Nuclear Power Plant Accident

    Science.gov (United States)

    Hayashi, Seiji; Takamura, Noriko

    2017-01-01

    To understand radiocesium transfer in the forest insect food web, we investigated the activity concentrations of radiocesium in forest insects in the Fukushima and Ibaraki Prefectures approximately 1.5–2.5 years after the Fukushima Dai-ichi Nuclear Power Plant. We analyzed 34 species of insects sampled from 4 orders and 4 feeding functional groups (herbivore, carnivore, omnivore, and detritivore) from three sites in each prefecture. 137Cs activity concentrations were lowest in herbivorous species and were especially high in detritivorous and omnivorous species that feed on forest litter and fungi. Radiocesium activity concentrations in any given species reflected the degree of contamination of that species’ primary food sources since radiocesium activity concentrations were found to be the lowest in leaves and grass and the highest in litter, bark, and fungi. This study confirmed that litter and other highly contaminated forest components such as fungi, decaying wood, bryophytes, and lichens serve as sources of 137Cs transfer into the forest insect community. PMID:28125745

  11. N-doped carbon dots derived from bovine serum albumin and formic acid with one- and two-photon fluorescence for live cell nuclear imaging.

    Science.gov (United States)

    Tan, Mingqian; Li, Xintong; Wu, Hao; Wang, Beibei; Wu, Jing

    2015-12-01

    Carbon dots with both one- and two-photon fluorescence have drawn great attention for biomedical imaging. Herein, nitrogen-doped carbon dots were facilely developed by one-pot hydrothermal method using bovine serum albumin and formic acid as carbon sources. They are highly water-soluble with strong fluorescence when excited with ultraviolet or near infrared light. The carbon dots have a diameter of ~8.32 nm and can emit strong two-photon induced fluorescence upon excitation at 750 nm with a femtosecond laser. X-ray photoelectron spectrometer analysis revealed that the carbon dots contained three components, C, N and O, corresponding to the peak at 285, 398 and 532 eV, respectively. The Fourier-transform infrared spectroscopy analysis revealed that there are carboxyl and carboxylic groups on the surface, which allowed further linking of functional molecules. pH stability study demonstrated that the carbon dots are able to be used in a wide range of pH values. The fluorescence mechanism is also discussed in this study. Importantly, these carbon dots are biocompatible and highly photostable, which can be directly applied for both one- and two-photon living cell imaging. After proper surface functionalization with TAT peptide, they can be used as fluorescent probes for live cell nuclear-targeted imaging.

  12. Interaction of bovine serum albumin (BSA) with novel gemini surfactants studied by synchrotron radiation scattering (SR-SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

    Science.gov (United States)

    Gospodarczyk, W; Szutkowski, K; Kozak, M

    2014-07-24

    The interaction of three dicationic (gemini) surfactants-3,3'-[1,6-(2,5-dioxahexane)]bis(1-dodecylimidazolium) chloride (oxyC2), 3,3'-[1,16-(2,15-dioxahexadecane)]bis(1-dodecylimidazolium) chloride (oxyC12), and 1,4-bis(butane)imidazole-1-yl-3-dodecylimidazolium chloride (C4)--with bovine serum albumin (BSA) has been studied by the use of small-angle X-ray scattering (SAXS), circular dichroism (CD), and (1)H nuclear magnetic resonance diffusometry. The results of CD studies show that the conformation of BSA was changed dramatically in the presence of all studied surfactants. The greater decrease (from 56 to 24%) in the α-helical structure of BSA was observed for oxyC2 surfactant. The radii of gyration estimated from SAXS data varied between 3 and 26 nm for the BSA/oxyC2 and BSA/oxyC12 systems. The hydrodynamic radius of the BSA/surfactant system estimated from NMR diffusometry varies between 5 and 11 nm for BSA/oxyC2 and 5 and 8 nm for BSA/oxyC12.

  13. Finite-thrust optimization of interplanetary transfers of space vehicle with bimodal nuclear thermal propulsion

    Science.gov (United States)

    Kharytonov, Oleksii M.; Kiforenko, Boris M.

    2011-08-01

    The nuclear thermal rocket (NTR) propulsion is one of the leading promising technologies for primary space propulsion for manned exploration of the solar system due to its high specific impulse capability and sufficiently high thrust-to-weight ratio. Another benefit of NTR is its possible bimodal design, when nuclear reactor is used for generation of a jet thrust in a high-thrust mode and (with an appropriate power conversion system) as a source of electric power to supply the payload and the electric engines in a low-thrust mode. The model of the NTR thrust control was developed considering high-thrust NTR as a propulsion system of limited power and exhaust velocity. For the proposed model the control of the thrust value is accomplished by the regulation of reactor thermal power and propellant mass flow rate. The problem of joint optimization of the combination of high- and low-thrust arcs and the parameters of bimodal NTR (BNTR) propulsion system is considered for the interplanetary transfers. The interplanetary trajectory of the space vehicle is formed by the high-thrust NTR burns, which define planet-centric maneuvers and by the low-thrust heliocentric arcs where the nuclear electric propulsion (NEP) is used. The high-thrust arcs are analyzed using finite-thrust approach. The motion of the corresponding dynamical system is realized in three phase spaces concerning the departure planet-centric maneuver by means of high-thrust NTR propulsion, the low-thrust NEP heliocentric maneuver and the approach high-thrust NTR planet-centric maneuver. The phase coordinates are related at the time instants of the change of the phase spaces due to the relations between the space vehicle masses. The optimal control analysis is performed using Pontryagin's maximum principle. The numerical results are analyzed for Earth-Mars "sprint" transfer. The optimal values of the parameters that define the masses of NTR and NEP subsystems have been evaluated. It is shown that the low

  14. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Müller Mathias

    2007-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

  15. Analysis on the revision of the United States authorizing procedure for the transfer of unclassified nuclear technology

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Sung-ho; Seo, Hana; Lee, Chansuh; Kim, Jong-sook [Korea Institute of Nuclear Nonproliferation and Control, Daejeon (Korea, Republic of)

    2015-10-15

    The DOE (Department Of Energy) has not comprehensively update 10CFR810 since 1986. Since then, the global civil nuclear market has expanded, particularly in China, the Middle East, and Eastern Europe, with vendors from France, Japan, the Republic of Korea, Russia, and Canada. In result, DOE issued revised 810 in respond to comments received from the public and commercial nuclear market changes. This regulation revision improves the efficiency of authorization process to promote national nuclear industry while maintaining nonproliferation control. Even though ROK has initiated a legal basis for Intangible technology transfer (ITT) for nuclear export control, working implementation system is not set up. This research proposes recommendable ITT implementation of the ROK according to the analysis result of the US regulation. In this revision, of 124 countries had been classified as general authorization under 10CFR810, 80 countries reclassified into the specific authorization. By remaining 'fast track' for specific authorization, in particular, time frames for internal DOE and interagency reviews are reduced. This means the US government actively copes with commercial nuclear market expands to promote their industry. Meanwhile, by remaining some of nuclear-weapon states (China, Russia, India) as specific authorization maintaining that the determinations are consistent with current US national security, diplomatic, and trade policy. By benchmarking the US regulation, Korea can improve the efficiency of the technology transfer authorization process easing the regulatory burden by reducing uncertainty and timelines while maintaining the highest level of nonproliferation control.

  16. TALEN-mediated modification of the bovine genome for large-scale production of human serum albumin.

    Science.gov (United States)

    Moghaddassi, Shaida; Eyestone, Will; Bishop, Colin E

    2014-01-01

    As an initial step towards creating genetically modified cattle as a biopharming source of recombinant human serum albumin (rHSA), we report modification of the bovine albumin (bA) locus by transcription activator-like effector nuclease (TALEN)-stimulated homology-directed repair (HDR). Pedigreed bovine fibroblasts were co-transfected with TALENs and an 11.5-kb human serum albumin (HSA) minigene donor construct, designed to simultaneously disrupt and replace bovine serum albumin (BSA) expression with controlled rHSA expression in both the liver and the milk. Targeted integration of the HSA minigene was confirmed in transfected fibroblasts at a frequency of approximately 11% and transgenic bovine embryos were produced from targeted fibroblasts using somatic cell nuclear transfer (SCNT). The research delineated here lays the foundation for the future generation of transgenic rHSA cattle with the potential to provide a large-scale, reliable, and quality-controlled source of rHSA.

  17. Novel approach for interlaboratory transfer of real-time PCR methods: detecting bovine meat and bone meal in feed.

    Science.gov (United States)

    Prado, Marta; Fumière, Olivier; Boix, Ana; Marien, Aline; Berben, Gilbert; von Holst, Christoph

    2009-07-01

    The availability of robust methods for the species-specific detection of meat and bone meal (MBM) in compound feedingstuffs is an important prerequisite to enforce current and upcoming European legislation on the use of processed animal proteins in animal nutrition. Among possible methods, those based on DNA turned out to be a reliable tool for this aim, since DNA is a quite thermostable molecule able to resist severe heat treatments applied in the manufacturing of animal meals. The application of such methods by control laboratories implies that the method has been validated including an assessment of its robustness. Successful transferability between laboratories is considered an important robustness criterion of the method. However, corresponding guidelines regarding the design of such a study relevant to this field are missing. Here, we demonstrate the feasibility of an alternative concept that was applied to check for the transferability of a qualitative assay for the detection of banned MBM in feedingstuffs at trace level based on real-time PCR. The concept was based on an experimental nested design applying analysis of variance (ANOVA) that was conducted independently in two laboratories and which allows for establishing major factors influencing the result of analysis. Statistical assessment of the results confirmed the importance of the DNA extraction/purification step utilised, whereas the PCR step turned out to be a minor factor regarding the overall variability of the results. Furthermore, blind samples comprised of compound feed adulterated with MBM at 0.1% and blank compound feed were correctly classified as "positive" or "negative" samples, thus confirming fitness of purpose for the method. This approach can be of interest for other research groups working in the development of real-time PCR methods and in their use by control laboratories.

  18. Coupled thermochemical, isotopic evolution and heat transfer simulations in highly irradiated UO2 nuclear fuel

    Science.gov (United States)

    Piro, M. H. A.; Banfield, J.; Clarno, K. T.; Simunovic, S.; Besmann, T. M.; Lewis, B. J.; Thompson, W. T.

    2013-10-01

    Predictive capabilities for simulating irradiated nuclear fuel behavior are enhanced in the current work by coupling thermochemistry, isotopic evolution and heat transfer. Thermodynamic models that are incorporated into this framework not only predict the departure from stoichiometry of UO2, but also consider dissolved fission and activation products in the fluorite oxide phase, noble metal inclusions, secondary oxides including uranates, zirconates, molybdates and the gas phase. Thermochemical computations utilize the spatial and temporal evolution of the fission and activation product inventory in the pellet, which is typically neglected in nuclear fuel performance simulations. Isotopic computations encompass the depletion, decay and transmutation of more than 2000 isotopes that are calculated at every point in space and time. These computations take into consideration neutron flux depression and the increased production of fissile plutonium near the fuel pellet periphery (i.e., the so-called “rim effect”). Thermochemical and isotopic predictions are in very good agreement with reported experimental measurements of highly irradiated UO2 fuel with an average burnup of 102 GW d t(U)-1. Simulation results demonstrate that predictions are considerably enhanced when coupling thermochemical and isotopic computations in comparison to empirical correlations. Notice: This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.

  19. Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

    Science.gov (United States)

    Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

    2017-03-01

    Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

  20. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  1. Nuclear overhauser enhancement mediated chemical exchange saturation transfer imaging at 7 Tesla in glioblastoma patients.

    Directory of Open Access Journals (Sweden)

    Daniel Paech

    Full Text Available BACKGROUND AND PURPOSE: Nuclear Overhauser Enhancement (NOE mediated chemical exchange saturation transfer (CEST is a novel magnetic resonance imaging (MRI technique on the basis of saturation transfer between exchanging protons of tissue proteins and bulk water. The purpose of this study was to evaluate and compare the information provided by three dimensional NOE mediated CEST at 7 Tesla (7T and standard MRI in glioblastoma patients. PATIENTS AND METHODS: Twelve patients with newly diagnosed histologically proven glioblastoma were enrolled in this prospective ethics committee-approved study. NOE mediated CEST contrast was acquired with a modified three-dimensional gradient-echo sequence and asymmetry analysis was conducted at 3.3 ppm (B1 = 0.7 µT to calculate the magnetization transfer ratio asymmetry (MTR(asym. Contrast enhanced T1 (CE-T1 and T2-weighted images were acquired at 3T and used for data co-registration and comparison. RESULTS: Mean NOE mediated CEST signal based on MTR(asym values over all patients was significantly increased (p<0.001 in CE-T1 tumor (-1.99 ± 1.22%, tumor necrosis (-1.36 ± 1.30% and peritumoral CEST hyperintensities (PTCH within T2 edema margins (-3.56 ± 1.24% compared to contralateral normal appearing white matter (-8.38 ± 1.19%. In CE-T1 tumor (p = 0.015 and tumor necrosis (p<0.001 mean MTR(asym values were significantly higher than in PTCH. Extent of the surrounding tumor hyperintensity was smaller in eight out of 12 patients on CEST than on T2-weighted images, while four displayed at equal size. In all patients, isolated high intensity regions (0.40 ± 2.21% displayed on CEST within the CE-T1 tumor that were not discernible on CE-T1 or T2-weighted images. CONCLUSION: NOE mediated CEST Imaging at 7 T provides additional information on the structure of peritumoral hyperintensities in glioblastoma and displays isolated high intensity regions within the CE-T1 tumor that cannot be acquired on CE-T1 or T2

  2. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.

    Science.gov (United States)

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-09-22

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

  3. Multiple horizontal transfers of nuclear ribosomal genes between phylogenetically distinct grass lineages

    Science.gov (United States)

    Mahelka, Václav; Krak, Karol; Kopecký, David; Fehrer, Judith; Šafář, Jan; Bartoš, Jan; Hobza, Roman; Blavet, Nicolas; Blattner, Frank R.

    2017-01-01

    The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley (Hordeum) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral. PMID:28137844

  4. Lymphoid lineage differentiation potential of mouse nuclear transfer embryonic stem cells.

    Science.gov (United States)

    Eslami-Arshaghi, Tarlan; Salehi, Mohammad; Soleimani, Masoud; Gholipourmalekabadi, Mazaher; Mossahebi-Mohammadi, Majid; Ardeshirylajimi, Abdolreza; Rajabi, Hoda

    2015-09-01

    Stem cells therapy is considered as an efficient strategy for the treatment of some diseases. Nevertheless, some obstacles such as probability of rejection by the immune system limit applications of this strategy. Therefore, several efforts have been made to overcome this among which using the induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cell (nt-ESCs) are the most efficient strategies. The objective of this study was to evaluate the differentiation potential of the nt-ESCs to lymphoid lineage in the presence of IL-7, IL-3, FLT3-ligand and TPO growth factors in vitro. To this end, the nt-ESCs cells were prepared and treated with aforementioned growth factors for 7 and 14 days. Then, the cells were examined for expression of lymphoid markers (CD3, CD25, CD127 and CD19) by quantitative PCR (q-PCR) and flow cytometry. An increased expression of CD19 and CD25 markers was observed in the treated cells compared with the negative control samples by day 7. After 14 days, the expression level of all the tested CD markers significantly increased in the treated groups in comparison with the control. The current study reveals the potential of the nt-ESCs in differentiation to lymphoid lineage in the presence of defined growth factors.

  5. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer.

    Science.gov (United States)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

    2014-10-03

    Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  6. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.

    Science.gov (United States)

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-11-06

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

  7. Growth and hematologic characteristics of cloned dogs derived from adult somatic cell nuclear transfer.

    Science.gov (United States)

    Park, Jung Eun; Kim, Min Kyu; Kang, Jung Taek; Oh, Hyun Ju; Hong, So Gun; Kim, Dae Young; Jang, Goo; Lee, Byeong Chun

    2010-04-01

    Three viable female dogs, which have the same genotype, have been successfully produced by somatic cell nuclear transfer (SCNT); however, data on the growth pattern of cloned dogs are lacking. Thus, the aim of this study was (1) to assess growth parameters among those cloned dogs with measurement of body weight, height, and radiographic analysis of skull size and bone plate, and (2) to compare hematologic characteristics among the donor dog, cloned dogs, and age-matched control dogs. The cloned dogs were kept in the same environmental conditions. The body weight increased from 0.52, 0.46, and 0.52 kg at birth to 21.9, 22.9, and 20.4 kg at 68 weeks of age for individual cloned dogs, respectively. The withers height increased from 34.5, 32.6, and 35.2 cm at 8 weeks of age to 67.1 cm at 68 weeks of age in the three clones. The radiographic data demonstrated that patterns of bone growth were similar among cloned dogs, and all measured parameters of matured cloned dogs were similar with that of the fully grown donor dog. An age-specific pattern was identified on hematologic and serum biochemical measurements in both cloned dogs and age-matched controls. The parameters examined were within the normal reference ranges for healthy dogs. In conclusion, three genetically identical cloned dogs showed similar growth characteristics and had normal hematological and serum biochemical parameters.

  8. Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer.

    Science.gov (United States)

    Koo, Ok Jae; Hossein, Mohammad Shamim; Hong, So Gun; Martinez-Conejero, Jose A; Lee, Byeong Chun

    2009-02-01

    Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents - dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) - on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

  9. Optical pumping and population transfer of nuclear-spin states of caesium atoms in high magnetic fields

    Institute of Scientific and Technical Information of China (English)

    Luo Jun; Sun Xian-Ping; Zeng Xi-Zhi; Zhan Ming-Sheng

    2007-01-01

    Nuclear-spin states of gaseous-state Cs atoms in the ground state are optically manipulated using a Ti:sapphire laser in a magnetic field of 1.516 T, in which optical coupling of the nuclear-spin states is achieved through hyperfine interactions between electrons and nuclei. The steady-state population distribution in the hyperfine Zeeman sublevels of the ground state is detected by using a tunable diode laser. Furthermore, the state population transfer among the of Cs in the ground state due to stochastic collisions between Cs atoms and buffer-gas molecules, is studied at different of the hyperfine interaction can strongly cause the state population transfer and spin-state interchange among the hyperfine Zeeman sublevels. The calculated results maybe explain the steady-state population in hyperfine Zeeman sublevels in terms of rates of optical-pumping, electron-spin flip, nuclear spin flip, and electron-nuclear spin flip-flop transitions among the hyperfine Zeeman sublevels of the ground state of Cs atoms. This method may be applied to the nuclear-spin-based solid-state quantum computation.

  10. Role of hydrogen-bonding and photoinduced electron transfer (PET) on the interaction of resorcinol based acridinedione dyes with Bovine Serum Albumin (BSA) in water

    Energy Technology Data Exchange (ETDEWEB)

    Kumaran, Rajendran, E-mail: kumaranwau@rediffmail.com [Department of Chemistry, Dwaraka Doss Goverdhan Doss, Vaishnav College (Autonomous), 833, Gokul Bagh, E.V.R. Periyar Road, Arumbakkam, Chennai 600106, Tamil Nadu (India); Vanjinathan, Mahalingam [Department of Chemistry, Dwaraka Doss Goverdhan Doss, Vaishnav College (Autonomous), 833, Gokul Bagh, E.V.R. Periyar Road, Arumbakkam, Chennai 600106, Tamil Nadu (India); Ramamurthy, Perumal [National Centre for Ultrafast Processes, University of Madras, Taramani Campus Chennai 600113, Tamil Nadu (India)

    2015-08-15

    Resorcinol based acridinedione (ADDR) dyes are a class of laser dyes and have structural similarity with purine derivatives, nicotinamide adenine dinucleotide (NADH) analogs. These dyes are classified into photoinduced electron transfer (PET) and non-photoinduced electron transfer dyes, and the photophysical properties of family of these dyes exhibiting PET behavior are entirely different from that of non-PET dyes. The PET process in ADDR dyes is governed by the solvent polarity such that an ADDR dye exhibits PET process through space in an aprotic solvent like acetonitrile and does not exhibit the same in protic solvents like water and methanol. A comparison on the fluorescence emission, lifetime and nature of interaction of various ADDR dyes with a large globular protein like Bovine Serum Albumin (BSA) was carried out in aqueous solution. The interaction of PET based ADDR dyes with BSA in water is found to be largely hydrophobic, but hydrogen-bonding interaction of BSA with dye molecule influences the fluorescence emission of the dye and shifts the emission towards red region. Fluorescence spectral studies reveal that the excited state properties of PET based ADDR dyes are largely influenced by the addition of BSA. The microenvironment around the dye results in significant change in the fluorescence lifetime and emission. Fluorescence enhancement with a red shift in the emission results after the addition of BSA to ADDR dyes containing free amino hydrogen in the 10th position of basic acridinedione dye. The amino hydrogen (N–H) in the 10th position of ADDR dye is replaced by methyl group (N–CH{sub 3}), a significant decrease in the fluorescence intensity with no apparent shift in the emission maximum was observed after the addition of BSA. The nature of interaction between ADDR dyes with BSA is hydrogen-bonding and the dye remains unbound even at the highest concentration of BSA. Circular Dichroism (CD) studies show that the addition of dye to BSA results in

  11. Early pregnancy diagnosis by serum progesterone and ultrasound in sheep carrying somatic cell nuclear transfer-derived pregnancies.

    Science.gov (United States)

    Alexander, B; Coppola, G; Mastromonaco, G F; St John, E; Reyes, E R; Betts, D H; King, W A

    2008-04-01

    Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)-derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus-synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post-estrus (day 7 sample) and 19 days post-estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.

  12. Factors Affecting the Efficiency of Embryonic Cell Nuclear Transfer in Rabbits

    Institute of Scientific and Technical Information of China (English)

    CUI Kui-qing; LIU Qing-you; XIE Ying; WEI Jing-wei; SHI De-shun

    2005-01-01

    Factors affecting the efficiency of nuclear transfer (NT) in rabbits were examined in the present study. When 100 V mm-1of pulse strength and 15 μs of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses (P<0.05), but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups (P> 0.05). When the duration and number of electronic pulse were fixed at 15 μs and 3 times, increase of pulse intensity from 100 V mm-1 to 150 V mm-1 and 200 V mm-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos (P < 0.05), although the fusion rate did not significantly differ among the three groups (P > 0.05). Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage (P < 0.05), although the fusion rate was similar (P > 0.05). Activation of cytoplasts prior to fusion increased the cleavage rate (P < 0.05) and blastocyst development (P < 0.05) of reconstructed embryos, but decreased the fusion rate (P < 0.05) compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM + 3% OCS at the first 48 h and then cultured in TCM199+ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199+ 10% FCS or TCM199+ 3% OCS (P < 0.05). When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.

  13. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer

    OpenAIRE

    Mingru Yin; Weihua Jiang; Zhenfu Fang; Pengcheng Kong; Fengying Xing; Yao Li; Xuejin Chen; Shangang Li

    2015-01-01

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT....

  14. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

    OpenAIRE

    Young-Hee Jeong; Hanlin Lu; Chi-Hun Park; Meiyan Li; Huijuan Luo; Joung Joo Kim; Siyang Liu; Kyeong Hee Ko; Shujia Huang; In Sung Hwang; Mi Na Kang; Desheng Gong; Kang Bae Park; Eun Ji Choi; Jung Hyun Park

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, e...

  15. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  16. Trichostatin A rescues the disrupted imprinting induced by somatic cell nuclear transfer in pigs.

    Directory of Open Access Journals (Sweden)

    Yanjun Huan

    Full Text Available Imprinting disorders induced by somatic cell nuclear transfer (SCNT usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA, and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals.

  17. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

    Science.gov (United States)

    Zhou, Xiaoqing; Xin, Jige; Fan, Nana; Zou, Qingjian; Huang, Jiao; Ouyang, Zhen; Zhao, Yu; Zhao, Bentian; Liu, Zhaoming; Lai, Sisi; Yi, Xiaoling; Guo, Lin; Esteban, Miguel A; Zeng, Yangzhi; Yang, Huaqiang; Lai, Liangxue

    2015-03-01

    The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

  18. Cloning missy: obtaining multiple offspring of a specific canine genotype by somatic cell nuclear transfer.

    Science.gov (United States)

    Hossein, Mohammad Shamim; Jeong, Yeon Woo; Park, Sun Woo; Kim, Joung Joo; Lee, Eugine; Ko, Kyeong Hee; Kim, Huen Suk; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hawthorne, Lou; Hwang, Woo Suk

    2009-03-01

    The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

  19. Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer.

    Science.gov (United States)

    Cong, Peiqing; Zhu, Kongju; Ji, Qianqian; Zhao, Haijing; Chen, Yaosheng

    2013-09-01

    Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

  20. Nuclear dynamics investigation of the initial electron transfer in the cyclobutane pyrimidine dimer lesion repair process by photolyases

    CERN Document Server

    Joubert-Doriol, Loic; Olivucci, Massimo; Izmaylov, Artur F

    2016-01-01

    Photolyases are proteins capable of harvesting the sunlight to repair DNA damages caused by UV light. In this work we focus on the first step in the repair process of the cyclobutane pyrimidine dimer photoproduct (CPD) lesion, which is an electron transfer (ET) from a flavine cofactor to CPD, and study the role of various nuclear degrees of freedom (DOF) in this step. The ET step has been experimentally studied using transient spectroscopy and the corresponding data provide excellent basis for testing the quality of quantum dynamical models. Based on previous theoretical studies of electronic structure and conformations of the protein active site, we present a procedure to build a diabatic Hamiltonian for simulating the ET reaction in a molecular complex mimicking the enzyme's active site. We generate a reduced nuclear dimensional model that provides a first non-empirical quantum dynamical description of the structural features influencing the ET rate. By varying the nuclear DOF parametrization in the model t...

  1. Conventional and Bimodal Nuclear Thermal Rocket (NTR) Artificial Gravity Mars Transfer Vehicle Concepts

    Science.gov (United States)

    Borowski, Stanley K.; McCurdy, David R.; Packard, Thomas W.

    2016-01-01

    A variety of countermeasures have been developed to address the debilitating physiological effects of zero-gravity (0-g) experienced by cosmonauts and astronauts during their approximately 0.5 to 1.2 year long stays in low Earth orbit (LEO). Longer interplanetary flights, combined with possible prolonged stays in Mars orbit, could subject crewmembers to up to approximately 2.5 years of weightlessness. In view of known and recently diagnosed problems associated with 0-g, an artificial gravity (AG) spacecraft offers many advantages and may indeed be an enabling technology for human flights to Mars. A number of important human factors must be taken into account in selecting the rotation radius, rotation rate, and orientation of the habitation module or modules. These factors include the gravity gradient effect, radial and tangential Coriolis forces, along with cross-coupled acceleration effects. Artificial gravity Mars transfer vehicle (MTV) concepts are presented that utilize both conventional NTR, as well as, enhanced bimodal nuclear thermal rocket (BNTR) propulsion. The NTR is a proven technology that generates high thrust and has a specific impulse (Isp) capability of approximately 900 s-twice that of today's best chemical rockets. The AG/MTV concepts using conventional Nuclear Thermal Propulsion (NTP) carry twin cylindrical International Space Station (ISS)- type habitation modules with their long axes oriented either perpendicular or parallel to the longitudinal spin axis of the MTV and utilize photovoltaic arrays (PVAs) for spacecraft power. The twin habitat modules are connected to a central operations hub located at the front of the MTV via two pressurized tunnels that provide the rotation radius for the habitat modules. For the BNTR AG/MTV option, each engine has its own closed secondary helium(He)-xenon (Xe) gas loop and Brayton Rotating Unit (BRU) that can generate 10s of kilowatts (kWe) of spacecraft electrical power during the mission coast phase

  2. Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.

  3. Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer.

    Science.gov (United States)

    Pan, DengKe; Zhang, Li; Zhou, YanRong; Feng, Chong; Long, Chuan; Liu, Xiao; Wan, Rong; Zhang, Jian; Lin, AiXing; Dong, EnQiu; Wang, ShuChen; Xu, HouGang; Chen, HongXing

    2010-04-01

    Omega-3(omega-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of omega-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.

  4. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

    Science.gov (United States)

    Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

    2003-01-01

    The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei

  5. Effect of histone acetylation modification with MGCD0103, a histone deacetylase inhibitor, on nuclear reprogramming and the developmental competence of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

    2017-01-01

    Cloning remains as an important technique to enhance the reconstitution and distribution of animal population with high-genetic merit. One of the major detrimental factors of this technique is the abnormal epigenetic modifications. MGCD0103 is known as a histone deacetylase inhibitor. In this study, we investigated the effect of MGCD0103 on the in vitro blastocyst formation rate in porcine somatic cell nuclear transferred (SCNT) embryos and expression in acetylation of the histone H3 lysine 9 and histone H4 lysine 12. We compared the in vitro embryonic development of SCNT embryos treated with different concentrations of MGCD0103 for 24 hours. Our results reported that treating with 0.2-μM MGCD0103 for 24 hours effectively improved the development of SCNT embryos, in comparison to the control group (blastocyst formation rate, 25.5 vs. 10.7%, P transferred into two surrogate sows, one of whom became pregnant and three fetuses developed. These results suggest that MGCD0103 can enhance the nuclear reprogramming and improve in vitro developmental potential of porcine SCNT embryos.

  6. Transfer of elements relevant to nuclear fuel cycle from soil to boreal plants and animals in experimental meso- and microcosms

    Energy Technology Data Exchange (ETDEWEB)

    Tuovinen, Tiina S., E-mail: tiina.tuovinen@uef.fi [Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Kasurinen, Anne; Häikiö, Elina [Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland); Tervahauta, Arja [Department of Biology, University of Eastern Finland, P.O. Box FI-70211, Kuopio (Finland); Makkonen, Sari; Holopainen, Toini; Juutilainen, Jukka [Department of Environmental Science, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio (Finland)

    2016-01-01

    Uranium (U), cobalt (Co), molybdenum (Mo), nickel (Ni), lead (Pb), thorium (Th) and zinc (Zn) occur naturally in soil but their radioactive isotopes can also be released into the environment during the nuclear fuel cycle. The transfer of these elements was studied in three different trophic levels in experimental mesocosms containing downy birch (Betula pubescens), narrow buckler fern (Dryopteris carthusiana) and Scandinavian small-reed (Calamagrostis purpurea ssp. Phragmitoides) as producers, snails (Arianta arbostorum) as herbivores, and earthworms (Lumbricus terrestris) as decomposers. To determine more precisely whether the element uptake of snails is mainly via their food (birch leaves) or both via soil and food, a separate microcosm experiment was also performed. The element uptake of snails did not generally depend on the presence of soil, indicating that the main uptake route was food, except for U, where soil contact was important for uptake when soil U concentration was high. Transfer of elements from soil to plants was not linear, i.e. it was not correctly described by constant concentration ratios (CR) commonly applied in radioecological modeling. Similar nonlinear transfer was found for the invertebrate animals included in this study: elements other than U were taken up more efficiently when element concentration in soil or food was low. - Highlights: • We studied transfer of elements in boreal food chain using meso- and microcosms. • Elements related to nuclear fuel cycle and mining were examined. • Higher uptake at lower soil concentrations was observed for primary producers. • Snails took up elements mainly from food but for U also soil was an element source. • Non-linear transfer of essential elements was observed for herbivore and decomposer.

  7. Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.

    Science.gov (United States)

    Lagutina, Irina; Lazzari, Giovanna; Duchi, Roberto; Turini, Paola; Tessaro, Irene; Brunetti, Dario; Colleoni, Silvia; Crotti, Gabriella; Galli, Cesare

    2007-01-01

    Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.

  8. Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Wei Deng

    Full Text Available Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3% formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.

  9. Production of hGFAP-DsRed transgenic Guangxi Bama mini-pigs via somatic cell nuclear transfer.

    Science.gov (United States)

    Hu, L L; Lu, Y Q; Xu, H Y; Yang, X G; Lu, S S; Lu, K H

    2015-12-08

    The mini-pig is a useful animal model for human biomedical research due to its physiological similarity to humans and the ease of handling. In order to optimize the efficiency of production of transgenic Bama mini-pigs through somatic cell nuclear transfer (SCNT), we examined the effects of contact inhibition, roscovitine treatment, and serum starvation on the cell cycle synchronization and transgenic cloned embryo development in vivo and in vitro after nuclear transfer. The analysis showed that the rates of G0/G1 stage cells in the contact inhibition (92.11%) and roscovitine treatment groups (89.59%) were significantly higher than in serum starvation group (80.82%). A higher rate of apoptosis was seen in the serum starvation group (14.13%) compared to the contact inhibition and roscovitine treatment groups (6.71 and 2.46% respectively, P transferred to surrogate sows; one pregnancy was established and three embryos from the roscovitine treatment group successfully completed gestation. These results indicate that the roscovitine treatment was more effective at synchronizing transgenic kidney cells in Bama mini-pigs and allowed reconstructed embryos to develop to full term.

  10. Sensors and nuclear power. Report by the Technology Transfer Sensors Task Team

    Energy Technology Data Exchange (ETDEWEB)

    1985-06-01

    The existing sensor systems for the basic process parameters in nuclear power plant operation have limitations with respect to accuracy, ease of maintenance and signal processing. These limitations comprise the economy of nuclear power generation. To reduce the costs and improve performance of nuclear power plant fabrication, operation, maintenance and repair we need to advance the sensor technology being applied in the nuclear industry. The economic viability and public acceptance of nuclear power will depend on how well we direct and apply technological advances to the industry. This report was prepared by a team with members representing a wide range of the nuclear industry embracing the university programs, national laboratories, architect engineers and reactor manufacturers. An intensive effort was made to survey current sensor technology, evaluate future trends and determine development needs. This included literature surveys, visits with utilities, universities, laboratories and organizations outside the nuclear industry. Several conferences were attended to take advantage of the access to experts in selected topics and to obtain opinions. Numerous telephone contacts and exchanges by mail supplemented the above efforts. Finally, the broad technical depth of the team members provided the basis for the stimulating working sessions during which this report was organized and drafted.

  11. Nuclear magnetic resonance study of charge transfer complex formation between Silver Nitrate and Benzylcyanide in Solvent Ethylene Glycol

    CERN Document Server

    Modarress, H

    2003-01-01

    The formation constant for charge transfer complexes between electron acceptor (AgNo sub 3) and electron donor benzylcyanide (C sub 6 H sub 5 -CH sub 2 -C ident to N) in solvent ethyleneglycol [(CH sub 2 OH) sub 2] has been evaluated by using the nuclear magnetic resonance chemical shifts of aromatic group of benzylcyanide measured against external references, tetramethylsilane, hexamethyldisilane and cyclohexane at 20 sup d ig sup C. The external referencing procedure eliminated the interference of internal reference in the course of complexation. The necessary bulk magnetic susceptibility corrections on the measured chemical shifts have been made. The solution nationalised and their effects on the formation constant have been considered and a new equation has been suggested to obtain the main ionic activity coefficient of AgNO sub 3 from nuclear magnetic resonance results. The mean ionic activity coefficient has been taken into account in the formation constant calculations. The results indicated that the a...

  12. Conventional and Bimodal Nuclear Thermal Rocket (NTR) Artificial Gravity Mars Transfer Vehicle Concepts

    Science.gov (United States)

    Borowski, Stanley K.; McCurdy, David R.; Packard, Thomas W.

    2014-01-01

    A variety of countermeasures have been developed to address the debilitating physiological effects of "zero-gravity" (0-g) experienced by cosmonauts and astronauts during their approximately 0.5-1.2 year long stays in LEO (Low Earth Orbit). Longer interplanetary flights, combined with possible prolonged stays in Mars orbit, could subject crewmembers to up to approximately 2.5 years of weightlessness. In view of known and recently diagnosed problems associated with 0-g, an artificial gravity spacecraft offers many advantages and may indeed be an enabling technology for human flights to Mars. A number of important human factors must be taken into account in selecting the rotation radius, rotation rate, and orientation of the habitation module or modules. These factors include the gravity gradient effect, radial and tangential Coriolis forces, along with cross-coupled acceleration effects. Artificial gravity (AG) Mars transfer vehicle (MTV) concepts are presented that utilize both conventional NTR, as well as, enhanced "bimodal" nuclear thermal rocket (BNTR) propulsion. The NTR is a proven technology that generates high thrust and has a specific impulse (I (sub sp)) capability of approximately 900 s - twice that of today's best chemical rockets. The AG/MTV concepts using conventional NTP carry twin cylindrical "ISS-type" habitation modules with their long axes oriented either perpendicular or parallel to the longitudinal spin axis of the MTV and utilize photovoltaic arrays (PVAs) for spacecraft power. The twin habitat modules are connected to a central operations hub located at the front of the MTV via two pressurized tunnels that provide the rotation radius for the habitat modules. For the BNTR AG/MTV option, each engine has its own "closed" secondary helium-xenon gas loop and Brayton rotating unit that can generate tens of kilowatts (kW (sub e)) of spacecraft electrical power during the mission coast phase eliminating the need for large PVAs. A single inflatable

  13. Engineering design elements of a two-phase thermosyphon to transfer nuclear thermal energy to a hydrogen plant

    Science.gov (United States)

    Sabharwall, Piyush

    Two hydrogen production processes, both powered by Next Generation Nuclear Plant (NGNP), are currently under investigation at the Idaho National Laboratory. The first is high-temperature steam electrolysis utilizing both heat and electricity and the second is thermo-chemical production through the sulfur-iodine process primarily utilizing heat. Both processes require high temperature (>850°C) for enhanced efficiency; temperatures indicative of NGNP. Safety and licensing mandates prudently dictate that the NGNP and the hydrogen production facility be physically isolated, perhaps requiring separation of over 100m. There are several options to transferring multi-megawatt thermal power over such a distance. One option is simply to produce only electricity, transfer by wire to the hydrogen plant, and then reconvert the electric energy to heat via Joule or induction heating. Electrical transport, however, suffers energy losses of 60-70% due to the thermal to electric conversion inherent in the Brayton cycle. A second option is thermal energy transport via a single-phase forced convection loop where a fluid is mechanically pumped between heat exchangers at the nuclear and hydrogen plants. High temperatures, however, present unique materials and pumping challenges. Single phase, low pressure helium is an attractive option for NGNP, but is not suitable for a single purpose facility dictated to hydrogen production because low pressure helium requires higher pumping power and makes the process very inefficient. A third option is two-phase heat transfer utilizing a high temperature thermosyphon. Heat transport occurs via evaporation and condensation, and the heat transport fluid is re-circulated by gravitational force. Thermosyphon has the capability to transport heat at high rates over appreciable distances, virtually isothermally and without any requirement for external pumping devices. For process heat, intermediate heat exchangers (IHX) are desired to transfer heat from

  14. The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells.

    Science.gov (United States)

    Zhou, X; Ringseis, R; Wen, G; Eder, K

    2015-06-01

    Carnitine uptake into tissues is mediated mainly by the novel organic cation transporter 2 (OCTN2), whose expression is upregulated in the liver of early-lactating dairy cows. It has been shown recently that pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), stimulate OCTN2 expression and carnitine uptake in intestinal cells and inflamed intestinal mucosa. Given that many early-lactating dairy cows show typical signs of hepatic and systemic inflammation, such as elevated concentrations of circulating TNFα and activation of the key regulator of inflammation, nuclear factor κB (NF-κB), in tissues, it is possible that upregulation of OCTN2 and increase of carnitine uptake by TNFα is mediated by NF-κB, a mechanism that might contribute to the upregulation of OCNT2 in the liver of early-lactating dairy cows. Thus, in the present study, we tested the hypothesis that TNFα stimulates OCTN2 gene expression and carnitine uptake via NF-κB in the bovine Madin-Darby bovine kidney (MDBK) cell line. Treatment with TNFα caused activation of NF-κB, increased the mRNA and protein concentration of OCTN2, and stimulated the uptake of carnitine in MDBK cells. In contrast, combined treatment of MDBK cells with TNFα and the NF-κB inhibitor BAY 11-7085 completely blocked the effect of TNFα on OCTN2 mRNA and protein concentration and uptake of carnitine. These findings suggest that the bovine OCTN2 gene and carnitine uptake are regulated by NF-κB. Future studies are required to show the in vivo relevance of this regulatory mechanism in cattle.

  15. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

    Science.gov (United States)

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.

  16. Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors.

    Science.gov (United States)

    Johannesson, Bjarki; Sagi, Ido; Gore, Athurva; Paull, Daniel; Yamada, Mitsutoshi; Golan-Lev, Tamar; Li, Zhe; LeDuc, Charles; Shen, Yufeng; Stern, Samantha; Xu, Nanfang; Ma, Hong; Kang, Eunju; Mitalipov, Shoukhrat; Sauer, Mark V; Zhang, Kun; Benvenisty, Nissim; Egli, Dieter

    2014-11-06

    The recent finding that reprogrammed human pluripotent stem cells can be derived by nuclear transfer into human oocytes as well as by induced expression of defined factors has revitalized the debate on whether one approach might be advantageous over the other. Here we compare the genetic and epigenetic integrity of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal, and adult origin. The two cell types showed similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs had comparable numbers of de novo coding mutations, but significantly more than parthenogenetic ESCs. As iPSCs, NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of derivation approach.

  17. Comparison of in vitro fertilization and nuclear transfer techniques in the production of buffalo pre-implantation embryos

    Directory of Open Access Journals (Sweden)

    L.C. Cruz

    2010-02-01

    Full Text Available This study was conducted to evaluate the timing of cleavage and blastocoel formation of in vitro fertilized and nuclear transfer embryos and to compare the efficiency of in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT techniques in the production of buffalo embryos under the same culture system. Cumulus oocyte complexes (COCs were matured in vitro for 22 and 24 hr for SCNT and IVF, respectively. For IVF, COCs were inseminated with frozen-thawed semen and cultured in modified synthetic oviductal fluid supplemented with amino acids for 7 days. For SCNT, ear skin fibroblasts were inserted individually into enucleated oocytes, fused electrically, and activated with ethanol followed by cycloheximide treatment for 6 hr before culture in the same condition as IVF embryos. The results showed that SCNT embryos cleaved and formed blastocoel earlier than IVF embryos. The cleavage rate is significantly higher in SCNT embryos; while the blastocyst formation rate of IVF embryos is significantly higher (P<0.01 than SCNT embryos. The shorter time taken from cleavage to blastocoel formation by NT embryos as compared to in vitro fertilized ones suggests a difference in their developmental kinetics. The difference in the developmental competence between NT and IVF embryos warrants further investigation.

  18. The Transfer and diffusion of Cesium 137 within forest ecosystem in Fukushima after the nuclear power plant accident

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Takahiro; Murakami, Masashi [Community Ecology Lab., Biology Course, Faculty of Science, Chiba University, Chiba, 263-8522 (Japan); Ishii, Nobuyoshi [National Institute of Radiological Sciences, Chiba, 263-8555 (Japan); Tanoi, Keitaro; Hirose, Atsushi; Ohte, Nobuhito [Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 (Japan)

    2014-07-01

    A large amount of radionuclides was released from the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident after the disastrous earthquake and subsequent tsunami of March 2011. Among the variety of radionuclides released from FDNPP, cesium 137 ({sup 137}Cs) is the most worrying radionuclide in the environment, with a half-life of 30 years. Since most of the Japanese land area is covered by forest, the distribution and transportation of radioactive materials within forest ecosystems should be conscientiously monitored. In Europe, many studies reported that the {sup 137}Cs deposition caused by the Chernobyl accident has still been distributed in the litter and soil layers and has become a source for the soil-to-plant transfer. Most of these studies emphasize the 'stability' of {sup 137}Cs within forest ecosystems, because {sup 137}Cs are considered to be strongly and immediately fixed in clay minerals. Even though there are many studies of the soil-to-plant transfer of {sup 137}Cs in forest after several years of Chernobyl accident, very initial distribution and transfer of {sup 137}Cs in food web within one to two years after the deposition in forest ecosystems have never been examined. The evaluation of the initial dynamics of {sup 137}Cs in forest ecosystems should be quite important because of the increasing stability of {sup 137}Cs after the deposition. The accumulation and transfer of {sup 137}Cs through food web within forest ecosystems were examined by collecting various organisms at forests in Fukushima. The {sup 137}Cs concentrations, natural Cs and K concentrations, and delta {sup 15}N of the specimens were measured to evaluate the occurrence of bioaccumulation or bio-diffusion of {sup 137}Cs through tropic interaction within forest ecosystem. {sup 137}Cs was highly concentrated on leaf litters which had been deposited in autumn 2010, before the accident. This accumulated {sup 137}Cs had transferred to higher trophic organisms mainly through

  19. Probing nuclear shell structure beyond the N=40 subshell using multiple Coulomb excitation and transfer experiments

    Energy Technology Data Exchange (ETDEWEB)

    Hellgartner, Stefanie Christine

    2015-11-13

    In this work, the N=40 subshell closure is investigated with two complementary methods using a radioactive {sup 72}Zn ISOLDE beam: One- and two-neutron transfer reactions and multiple Coulomb excitation. In the one-neutron transfer reaction, two new levels of {sup 73}Zn were discovered. The two-neutron transfer channel allowed to study the differential cross section of the ground state and the 2{sup +}{sub 1} state of {sup 74}Zn. In the Coulomb excitation experiment, the measured B(E2) values and quadrupole moments of {sup 72}Zn showed that the yrast states 0{sup +}{sub 1}, 2{sup +}{sub 1} and 4{sup +}{sub 1} are moderately collective. Contrary, the 0{sup +}{sub 2} state has a different structure, since it features a stronger closed N=40 configuration compared to the ground state.

  20. Higher resolution and higher momentum transfer; New insight into nuclear structure

    Science.gov (United States)

    Heisenberg, J. H.

    1987-10-01

    The measurements of high momentum transfer data allows extraction of properties of very specific orbits due to the characteristic momentum transfer dependence of their respective form factors. To fully use this sensitivity, form factors need to be mapped out up to >3.5 /fm. The bulk of the new results comes from weaker levels where resolution is essential not only to separate the levels from neighboring states but also to enhance the peak to background ratio. Progress in both of these criteria have produced experimental results that force us to go beyond the mean-field HF-approach. (AIP)

  1. Nuclear fragmentation energy and momentum transfer distributions in relativistic heavy-ion collisions

    Science.gov (United States)

    Khandelwal, Govind S.; Khan, Ferdous

    1989-01-01

    An optical model description of energy and momentum transfer in relativistic heavy-ion collisions, based upon composite particle multiple scattering theory, is presented. Transverse and longitudinal momentum transfers to the projectile are shown to arise from the real and absorptive part of the optical potential, respectively. Comparisons of fragment momentum distribution observables with experiments are made and trends outlined based on our knowledge of the underlying nucleon-nucleon interaction. Corrections to the above calculations are discussed. Finally, use of the model as a tool for estimating collision impact parameters is indicated.

  2. Transfer of elements relevant to nuclear fuel cycle from soil to boreal plants and animals in experimental meso- and microcosms.

    Science.gov (United States)

    Tuovinen, Tiina S; Kasurinen, Anne; Häikiö, Elina; Tervahauta, Arja; Makkonen, Sari; Holopainen, Toini; Juutilainen, Jukka

    2016-01-01

    Uranium (U), cobalt (Co), molybdenum (Mo), nickel (Ni), lead (Pb), thorium (Th) and zinc (Zn) occur naturally in soil but their radioactive isotopes can also be released into the environment during the nuclear fuel cycle. The transfer of these elements was studied in three different trophic levels in experimental mesocosms containing downy birch (Betula pubescens), narrow buckler fern (Dryopteris carthusiana) and Scandinavian small-reed (Calamagrostis purpurea ssp. Phragmitoides) as producers, snails (Arianta arbostorum) as herbivores, and earthworms (Lumbricus terrestris) as decomposers. To determine more precisely whether the element uptake of snails is mainly via their food (birch leaves) or both via soil and food, a separate microcosm experiment was also performed. The element uptake of snails did not generally depend on the presence of soil, indicating that the main uptake route was food, except for U, where soil contact was important for uptake when soil U concentration was high. Transfer of elements from soil to plants was not linear, i.e. it was not correctly described by constant concentration ratios (CR) commonly applied in radioecological modeling. Similar nonlinear transfer was found for the invertebrate animals included in this study: elements other than U were taken up more efficiently when element concentration in soil or food was low.

  3. Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

    Science.gov (United States)

    Kim, Sue; Park, Sun Woo; Hossein, Mohammad Shamim; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Eugine; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-05-01

    To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.

  4. High Power Nuclear Electric Propulsion (NEP) for Cargo and Propellant Transfer Missions in Cislunar Space

    Science.gov (United States)

    Falck, Robert D.; Borowski, Stanley K.

    2003-01-01

    The performance of Nuclear Electric Propulsion (NEP) in transporting cargo and propellant from Low Earth Orbit (LEO) to the first Earth-Moon Lagrange point (EML1) is examined. The baseline NEP vehicle utilizes a fission reactor system with Brayton power conversion for electric power generation to power multiple liquid hydrogen magnetoplasmadynamic (MPD) thrusters. Vehicle characteristics and performance levels are based on technology availability in a fifteen to twenty year timeframe. Results of numerical trajectory analyses are also provided.

  5. Desarrollo de embriones de bovino obtenidos por fecundación in vitro cultivados con células oviductales o medio condicionado y transferidos a hembras receptoras Bovine embryo development produced by in vitro fertilization cultured with oviductal cell or conditioned medium and transfer to recipients

    Directory of Open Access Journals (Sweden)

    M RATTO

    1999-01-01

    Full Text Available Se comparó el desarrollo in vitro de ovocitos obtenidos de ovarios de vaca de matadero, madurados, fecundados y cultivados in vitro bajo dos sistemas. Los ovocitos fueron cultivados en un medio de maduración a 39 °C, 5 % de CO2 y humedad relativa de 95 % durante 22 horas. Posteriormente, fueron incubados con espermatozoides seleccionados a través de una gradiente discontinua de Percoll. La tasa de maduración nuclear y fecundación fueron de 93,7 % (74/79 y 76,9 % (50/65 respectivamente. Un total de 252 ovocitos fecundados fueron cultivados in vitro. El porcentaje de desarrollo in vitro a las 2 días post-inseminación (embriones de 4-8 células fue de 62,7 % (64/102 para los cigotos cultivados con células oviductales y de 67 % (100/150 para los cultivados en medio condicionado (P0,05. El porcentaje de desarrollo de mórulas fue de 17,6 % (18/102 para los cigotos cultivados con células oviductales y de 13,3 % (20/150 para los cultivados con medio condicionado (P0,05. Se obtuvo una tasa de desarrollo del 15,7 % (16/102 de blastocistos para aquellos cigotos cultivados con células oviductales. No se obtuvo blastocistos a partir de cigotos cultivados en medio condicionado. Cuatro blastocistos fueron transferidos a dos hembras receptoras. A los 42 y 57 días se encontró la presencia de un feto en cada hembraThe in vitro development of matured and fertilized bovine oocytes was compared between two culture systems. Oocytes were collected by aspiration of follicles of 3-8 mm in diameter using an 18g needle. After morphological selection the oocytes were incubated at 39 0C, 5 % C02 y 95 % relative humidity, during 22 hours. Afterwards, oocytes were incubated with spermatozoa selected by Percoll gradient system. The rate of nuclear maturation and fertilization was 93,7 % (74/79 and 76,9 % (50/65, respectively. A total of 252 zygotes were cultured, 102 with oviductal cells and 150 in conditioned medium. The in vitro development on day 2 of culture

  6. Radiocesium transfer from hillslopes to the Pacific Ocean after the Fukushima Nuclear Power Plant accident: A review.

    Science.gov (United States)

    Evrard, Olivier; Laceby, J Patrick; Lepage, Hugo; Onda, Yuichi; Cerdan, Olivier; Ayrault, Sophie

    2015-10-01

    The devastating tsunami triggered by the Great East Japan Earthquake on March 11, 2011 inundated the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) resulting in a loss of cooling and a series of explosions releasing the largest quantity of radioactive material into the atmosphere since the Chernobyl nuclear accident. Although 80% of the radionuclides from this accidental release were transported over the Pacific Ocean, 20% were deposited over Japanese coastal catchments that are subject to frequent typhoons. Among the radioisotopes released during the FDNPP accident, radiocesium ((134)Cs and (137)Cs) is considered the most serious current and future health risk for the local population. The goal of this review is to synthesize research relevant to the transfer of FDNPP derived radiocesium from hillslopes to the Pacific Ocean. After radiocesium fallout deposition on vegetation and soils, the contamination may remain stored in forest canopies, in vegetative litter on the ground, or in the soil. Once radiocesium contacts soil, it is quickly and almost irreversibly bound to fine soil particles. The kinetic energy of raindrops instigates the displacement of soil particles, and their bound radiocesium, which may be mobilized and transported with overland flow. Soil erosion is one of the main processes transferring particle-bound radiocesium from hillslopes through rivers and streams, and ultimately to the Pacific Ocean. Accordingly this review will summarize results regarding the fundamental processes and dynamics that govern radiocesium transfer from hillslopes to the Pacific Ocean published in the literature within the first four years after the FDNPP accident. The majority of radiocesium is reported to be transported in the particulate fraction, attached to fine particles. The contribution of the dissolved fraction to radiocesium migration is only relevant in base flows and is hypothesized to decline over time. Owing to the hydro-meteorological context of the

  7. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  8. Experimental study on the heat transfer characteristics of a nuclear reactor containment wall cooled by gravitationally falling water

    Science.gov (United States)

    Pasek, Ari D.; Umar, Efrison; Suwono, Aryadi; Manalu, Reinhard E. E.

    2012-06-01

    Gravitationally falling water cooling is one of mechanism utilized by a modern nuclear Pressurized Water Reactor (PWR) for its Passive Containment Cooling System (PCCS). Since the cooling is closely related to the safety, water film cooling characteristics of the PCCS should be studied. This paper deals with the experimental study of laminar water film cooling on the containment model wall. The influences of water mass flow rate and wall heat rate on the heat transfer characteristic were studied. This research was started with design and assembly of a containment model equipped with the water cooling system, and calibration of all measurement devices. The containment model is a scaled down model of AP 1000 reactor. Below the containment steam is generated using electrical heaters. The steam heated the containment wall, and then the temperatures of the wall in several positions were measure transiently using thermocouples and data acquisition. The containment was then cooled by falling water sprayed from the top of the containment. The experiments were done for various wall heat rate and cooling water flow rate. The objective of the research is to find the temperature profile along the wall before and after the water cooling applied, prediction of the water film characteristic such as means velocity, thickness and their influence to the heat transfer coefficient. The result of the experiments shows that the wall temperatures significantly drop after being sprayed with water. The thickness of water film increases with increasing water flow rate and remained constant with increasing wall heat rate. The heat transfer coefficient decreases as film mass flow rate increase due to the increases of the film thickness which causes the increasing of the thermal resistance. The heat transfer coefficient increases slightly as the wall heat rate increases. The experimental results were then compared with previous theoretical studied.

  9. Development of the process of energy transfer from a nuclear Power Plant to an intermediate temperature electrolyse; Desarrollo del proceso de transferencia de energia desde una central nuclear a un electrolizador de temperatura intermedia

    Energy Technology Data Exchange (ETDEWEB)

    Munoz Cervantes, A.; Cuadrado Garcia, P.; Soraino Garcia, J.

    2013-07-01

    Fifty million tons of hydrogen are consumed annually in the world in various industrial processes. Among them, the ammonia production, oil refining and the production of methanol. One of the methods to produce it is the electrolysis of water, oxygen and hydrogen. This process needs electricity and steam which a central nuclear It can be your source; Hence the importance of developing the transfer process energy between the two. The objective of the study is to characterize the process of thermal energy transfer from a nuclear power plant to an electrolyzer of intermediate temperature (ITSE) already defined. The study is limited to the intermediate engineering process, from the central to the cell.

  10. Sensitizing solid state nuclear magnetic resonance of dilute nuclei by spin-diffusion assisted polarization transfers.

    Science.gov (United States)

    Lupulescu, Adonis; Frydman, Lucio

    2011-10-01

    Recent years have witnessed efforts geared at increasing the sensitivity of NMR experiments, by relying on the suitable tailoring and exploitation of relaxation phenomena. These efforts have included the use of paramagnetic agents, enhanced (1)H-(1)H incoherent and coherent transfers processes in 2D liquid state spectroscopy, and homonuclear (13)C-(13)C spin diffusion effects in labeled solids. The present study examines some of the opportunities that could open when exploiting spontaneous (1)H-(1)H spin-diffusion processes, to enhance relaxation and to improve the sensitivity of dilute nuclei in solid state NMR measurements. It is shown that polarization transfer experiments executed under sufficiently fast magic-angle-spinning conditions, enable a selective polarization of the dilute low-γ spins by their immediate neighboring protons. Repolarization of the latter can then occur during the time involved in monitoring the signal emitted by the low-γ nuclei. The basic features involved in the resulting approach, and its potential to improve the effective sensitivity of solid state NMR measurements on dilute nuclei, are analyzed. Experimental tests witness the advantages that could reside from utilizing this kind of approach over conventional cross-polarization processes. These measurements also highlight a number of limitations that will have to be overcome for transforming selective polarization transfers of this kind into analytical methods of choice.

  11. Transference patron for the calibration of activemeters for use in nuclear medicine; Patron de transferencia para la calibracion de activimetros de uso en medicina nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Cortes P, A.; Garcia D, O.C.; Ortiz P, I.; Becerril V, A. [Instituto nacional de Investigaciones Nucleares, Laboratorio de Patrones Radiactivos, Departamento de Metrologia, A.P. 18-1027. C.P. 11801 Mexico D.F. (Mexico)

    2000-07-01

    With the object to improve the calibration service of the activemetersof the users (nuclear medicine centres) so internal as external users at ININ and initiating the establishment of the traceability of the activity measures that carry out in the country, with respect to the National Patron of Nuclear Activity No. ININ-PNM-2, it is initiated the characterization and the performance control of the activemeter make Capintec, model CRC-7BT of the laboratory of Radioactive Patrons (LPR) of the Department of Metrology for to convert it in a transference patron. This characterization and control are based in the sustained review of the activemeter calibration for the I-131, Mo-99 and Sm-153 radioisotopes with activities from 100 mCi until some {mu} Ci and the Tc-99m, In-111, Tl-201, Ga-67, Cs-137, Co-60, and Ba-133 with activities of the range of some {mu} Ci. To verify the good performance of the instrument it was revised the linearity of its scale, the stability of readings, the variation of readings respect of the source position inside the activemeter well and it was determined the calibration factors for each one of the radiosotopes mentionated. The radioactive sources of mid-short life that are used which consist in radioactive solutions of 10 cm{sup 3} contained in polyethylene small bottles of 25 cm{sup 3} elaborated in the LPR and whose activity is measured with respect to the National Patron ININ-PNM-2. The sources of mid-large life are ampoules with 5 cm{sup 3} of radioactive solution, calibrated in activity by the primary laboratory, LNHB (formerly LMRI) of France. (Author)

  12. Corrigendum to "Coupled thermochemical, isotopic evolution and heat transfer simulations in highly irradiated UO2 nuclear fuel"

    Science.gov (United States)

    Piro, M. H. A.; Banfield, J.; Clarno, K.; Simunovic, S.; Besmann, T. M.; Lewis, B. J.; Thompson, W. T.

    2016-09-01

    Figs. 7-9 in "Coupled thermochemical, isotopic evolution and heat transfer simulations in highly irradiated UO2 nuclear fuel" [1] have a consistent error corresponding to the relative proportions of iodine. Reported concentrations of iodine in the original manuscript are approximately ten times higher than expected, and are comparable in atomic proportions to cesium. One would expect that the amount of cesium would be about one order of magnitude greater than iodine based on the difference in fission yields of 235U and 239Pu. A practical consequence of this error would affect the predicted quantity and chemical composition of iodine on the fuel surface, which is related to iodine-induced stress corrosion cracking [2].

  13. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    Science.gov (United States)

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  14. Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal.

    Science.gov (United States)

    Lee, Eunsong; Song, Kilyoung

    2007-12-01

    The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.

  15. Development capacity of pre- and postpubertal pig oocytes evaluated by somatic cell nuclear transfer and parthenogenetic activation

    DEFF Research Database (Denmark)

    Skovsgaard, Hanne; Li, Rong; Liu, Ying;

    2013-01-01

    Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear...... transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible...... perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6...

  16. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY... live bovines and products derived from bovines with regard to bovine spongiform encephalopathy. This... with regard to bovine spongiform encephalopathy. Comments on the proposed rule were required to......

  17. Nuclear thermal source transfer unit, post-blast soil sample drying system

    Energy Technology Data Exchange (ETDEWEB)

    Wiser, Ralph S. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Valencia, Matthew J [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-01-03

    Los Alamos National Laboratory states that its mission is “To solve national security challenges through scientific excellence.” [2] The Science Undergraduate Laboratory Internship (SULI) programs exists to engage undergraduate students in STEM work by providing opportunity to work at DOE facilities. [5] As an undergraduate mechanical engineering intern under the SULI program at Los Alamos during the fall semester of 2016, I had the opportunity to contribute to the mission of the Laboratory while developing skills in a STEM discipline. I worked with Technology Applications, an engineering group that supports non-proliferation, counter terrorism, and emergency response missions. This group specializes in tool design, weapons engineering, rapid prototyping, and mission training. I assisted with two major projects during my appointment Los Alamos. The first was a thermal source transportation unit, intended to safely contain a nuclear thermal source during transit. The second was a soil drying unit for use in nuclear postblast field sample collection. These projects have given me invaluable experience working alongside a team of professional engineers. Skills developed include modeling, simulation, group design, product and system design, and product testing.

  18. Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes).

    Science.gov (United States)

    Bubenshchikova, Ekaterina; Kaftanovskaya, Elena; Motosugi, Nami; Fujimoto, Takafumi; Arai, Katsutoshi; Kinoshita, Masato; Hashimoto, Hisashi; Ozato, Kenjiro; Wakamatsu, Yuko

    2007-12-01

    Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

  19. Mode of encapsulation of linezolid by β-cyclodextrin and its role in bovine serum albumin binding.

    Science.gov (United States)

    Natesan, Sudha; Sowrirajan, Chandrasekaran; Yousuf, Sameena; Enoch, Israel V M V

    2015-01-22

    We describe, in this article, the associative interaction between Linezolid and β-Cyclodextrin, and the influence of β-Cyclodextrin on Linezolid's binding to Bovine serum albumin. β-Cyclodextrin forms a 1:1 inclusion complex with Linezolid, with a binding constant value of 3.51×10(2)M(-1). The binding is studied using ultraviolet-visible absorption, fluorescence, nuclear magnetic resonance, and rotating-frame overhauser effect spectroscopic techniques. The amide substituent on the oxazolidinone ring of Linezolid is involved in its binding to β-Cyclodextrin. The binding of the Linezolid to bovine serum albumin, in the absence and the presence of β-Cyclodextrin, is studied by analyzing the fluorescence quenching and Förster resonance energy transfer. The Stern-Volmer quenching constant, the binding constant, and energy transfer occurring on the interaction of the Linezolid with BSA are found to be smaller in the presence of β-Cyclodextrin than in water.

  20. Photoprotection and triplet energy transfer in higher plants: the role of electronic and nuclear fluctuations.

    Science.gov (United States)

    Cupellini, Lorenzo; Jurinovich, Sandro; Prandi, Ingrid G; Caprasecca, Stefano; Mennucci, Benedetta

    2016-04-28

    Photosynthetic organisms employ several photoprotection strategies to avoid damage due to the excess energy in high light conditions. Among these, quenching of triplet chlorophylls by neighboring carotenoids (Cars) is fundamental in preventing the formation of singlet oxygen. Cars are able to accept the triplets from chlorophylls by triplet energy transfer (TET). We have here studied TET rates in CP29, a minor light-harvesting complex (LHC) of the Photosystem II in plants. A fully atomistic strategy combining classical molecular dynamics of the LHC in its natural environment with a hybrid time-dependent density functional theory/polarizable MM description of the TET is used. We find that the structural fluctuations of the pigment-protein complex can largely enhance the transfer rates with respect to those predicted using the crystal structure, reducing the triplet quenching times in the subnanosecond scale. These findings add a new perspective for the interpretation of the photoprotection function and its relation with structural motions of the LHC.

  1. Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

    Institute of Scientific and Technical Information of China (English)

    Wang ErYao; YU Yang; Li XueMei; JIAO LiHong; Wang Liu

    2007-01-01

    A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse activation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.

  2. A Multi-Dimensional Heat Transfer Model of a Tie-Tube and Hexagonal Fuel Element for Nuclear Thermal Propulsion

    Science.gov (United States)

    Gomez, C. F.; Mireles, O. R.; Stewart, E.

    2016-01-01

    The Space Capable Cryogenic Thermal Engine (SCCTE) effort considers a nuclear thermal rocket design based around a Low-Enriched Uranium (LEU) design fission reactor. The reactor core is comprised of bundled hexagonal fuel elements that directly heat hydrogen for expansion in a thrust chamber and hexagonal tie-tubes that house zirconium hydride moderator mass for the purpose of thermalizing fast neutrons resulting from fission events. Created 3D steady state Hex fuel rod model with 1D flow channels. Hand Calculation were used to set up initial conditions for fluid flow. The Hex Fuel rod uses 1D flow paths to model the channels using empirical correlations for heat transfer in a pipe. Created a 2-D axisymmetric transient to steady state model using the CFD turbulent flow and Heat Transfer module in COMSOL. This model was developed to find and understand the hydrogen flow that might effect the thermal gradients axially and at the end of the tie tube where the flow turns and enters an annulus. The Hex fuel rod and Tie tube models were made based on requirements given to us by CSNR and the SCCTE team. The models helped simplify and understand the physics and assumptions. Using pipe correlations reduced the complexity of the 3-D fuel rod model and is numerically more stable and computationally more time-efficient compared to the CFD approach. The 2-D axisymmetric tie tube model can be used as a reference "Virtual test model" for comparing and improving 3-D Models.

  3. [The radioecology of the grapevine. 1. The transfer of nuclear weapons fallout from the soil into wine].

    Science.gov (United States)

    Hellmuth, K H; Wagner, A; Fischer, E

    1989-04-01

    In a field investigation (1983-1985) comprising eight places of the most important viticultural regions in the Federal Republic of Germany, the contents of the radionuclides tritium (3H), carbon-14 (14C), strontium-90 (90Sr), and cesium-137 (137Cs) in air, soils, leaves of the vine, grapes and wine were measured and site-specific transfer factors were calculated. Data concerning soil parameters, climatic conditions, cultivation and vinification were collected. The tritium content of all samples was 10 Bq/l water of combustion, independent of location and year. The specific activity of 14C in the atmosphere and in biological material was 0.22 Bq/g carbon, independent of site and year. 90Sr contents of soils fluctuated between 0.7 and 3.5 Bq/kg dry matter. The mean content of leaves was 2 Bq/kg fresh material, of grapes 0.035 Bq/kg and of wine 0.008 Bq/l. 137Cs content of soils fluctuated between 1.3 and 7.9 Bq/kg dry matter. The mean content of leaves was 0.098 Bq/kg fresh material, of grapes 0.021 Bq/kg and of wine 0.0085 Bq/l. A relation between transfer of radionuclides and soil parameters and between the contents of grapes and wine was not recognizable. While cultivar-specific differences were not observed in grapes, red wines contained somewhat more 137Cs than white wines. Transfer factors soil grapes were 0.027 for 90Sr and 0.0057 for 137Cs. Site-specific influences such as soil parameters, climate, cultivation, vinification and differences between years led to a relatively small fluctuation of values. An influence of the nuclear power station Neckarwestheim has not been found in any of the radionuclides.

  4. Heat transfer in the core graphite structures of RBMK nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Knoglinger, E., E-mail: ernst.knoglinger@a1.net [Am Winklerwald 15, A 4020 Linz (Austria); Wölfl, H., E-mail: herbert.woelfl@tele2.at [Berg, Im Weideland 19, A 4060 Linz (Austria); Kaliatka, A., E-mail: algirdas.kaliatka@lei.lt [Laboratory of Nuclear Installation Safety, Lithuanian Energy Institute, Breslaujos 3, LT-44403 Kaunas (Lithuania)

    2015-11-15

    Highlights: • Proposed solution of heat transfer model from a hollow cylinder to a fluid through narrow duct. • Thermal conductance of annular gaps, filled by two component gas was discussed. • Xenon transient preceding the Chernobyl Accident was analyzed. • Reactivity balance during power manoeuvres and potenrial causes of the accident were discussed. - Abstract: Conductive and combined radiative/conductive gap conductance models are presented and discussed in great detail. The heat resistance concept and an exact solution to the one dimensional heat conduction equation for a 3-region composite hollow cylinder are used to calculate gap conductance in function of gap gas composition and fuel burn up. The study includes the back calculation of a reactor experiment performed at the Ignalina NPP Unit-1 which provides some insight in the function of the RBMK nitrogen supply and regulating device and an investigation of the role the graphite temperature played during the power manoeuvres preceding the Chernobyl Accident.

  5. Energy transfer and thermal conductivity through inert matrix and nuclear fuel analogous materials

    Energy Technology Data Exchange (ETDEWEB)

    Degueldre, C.; Heimgartner, P.; Graber, T. [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Takano, Masahide; Ohmichi, Toshihiko; Fukuda, Kousaku

    1997-11-01

    Thermal conductivity of zirconia based inert matrix and analogous fuel materials were measured and modelled. Measurements were performed using the laser flash method and systematically applied for binary and ternary zirconia-yttria-erbia as well as quaternary systems including thoria or ceria. Measurements were carried out from room temperature to 1250 K. Thermal conductivity was also modelled taking into account the dopant effect on the lattice parameter of the cubic solid solution, the oxygen vacancy size and their concentration. Experimental and modelled lattice parameter values are compared prior to full justification of the results. In the temperature range from 300 to 950 K, the thermal conductivity of the single phase solid solution with yttria, erbia and ceria as analogous fuel material is about 2 W{center_dot}m{sup -1}K{sup -1}. Energy transfer in these materials is discussed on the basis of both photonic and phononic conductivities. (author)

  6. Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning.

    Science.gov (United States)

    Yan, Hao; Yan, Zhonghai; Ma, Qingwen; Jiao, Fei; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2011-01-01

    Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.

  7. Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning

    Institute of Scientific and Technical Information of China (English)

    Hao Yan; Zhonghai Yan; Qingwen Ma; Fei Jiao; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2011-01-01

    Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.

  8. High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

    DEFF Research Database (Denmark)

    Li, J.; Østrup, Olga; Villemoes, Klaus

    2008-01-01

    Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim...

  9. Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    Qiuyan Li; Hengxi Wei; Ying Guo; Yan Li; Rui Zhao; Yufang Ma; Zhengquan Yu; Bo Tang; Lei Zhang; Yunping Dai; Ning Li

    2009-01-01

    Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, s1w3 and s1w6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after acti-vation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

  10. Hyperpolarization of 29Si by Resonant Nuclear Spin Transfer from Optically Hyperpolarized 31P Donors

    Science.gov (United States)

    Dluhy, Phillip; Salvail, Jeff; Saeedi, Kamyar; Thewalt, Mike; Simons, Stephanie

    2014-03-01

    Recent developments in nanomedicine have allowed nanoparticles of silicon containing hyperpolarized 29Si to be imaged in vivo using magnetic resonance imaging. The extremely long relaxation times and isotropy of the Si lattice make polarized 29Si isotopes ideal for these sorts of imaging methods. However, one of the major difficulties standing in the path of widespread adoption of these techniques is the slow rate at which the 29Si is hyperpolarized and the limited maximum hyperpolarization achievable. In this talk, I will describe an effective method for hyperpolarization of the 29Si isotopes using resonant optical pumping of the donor bound exciton transitions to polarize the 31P donor nuclei, and a choice of static magnetic field that conserves energy during spin flip flops between donor nuclear and 29Si spins to facilitate diffusion of this polarization. Using this method, we are able to polarize greater than 10% of the 29Si centers in 64 hours without seeing saturation of the 29Si polarization.

  11. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.

  12. Heat Transfer Salts for Nuclear Reactor Systems - Chemistry Control, Corrosion Mitigation, and Modeling

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Mark [Univ. of Wisconsin, Madison, WI (United States); Sridharan, Kumar [Univ. of Wisconsin, Madison, WI (United States); Morgan, Dane [Univ. of Wisconsin, Madison, WI (United States); Peterson, Per [Univ. of Wisconsin, Madison, WI (United States); Calderoni, Pattrick [Univ. of Wisconsin, Madison, WI (United States); Scheele, Randall [Univ. of Wisconsin, Madison, WI (United States); Casekka, Andrew [Univ. of Wisconsin, Madison, WI (United States); McNamara, Bruce [Univ. of Wisconsin, Madison, WI (United States)

    2015-01-22

    The concept of a molten salt reactor has existed for nearly sixty years. Previously all work was done during a large collaborative effort at Oak Ridge National Laboratory, culminating in a research reactor which operated for 15,000 hours without major error. This technical success has garnished interest in modern, high temperature, reactor schemes. Research using molten fluoride salts for nuclear applications requires a steady supply of high grade molten salts. There is no bulk supplier of research grade fluoride salts in the world, so a facility which could provide all the salt needed for testing at the University of Wisconsin had to be produced. Two salt purification devices were made for this purpose, a large scale purifier, and a small scale purifier, each designed to clean the salts from impurities and reduce their corrosion potential. As of now, the small scale has performed with flibe salt, hydrogen, and hydrogen fluoride, yielding clean salt. This salt is currently being used in corrosion testing facilities at the Massachusetts Institute of Technology and the University of Wisconsin. Working with the beryllium based salts requires extensive safety measures and health monitoring to prevent the development of acute or chronic beryllium disease, two pulmonary diseases created by an allergic reaction to beryllium in the lungs. Extensive health monitoring, engineering controls, and environment monitoring had to be set up with the University of Wisconsin department of Environment, Health and Safety. The hydrogen fluoride required for purification was also an extreme health hazard requiring thoughtful planning and execution. These dangers have made research a slow and tedious process. Simple processes, such as chemical handling and clean-up, can take large amounts of ingenuity and time. Other work has complemented the experimental research at Wisconsin to advance high temperature reactor goals. Modeling work has been performed in house to re

  13. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

  14. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    Directory of Open Access Journals (Sweden)

    Byungkuk Min

    2016-05-01

    Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

  15. Effect of radiocesium transfer on ambient dose rate in forest environments affected by the Fukushima Nuclear Power Plant accident

    Science.gov (United States)

    Kato, H.

    2015-12-01

    We investigated the transfer of canopy-intercepted radiocesium to the forest floor during 3 years following the Fukushima Daiichi Nuclear Power Plant accident. The cesium-137 (Cs-137) contents in throughfall, stemflow, and litterfall were monitored in two coniferous stands (plantation of Japanese cedar) and a deciduous broad-leaved forest stand (Japanese oak with red pine). We also measured the ambient dose rate (ADR) at different heights in the forest using a survey meter and a portable Ge gamma-ray detector. Total Cs-137 deposition flux from the canopy to forest floor for the mature cedar, young cedar, and the mixed broad-leaved stands were 166 kBq/m2, 174 kBq/m2, and 60 kBq/m2, respectively. These values correspond to 38%, 40% and 13% of total atmospheric input after the accident. The ambient dose rate in forest exhibited height dependency and its vertical distribution varied with forest type and stand age. The ambient dose rate showed an exponential decrease with time for all the forest sites, however the decreasing trend differed depending on the height of dose measurement and forest type. The ambient dose rate at the canopy (approx. 10 m-height) decreased faster than that expected from physical decay of the two radiocesium isotopes, whereas those at the forest floor varied between the three forest stands. The radiocesium deposition via throughfall seemed to increase ambient dose rate during the first 200 days after the accident, however there was no clear relationship between litterfall and ambient dose rate since 400 days after the accident. These data suggested that the ambient dose rate in forest environment varied both spatially and temporally reflecting the transfer of radiocesium from canopy to forest floor. However, further monitoring investigation and analysis are required to determine the effect of litterfall on long-term trend of ambient dose rate in forest environments.

  16. Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

    Science.gov (United States)

    Edwards, J L; Schrick, F N; McCracken, M D; van Amstel, S R; Hopkins, F M; Welborn, M G; Davies, C J

    2003-08-01

    In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

  17. Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs

    Directory of Open Access Journals (Sweden)

    Timothy P. Sheets

    2016-12-01

    Full Text Available The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR and associated nuclease Cas9 (CRISPR/Cas9, it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100% generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.

  18. An ideal oocyte activation protocol and embryo culture conditions for somatic cell nuclear transfer using sheep oocytes.

    Science.gov (United States)

    Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa

    2014-10-01

    Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.

  19. Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

    Institute of Scientific and Technical Information of China (English)

    Dongshan Yang; Jiangtian Tian; Feng Li; Jifeng Zhang; Lin Chang; Duanqing Pei; Y Eugene Chen; Liangxue Lai; Huaqiang Yang; Wei Li; Bentian Zhao; Zhen Ouyang; Zhaoming Liu; Yu Zhao; Nana Fan; Jun Song

    2011-01-01

    @@ Dear Editor, Gene targeting in mouse embryonic stem (ES) cells has revolutionized the field of mouse genetics and allowed for the analysis of diverse aspects of gene function in vivo.For more than two decades,researchers have been actively searching for ES cells from large animals such as pigs and cattle.Unfortunately,to date,no ES cell lines from large animals have passed the crucial test of germ line contribution.The sole method of gene targeting to date in these species remains somatic cell nuclear transfer (SCNT) combined with DNA homologous recombination (HR).Due to the limited proliferation competency and extremely low frequency of HR in somatic cells (less than 10-6),this process is highly inefficient and only a few successful examples have been achieved,even though enrichment strategies such as positivenegative marker selection,promoter-trap and adenoassociated viral delivery were previously used [1-3].The low efficiency of gene targeting in cultured somatic cells is the main barrier for gene targeting in large animals.Recently,zinc-finger nuclease (ZFN) technology has emerged as a powerful tool for genome editing.The success of ZFN technology for gene targeting in fruit flies,zebra fish,rodents as well as human cell lines encouraged us to establish a high-efficiency gene-targeting platform in large animals such as pigs [4-8].

  20. Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs.

    Science.gov (United States)

    Sheets, Timothy P; Park, Chi-Hun; Park, Ki-Eun; Powell, Anne; Donovan, David M; Telugu, Bhanu P

    2016-12-03

    The domestic pig is an ideal "dual purpose" animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.

  1. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...... nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four-cell...... and active cells at the eight-cell, 16-cell and blastocyst stage, respectively. In the 143 SCNT embryos investigated, all two-cell embryos (n = 34) and early four-cell embryos (n = 12) were also transcriptionally inactive. At the late four-cell stage (n = 33) and at the eight-cell stage (n = 24) there were...

  2. Day-3 Medium Changes can Affect Developmental Potential of Porcine Somatic Cell Nuclear Transfer and Parthenogenesis Embryos In Vitro

    Directory of Open Access Journals (Sweden)

    Dibyendu Biswas and Sang Hwan Hyun*

    2011-01-01

    Full Text Available The aim of the present study was to compare the developmental competence of porcine parthenotes and somatic cell nuclear transfer (SCNT embryos after day-3 medium change with fresh embryo culture medium to that of embryos that did not have a medium change (monoculture system. The parthenogenetic and SCNT blastocyst formation rates were significantly (P<0.05 higher in the no-medium-change group (43.3±2.3, 18.5±1.1%, respectively compared with the day-3 medium-change group (35.9±2.4, 7.9±0.9%, respectively. Total cell number in parthenotes and SCNT blastocysts was also significantly (P<0.05 higher in the no-media-change group (92.0±4.2, 66.9±7.7, respectively compared with the media-change group (81.5±3.1, 46.6±4.9, respectively. No significant difference in cleavage rate was found in either group for parthenotes or SCNT embryos. This result suggests that day-3 medium changes have negative effects on porcine parthenotes and SCNT embryos in vitro.

  3. Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells.

    Science.gov (United States)

    Wei, Hongjiang; Qing, Yubo; Pan, Weirong; Zhao, Hongye; Li, Honghui; Cheng, Wenmin; Zhao, Lu; Xu, Chengsheng; Li, Hong; Li, Si; Ye, Lei; Wei, Taiyun; Li, Xiaobing; Fu, Guowen; Li, Wengui; Xin, Jige; Zeng, Yangzhi

    2013-01-01

    Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4%) and newborn (76.4% and 21.8%) fibroblasts of the Banna miniature inbred pig (P>0.05). However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; Pcells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells.

  4. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-08-09

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

  5. Morphological abnormalities, impaired fetal development and decrease in myostatin expression following somatic cell nuclear transfer in dogs.

    Science.gov (United States)

    Hong, Il-Hwa; Jeong, Yeon-Woo; Shin, Taeyoung; Hyun, Sang-Hwan; Park, Jin-Kyu; Ki, Mi-Ran; Han, Seon-Young; Park, Se-Il; Lee, Ji-Hyun; Lee, Eun-Mi; Kim, Ah-Young; You, Sang-Young; Hwang, Woo-Suk; Jeong, Kyu-Shik

    2011-05-01

    Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT-cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down-regulated at the mRNA level in tongues and skeletal muscles of SCNT-cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT-cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates.

  6. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

  7. Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy.

    Science.gov (United States)

    Kowalik, Magdalena K; Slonina, Dominika; Rekawiecki, Robert; Kotwica, Jan

    2013-03-01

    Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P<0.001) in pregnant animals. SERBP1 mRNA expression was increased (P<0.05), while the level of this protein was decreased (P<0.05) on days 11-16 of the estrous cycle. The expression of PGR mRNA was higher (P<0.01) on days 17-20 compared to days 6-10 and 11-16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1-5 and 17-20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy.

  8. Effects of donor cells on in vitro development of cloned bovine embryos

    Institute of Scientific and Technical Information of China (English)

    Jing Fu; Pengfei Guan; Leiwen Zhao; Hua Li; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2008-01-01

    The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

  9. DNA methylation in porcine preimplantation embryos developed in-vivo or produced by in-vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  10. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  11. DNA methylation in porcine preimplantation embryos developed in vivo or produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV......), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  12. Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa).

    Science.gov (United States)

    Zhang, Ting-Yu; Dai, Jian-Jun; Wu, Cai-Feng; Gu, Xiao-Long; Liu, Liang; Wu, Zhi-Qiang; Xie, Yi-Ni; Wu, Bin; Chen, Hui-Lan; Li, Yao; Chen, Xue-Jin; Zhang, De-Fu

    2012-02-01

    To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

  13. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    Science.gov (United States)

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

  14. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  15. Production of wild buffalo (Bubalus arnee) embryos by interspecies somatic cell nuclear transfer using domestic buffalo (Bubalus bubalis) oocytes.

    Science.gov (United States)

    Priya, D; Selokar, N L; Raja, A K; Saini, M; Sahare, A A; Nala, N; Palta, P; Chauhan, M S; Manik, R S; Singla, S K

    2014-04-01

    The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p cell proliferation rate was significantly (p cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

  16. Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer.

    Science.gov (United States)

    Li, Juan; Gao, Yu; Petkov, Stoyan; Purup, Stig; Hyttel, Poul; Callesen, Henrik

    2014-06-10

    Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages. In addition, the expression level of H3K27me3 functional methylase EZH2 increased while no changes were observed on H3K27me3 demethylase JMJD3 in relation to passage number. Moreover, the expression levels of both the H3K4me3 methylase MLL1 and the H3K4me3 demethylase RBP2 were increased at high passages. By using lower passage (numbers 3-5) EG cells as donor cells, the SCNT efficiency was significantly lower compared with use of fetal fibroblast donor cells. However, similar blastocyst rates were achieved when using higher passage (numbers 9-12) EG cells as donor cells. In conclusion, the present study suggests that the epigenetic status of EG cells change with increasing passage numbers, and that higher passage number EG cells are better primed for SCNT.

  17. Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells.

    Directory of Open Access Journals (Sweden)

    Hongjiang Wei

    Full Text Available Somatic cell nuclear transfer (SCNT is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4% and newborn (76.4% and 21.8% fibroblasts of the Banna miniature inbred pig (P>0.05. However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; P<0.05. The pregnancy rates of the recipients in the fetal and newborn fibroblast groups (60% and 80%, respectively were higher than those in the adult fibroblast group. Eight, three, and one cloned piglet were obtained from reconstructed embryos of the fetal, newborn, and adult fibroblasts, respectively. Microsatellite analyses results indicated that the genotypes of all cloning piglets were identical to their donor cells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells.

  18. Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

    Science.gov (United States)

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Guo, Zhen-Hua; Zhu, Meng; Bai, Jing

    2016-01-01

    Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

  19. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

    Science.gov (United States)

    JEON, Yubyeol; NAM, Yeong-Hee; CHEONG, Seung-A; KWAK, Seong-Sung; LEE, Eunsong; HYUN, Sang-Hwan

    2016-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  20. Revival of extinct species using nuclear transfer: hope for the mammoth, true for the Pyrenean ibex, but is it time for "conservation cloning"?

    Science.gov (United States)

    Piña-Aguilar, Raul E; Lopez-Saucedo, Janet; Sheffield, Richard; Ruiz-Galaz, Lilia I; Barroso-Padilla, Jose de J; Gutiérrez-Gutiérrez, Antonio

    2009-09-01

    Recent accomplishments in the fields of nuclear transfer and genomics, such as the cloned offspring production from frozen mouse cells, cryopreserved at not too low temperatures without cryoprotectors; or the sequencing of wooly mammoth genome, have opened the opportunity for the revival of extinct species. As expected, they are receiving a lot of publicity in the media and also scientific attention. Furthermore, it was recently published the "revival" of the first extinct subspecie: the Pyrenean ibex (Capra pyrenaica pyrenaica), a wild goat extinct in 2000. This strengthens the field of cloning as it had been tarnished by induced pluripotent stem cells (iPS) and other methods of reprogramming. However, for biological conservation purposes, cloning is not generally accepted as an alternative for animal conservation, and there is an ongoing debate between reproductive scientists and conservation specialists. Although we believe that nuclear transfer technologies have an opportunity in conservation efforts for some species that are on the brink of extinction and that population status, geographical isolation, reproductive characteristics, and human pressure create a situation that is almost unsustainable. In this article we discuss the barriers in cloning mammoths and cloning controversies in conservation from a zoological perspective, citing the species that might benefit from nuclear transfer techniques in the arduous journey so as not to disappear forever from this, our world.

  1. Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice.

    Science.gov (United States)

    Galli, Cesare; Duchi, Roberto; Colleoni, Silvia; Lagutina, Irina; Lazzari, Giovanna

    2014-01-01

    Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.

  2. Efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos Effect of ibuprofen administered one hour before the bovine embryo transfer

    Directory of Open Access Journals (Sweden)

    H.J. Narváez

    2010-06-01

    Full Text Available Avaliou-se o efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos, com o objetivo de melhorar a taxa de prenhez. Após a avaliação da resposta ao protocolo de sincronização do estro, 76 fêmeas selecionadas como receptoras de embriões foram distribuídas em três grupos (G experimentais: G1 (n=25 receptoras usadas como controle, G2 (n=30 receptoras que receberam ibuprofeno 5mg/kg, I.M, uma hora antes da inovulação dos embriões, e G3 (n=21 receptoras que receberam uma matriz polimérica de liberação controlada de ibuprofeno administrado por via subcutânea. As taxas de prenhez foram de 16% (4/25, 43,3% (13/30 e 14,2% (3/21, para G1, G2 e G3, respectivamente. Observou-se diferença (PThe effect of the administered ibuprofen was evaluated one hour before the embryo transfer of bovine embryos in order to improve pregnancy rates. After evaluating the response to protocol synchronization of estrus, 76 Females selected as the recipients of embryos were distributed into three experimental groups: G1 (n = 25 surrogate cows used as control, G2 (n = 30 surrogate cows that received 5mg/kg ibuprofen, IM, one hour before the embryo transfer, and G3 (n = 20 surrogate cows that received an array polymeric release of controlled ibuprofen subcutaneously administered. The pregnancy rates were 16% (4/25, 43.3% (13/30, and 14.2% (3/21 for G1, G2, and G3, respectively. There was statistical difference (P<0.024 on pregnancy rate of G2, in comparison with those of G1 and G3. The administration of ibuprofen intramuscularly one hour before the embryo transfer resulted in better pregnancy rate in Nellore surrogate cows.

  3. Radioecological transfer of {sup 137}Cs from ground deposition to man from Chernobyl debris and from nuclear weapons fallout in different Swedish populations

    Energy Technology Data Exchange (ETDEWEB)

    Raeaef, C.L. [Malmoe Univ. Hospital, Lund Univ., Dept. of Radiation Physics, Malmoe (Sweden)

    2005-07-01

    A comparison of the estimated committed effective dose per unit activity deposition on ground was made between different critical groups in Sweden. The time-integrated aggregate transfer of {sup 137}Cs for the global fallout was 2-3 times higher than from Chernobyl debris for Swedish urban populations. For reindeer herders this difference is even more marked, with a factor of three to four higher time-integrated transfer factor of nuclear weapons fallout. Considering the transfer of Chernobyl {sup 137}Cs debris the time-integrated transfer factor appears to be more than 25 times higher for reindeer herders in Sweden than for the urban reference groups. An even more pronounced relative difference between the time integrated aggregate transfer was observed between reindeer herders and urban reference populations for the pre-Chernobyl fallout (a factor of 30). The projected committed effective dose from internal contamination of Chernobyl {sup 137}Cs per unit activity deposition is observed to be 2030 {mu}Sv/kBq m{sup -2}. The highest values in Sweden are obtained for reindeer herders with an estimated radioecological transfer of 0.5 mSv/kBq m{sup -2}. (au)

  4. Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock.

    Science.gov (United States)

    Pavani, Krishna C; Baron, Erica; Correia, Pedro; Lourenço, Joana; Bettencourt, Bruno Filipe; Sousa, Madalena; da Silva, Fernando Moreira

    2016-10-01

    Three assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

  5. 77 FR 20319 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-04-04

    ...; ] DEPARTMENT OF AGRICULTURE Animal and Plant Health Inspection Service 9 CFR Part 93 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Correction In proposed rule...

  6. 78 FR 73993 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2013-12-10

    ... Health Inspection Service 9 CFR Parts 92, 93, 94, 95, 96, and 98 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Corrections In rule document 2013-28228 appearing...

  7. Thermal-Hydraulic Analyses of Heat Transfer Fluid Requirements and Characteristics for Coupling A Hydrogen Production Plant to a High-Temperature Nuclear Reactor

    Energy Technology Data Exchange (ETDEWEB)

    C. B. Davis; C. H. Oh; R. B. Barner; D. F. Wilson

    2005-06-01

    The Department of Energy is investigating the use of high-temperature nuclear reactors to produce hydrogen using either thermochemical cycles or high-temperature electrolysis. Although the hydrogen production processes are in an early stage of development, coupling either of these processes to the hightemperature reactor requires both efficient heat transfer and adequate separation of the facilities to assure that off-normal events in the production facility do not impact the nuclear power plant. An intermediate heat transport loop will be required to separate the operations and safety functions of the nuclear and hydrogen plants. A next generation high-temperature reactor could be envisioned as a single-purpose facility that produces hydrogen or a dual-purpose facility that produces hydrogen and electricity. Early plants, such as the proposed Next Generation Nuclear Plant, may be dual-purpose facilities that demonstrate both hydrogen and efficient electrical generation. Later plants could be single-purpose facilities. At this stage of development, both single- and dual-purpose facilities need to be understood. Seven possible configurations for a system that transfers heat between the nuclear reactor and the hydrogen and/or electrical generation plants were identified. These configurations included both direct and indirect cycles for the production of electricity. Both helium and liquid salts were considered as the working fluid in the intermediate heat transport loop. Methods were developed to perform thermalhydraulic and cycle-efficiency evaluations of the different configurations and coolants. The thermalhydraulic evaluations estimated the sizes of various components in the intermediate heat transport loop for the different configurations. The relative sizes of components provide a relative indication of the capital cost associated with the various configurations. Estimates of the overall cycle efficiency of the various configurations were also determined. The

  8. Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

    Science.gov (United States)

    Sampaio, R V; Chiaratti, M R; Santos, D C N; Bressan, F F; Sangalli, J R; Sá, A L A; Silva, T V G; Costa, N N; Cordeiro, M S; Santos, S S D; Ambrosio, C E; Adona, P R; Meirelles, F V; Miranda, M S; Ohashi, O M

    2015-01-15

    Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

  9. Analysis of Heat Transfer Characteristics Based on Design Factors for Determining the Internal Geometry of Metal Insulation in Nuclear Power Plant

    Energy Technology Data Exchange (ETDEWEB)

    Song, Ki O; Yu, Jeong Ho; Lee, Tae Ho; Jeon, Hyun Ik; Ha, Seung Woo; Cho, Sun Young [KLES Inc., Seoul (Korea, Republic of)

    2015-11-15

    A heat insulating material used in the industrial site normally derives its heat insulating performance by using a low thermal conductivity material such as glass fiber. In case of the metal insulation for nuclear power plant, in contrast, only TP 304 stainless steel foil having high thermal conductivity is the only acceptable material. So, it is required to approach in structural aspect to ensure the insulation performance. In this study, the design factors related to the metal insulation internal structure were determined considering the three modes of heat transfer, i.e., conduction, convection, and radiation. The analysis of heat flow was used to understand the ratio of the heat transfer from each factor to the overall heat transfer from all the factors. Based on this study, in order to minimize the convection phenomenon caused by the internal insulation, a multiple foil was inserted in the insulation. The increase in the conduction heat transfer rate was compared, and the insulation performance under the three modes of heat transfer was analyzed in order to determine the internal geometry.

  10. Analysis of heat transfer characteristics based on design factors for determining the internal geometry of metal insulation in nuclear power plant

    Energy Technology Data Exchange (ETDEWEB)

    Song, Ki O; Yu, Jeong Ho; Lee, Tae Ho; Jeon, Hyun Ik; Ha, Seung Woo; Cho, Sun Young [KLES Inc., Daejeon (Korea, Republic of)

    2015-11-15

    A heat insulating material used in the industrial site normally derives its heat insulating performance by using a low thermal conductivity material such as glass fiber. In case of the metal insulation for nuclear power plant, in contrast, only TP 304 stainless steel foil having high thermal conductivity is the only acceptable material. So, it is required to approach in structural aspect to ensure the insulation performance. In this study, the design factors related to the metal insulation internal structure were determined considering the three modes of heat transfer, i.e., conduction, convection, and radiation. The analysis of heat flow was used to understand the ratio of the heat transfer from each factor to the overall heat transfer from all the factors. Based on this study, in order to minimize the convection phenomenon caused by the internal insulation, a multiple foil was inserted in the insulation. The increase in the conduction heat transfer rate was compared, and the insulation performance under the three modes of heat transfer was analyzed in order to determine the internal geometry.

  11. Technical aspects of the piezo, laser-assisted, and conventional methods for nuclear transfer of mouse oocytes and their efficiency and efficacy: Piezo minimizes damage of the ooplasmic membrane at injection.

    Science.gov (United States)

    Chen, Shee-Uan; Chao, Kuang-Han; Chang, Chia-Yi; Hsieh, Fon-Jou; Ho, Hong-Nerng; Yang, Yu-Shih

    2004-04-01

    Assessment of the advantages and disadvantages of the piezo, laser, and conventional methods for nuclear transfer has remained elusive. Furthermore, although the piezo method had been used by some investigators for research of sperm injection and nuclear transfer for several years, many researchers have failed to operate the technique smoothly and achieve reproducible results. The procedures of nuclear transfer using piezo were ascertained and described in detail. Mouse oocytes were enucleated, and injected with cumulus cells using the piezo, laser, or conventional methods. We investigated the time needed and survival of nuclear transfer. Development was compared among the three methods and parthenogenetic control specimens. The average time of nuclear transfer for each oocyte was significantly shorter using the piezo (118 +/- 9 s) and laser methods (120 +/- 11 s) than using the conventional method (170 +/- 11 s). The damage rate was smaller for the piezo group (10%) than the laser (37%) and conventional (40%) groups. The percentages of blastocyst formation (14%, 12%, and 11%) and the number of nuclei of blastocysts (54 +/- 13, 51 +/- 11, and 52 +/- 12) were similar among the piezo, laser, and conventional groups, but significantly lower than for the control group (83%, 105 +/- 14). The piezo technique is more efficient than the conventional method for nuclear transfer. The laser method is easy to operate, but the equipment is expensive. In addition, piezo induced fewer traumas while breaking the membrane than the aspiration techniques used in the laser and conventional methods.

  12. Effect of Embryo Transfer on the Bovine Estrus Synchronization and Conception%胚胎移植受体牛同期发情及受胎效果的研究

    Institute of Scientific and Technical Information of China (English)

    李刚

    2012-01-01

    The method of CIDR+E2 and twice use of uterine PGF2α infusion method were used in this study, embryo transfer for cattle estrus synchronization and fresh embryo transfer pregnancy was observed. The results showed that receptor bovine estrous rate at 24--48h was 85.5% and 51.6%, there was significantly difference between them (P〈0.05). Cattle before transplantation luteal receptor inspection pass rate was 79.3%, 77.8%, there was no significant difference (P〉0.05). Conception rate was 45.8% and 42.4%, the difference was not significant (P〉0.05). Estrus observed after 24h, 6.5d for direct examination Cattle with luteinizing for A, B grade can be used for transplantation.%本试验分别采用CIDR+E2法和两次PGF2α子宫输注法,对胚胎移植受体牛做同期发情处理,鲜胚移植观察其妊娠情况。试验结果表明两种处理方法的受体牛在24-48h发情率分别为85.5%和51.6%,两者之间差异显著(P〈0.05)。移植前对受体牛进行黄体检查,黄体合格率分另11为79.3%和77.8%,差异不显著(P〉0.05);受胎率分别为45.8%和42.4%,差异不显著(P〉0.05)。24h后观察发情,6.5d直检,黄体A、B级者用于移植。

  13. Unlocking the bovine genome

    Directory of Open Access Journals (Sweden)

    Worley Kim C

    2009-04-01

    Full Text Available Abstract The draft genome sequence of cattle (Bos taurus has now been analyzed by the Bovine Genome Sequencing and Analysis Consortium and the Bovine HapMap Consortium, which together represent an extensive collaboration involving more than 300 scientists from 25 different countries.

  14. Camel and bovine chymosin

    DEFF Research Database (Denmark)

    Langholm Jensen, Jesper; Mølgaard, Anne; Navarro Poulsen, Jens Christian;

    2013-01-01

    Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined...... interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk....

  15. Space transfer concepts and analysis for exploration missions. Implementation plan and element description document (draft final). Volume 3: Nuclear thermal rocket vehicle

    Science.gov (United States)

    1991-01-01

    This document presents the nuclear thermal rocket (NTR) concept design developed in support of the Space Transfer Concepts and Analysis for Exploration Missions (STCAEM) study. The evolution of the NTR concept is described along with the requirements, guidelines and assumptions for the design. Operating modes and options are defined and a systems description of the vehicle is presented. Artificial gravity configuration options and space and ground support systems are discussed. Finally, an implementation plan is presented which addresses technology needs, schedules, facilities and costs.

  16. Uso de inibidores das histonas deacetilases na transferência nuclear de células somáticas em bovinos

    OpenAIRE

    2016-01-01

    A clonagem de mamíferos por transferência nuclear de células somáticas (TNCS) ainda é afetada pela baixa eficiência. As modificações epigenéticas estabelecidas durante o processo de diferenciação celular estão entre os principais fatores. Uma vez que estas modificações atuam como barreiras epigenéticas restringindo a reprogramação do núcleo somático. Considerando isso, a maioria dos fatores que promovem a descondensação da cromatina, incluindo os inibidores das Histonas Deacetilases (HDACis),...

  17. Interspecies nuclear transfer using fibroblasts from leopard, tiger, and lion ear piece collected postmortem as donor cells and rabbit oocytes as recipients.

    Science.gov (United States)

    Yelisetti, Uma Mahesh; Komjeti, Suman; Katari, Venu Charan; Sisinthy, Shivaji; Brahmasani, Sambasiva Rao

    2016-06-01

    Skin fibroblast cells were obtained from a small piece of an ear of leopard, lion, and tiger collected postmortem and attempts were made to synchronize the skin fibroblasts at G0/G1 of cell cycle using three different approaches. Efficiency of the approaches was tested following interspecies nuclear transfer with rabbit oocytes as recipient cytoplasm. Fluorescence-activated cell sorting revealed that the proportion of G0/G1 cells increased significantly (P tiger were successfully synchronized and used for the development of blastocysts using rabbit oocytes as recipient cytoplasm.

  18. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

    Science.gov (United States)

    Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

    2007-03-01

    Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

  19. Assessment of the TiO{sub 2}/water nanofluid effects on heat transfer characteristics in VVER-1000 nuclear reactor using CFD modeling

    Energy Technology Data Exchange (ETDEWEB)

    Mousavizadeh, Seyed Mohammad; Ansarifar, Gholam Reza; Talebi, Mansour [Dept. of Nuclear Engineering, Faculty of Advanced Sciences and Technology, University of Isfahan, Isfahan (Iran, Islamic Republic of)

    2015-12-15

    The most important advantage of nanoparticles is the increased thermal conductivity coefficient and convection heat transfer coefficient so that, as a result of using a 1.5% volume concentration of nanoparticles, the thermal conductivity coefficient would increase by about twice. In this paper, the effects of a nanofluid (TiO2/water) on heat transfer characteristics such as the thermal conductivity coefficient, heat transfer coefficient, fuel clad, and fuel center temperatures in a VVER-1000 nuclear reactor are investigated. To this end, the cell equivalent of a fuel rod and its surrounding coolant fluid were obtained in the hexagonal fuel assembly of a VVER-1000 reactor. Then, a fuel rod was simulated in the hot channel using Computational Fluid Dynamics (CFD) simulation codes and thermohydraulic calculations (maximum fuel temperature, fluid outlet, Minimum Departure from Nucleate Boiling Ratio (MDNBR), etc.) were performed and compared with a VVER-1000 reactor without nanoparticles. One of the most important results of the analysis was that heat transfer and the thermal conductivity coefficient increased, and usage of the nanofluid reduced MDNBR.

  20. Energy metabolism during microsurgical transfer of human skeletal muscle assessed by high-pressure liquid chromatography and by 31P-nuclear magnetic resonance.

    Science.gov (United States)

    Lundberg, Jonas; Elander, Anna; Rakotonirainy, Olivier; Zetterlund, Therese; Fogdestam, Ingemar; Soussi, Bassam

    2002-01-01

    The effect of ischaemia and reperfusion on human skeletal muscle was studied during free vascularised muscle transfer. Muscle biopsy specimens were taken from patients having microsurgical muscle transfer, 18 cases (17 patients; 12 men, 5 women). The biopsies were taken three times: before transfer of the muscle (control), at maximum ischaemic time, and one hour after revascularisation. The biopsy specimens were analysed for purine nucleotides, by high-pressure liquid chromatography (HPLC), and by nuclear magnetic resonance (NMR) at 500 MHz. Phosphocreatine (PCr) recovered only partially (79%) and adenosine triphosphate (ATP) did not differ significantly from normal control after revascularisation and a mean ischaemic time of 114 minutes. NMR measurements showed an accumulation of glucose-6-phosphate (G-6-P) during the ischaemic period, indicating anaerobic metabolism. After three hours of ischaemia and one hour of reperfusion the PCr recovery was less than 60% (r = 0.7). The results confirm those of previous animal studies, which set three hours normothermic ischaemia as a safe limit for tissue preservation when transferring skeletal muscle. Longer ischaemic times may cause serious postoperative healing problems and reduced muscle function.

  1. Bovine Herpesvirus 4 infections and bovine mastitis

    NARCIS (Netherlands)

    Wellenberg, Gerardus Johannus

    2002-01-01

    Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown aetiol

  2. Economic optimization of the number of recipients in bovine embryo transfer programs Otimização econômica do número de receptoras em programas de transferência de embriões em bovinos

    Directory of Open Access Journals (Sweden)

    Renato Travassos Beltrame

    2007-06-01

    Full Text Available Purchase and maintenance of recipient females account for a large proportion of the costs and determine the number of calves that can be produced in an embryo transfer program. However, the large variability of embryo production by the donors and the need to purchase and synchronize the recipients before knowing the number of embryos collected make it difficult for the decision maker to identify the ideal number of recipient females to allocate. An ex-ante evaluation to determine the optimal number of recipient females was carried out through a sensitivity analysis for the ratio between the number of recipients and donors in a simulation model. The variability for the number of embryos collected was accounted for by applying the Monte Carlo simulation technique, assuming normal distribution and known values for mean and variance. The simulation considered monthly intervals between collections, during a 24 months program. The effect of embryo freezing on the number of pregnancies was considered by introducing a stock of frozen embryos into the mathematical model. Optimal recipient/donor ratio and the cost per pregnancy were compared for three recipient synchronization protocols (prostaglandin, progesterone - P4 and Ovsynch, based on the expected performance for synchronization, conception and transfer/treated rates for each protocol. Stochastic simulation associated with sensitivity analysis was effective in identifying the optimal donor to recipient ratio. Freezing embryos is effective to reduce the operational costs per pregnancy. The estimated optimal recipient/donor ratio was 20 for prostaglandin and 16.7 for the other protocols. The P4 protocol, although the most expensive, resulted in the lowest pregnancy cost estimation followed by prostaglandin and Ovsynch.A aquisição e manutenção de receptoras representam grande proporção dos custos e determinam o número de produtos gerados em um programa de transferência de embriões. Entretanto

  3. Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

  4. Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

    Institute of Scientific and Technical Information of China (English)

    Hengxi Wei; Qiuyan Li; Jun Li; Yan Li; Yunping Dai; Yufang Ma; Kai Xue; Ning Li

    2008-01-01

    Cloning pigs by somatic cell nuclear transfer (SCNT) has wide applications in basic research,human medicine and agricultural production.To improve cloning efficiency,the effect of two basic maturation media,NCSU-23 and TCMI99,was compared,and TCM199 was selected for the following experiments with leptin.We systematically studied the effects of leptin supplementation on oocytes in vitro maturation (IVM),in vitro development of parthenogenetically activated (Phi) and SCNT embryos and/n vivo develop-ment of SCNT embryos after embryo transfer (ET).The results showed that supplementation of 100 or 200 ng/ml leptin into the mat-uration medium did not greatly affect nuclear maturation of oocytes,or cleavage rates of PA and SCNT (P<0.05).Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium,and the number of cells in PA blastocysts was also improved (P<0.05).The number of cells in blastocyst of SCNT was improved,when 100 ng/ml leptin was added (P<0.05).Furthermore,supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate in pig cloning.

  5. Epigenetic modification related to somatic nuclear transfer%体细胞核移植诱导的表观遗传学变化

    Institute of Scientific and Technical Information of China (English)

    杨东山; 邓为; 樊娜娜; 赖良学

    2009-01-01

    Somatic cell nuclear transfer is a technology by which a somatic nucleus is put into an enucleated oocyte to make a reconstructed embryo, which subsequently develops to an offspring with the same genetics as donor cells. So far cloned animals have been produced in many species by somatic nuclear transfer. However, there are many problems need to be solved before its potential applications become fully practical, such as low efficiency, abnormal phenotypes and premature death, which are probably caused by incomplete or aberrant epigenetic reprogramming of donor cells. In recent decade, reprogramming after somatic nuclear transfer has been extensively studied, mainly focusing on remodeling of structures in or out of nucleus, modification of DNA methylation, X chromosome inactivation, modification of histone acetylation and recovery of telomere length. Those efforts are expected to lead to establish an approach artificially intervening the reprogramming of donor cell in the process of nuclear transfer and eventually improve the efficiency of somatic nuclear transfer.%体细胞核移植技术是指将一个分化的体细胞核置入去核的卵母细胞中,并发育产生与供体细胞遗传背景一致的克隆后代的技术.目前,世界上通过体细胞核移植技术已经产生了许多的克隆动物.但克隆过程中还存在着很多问题,比如,克隆效率太低、克隆个体常伴有表型异常和早亡等,从而使该技术应有的应用潜力不能得到充分的发挥.体细胞表观遗传学重编程的不完全或紊乱是造成核移植诸多问题的主要原因.近十多年来,人们对体细胞核移植后的重编程进行了广泛的研究,其核心内容包括核及核外结构的重塑、DNA甲基化模式的重建、基因印迹和X染色体失活、组蛋白乙酰化模式的重建、端粒长度恢复等,以期能够对其重编程加以人为干预,从而提高动物克隆效率.本文拟对体细胞核移植诱导的重编程研

  6. Somatic cell nuclear transfer in its first and the second decade: sussesses, setbacks, paradoxes and perspectives

    DEFF Research Database (Denmark)

    Vajta, Gabor

    2007-01-01

    The present review gives a subjective outline of the past and future of somatic cell nuclear trensfer (SCNT). The first decade was full of contradictions: amazing successes were followed by frustrating fiascos. Although the possibility of reversing somatic cell differentiation completely is a mor...

  7. Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer.

    Science.gov (United States)

    Jin, J X; Lee, S; Khoirinaya, C; Oh, A; Kim, G A; Lee, B C

    2016-03-01

    Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.

  8. In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells.

    Science.gov (United States)

    Zhang, Yun-Hai; Pan, Deng-Ke; Sun, Xiu-Zhu; Sun, Guo-Jie; Liu, Xiao-Hui; Wang, Xiao-Bo; Tian, Xing-Hua; Li, Yan; Dai, Yun-Ping; Li, Ning

    2006-08-01

    The present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at -4 degrees C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at -196 degrees C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

  9. A protocol for adult somatic cell nuclear transfer in medaka fish (Oryzias latipes) with a high rate of viable clone formation.

    Science.gov (United States)

    Bubenshchikova, Ekaterina; Kaftanovskaya, Elena; Adachi, Tomoko; Hashimoto, Hisashi; Kinoshita, Masato; Wakamatsu, Yuko

    2013-12-01

    Previously, we successfully generated fully grown, cloned medaka (the Japanese rice fish, Oryzias latipes) using donor nuclei from primary culture cells of adult caudal fin tissue and nonenucleated recipient eggs that were heat shock-treated to induce diploidization of the nuclei. However, the mechanism of clone formation using this method is unknown, and the rate of adult clone formation is not high enough for studies in basic and applied sciences. To gain insight into the mechanism and increase the success rate of this method of clone formation, we tested two distinct nuclear transfer protocols. In one protocol, the timing of transfer of donor nuclei was changed, and in the other, the size of the donor cells was changed; each protocol was based on our original methodology. Ultimately, we obtained an unexpectedly high rate of adult clone formation using the protocol that differed with respect to the timing of donor nuclei transfer. Specifically, 17% of the transplants that developed to the blastula stage ultimately developed into adult clones. The success rate with this method was 13 times higher than that obtained using the original method. Analyses focusing on the reasons for this high success rate of clone formation will help to elucidate the mechanism of clone formation that occurs with this method.

  10. Effects of transient and non-uniform distribution of heat flux on intensity of heat transfer and burnout conditions in the channels of nuclear reactors

    Energy Technology Data Exchange (ETDEWEB)

    Vitaly Osmachkin [Russian Research Center ' Kurchatov Institute' 1, Kurchatov sq, Moscow 123182 (Russian Federation)

    2005-07-01

    Full text of publication follows: The influence of power transient, changes of flow rate, inlet temperatures or pressure in cores of nuclear reactors on heat transfer and burnout conditions in channels depend on rate of such violations. Non-uniform distribution of the heat flux is also important factor for heat transfer and development of crisis phenomenon. Such effects may be significant for NPPs safety. But they have not yet generally accepted interpretation. Steady state approach is often recommended for use in calculations. In the paper a review of experimental observed so-called non-equilibrium effects is presented. The effects of space and time factors are displaying due delay in reformation turbulence intensity, velocity, temperatures or void fraction profiles, water film flow on the surface of heated channels. For estimation of such effect different methods are used. Modern computer codes based on two or three fluids approaches are considered as most effective. But simple and clear correlations may light up the mechanics of effects on heat transfer and improve general understanding of scale and significance of the transient events. In the paper the simplified methods for assessment the influence of lags in the development of distributions of parameters of flow, the relaxation of temporal or space violations are considered. They are compared with more sophisticated approaches. Velocities of disturbance fronts moving along the channels are discussed also. (author)

  11. Taking a fresh Look at boiling heat transfer on the road to improved nuclear economics and efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Baglietto, E.; Pointer, W. D.

    2016-08-01

    In the effort to reinvigorate innovation in the way we design, build, and operate the nuclear power generating stations of today and tomorrow, nothing can be taken for granted. Not even the seemingly familiar physics of boiling water. The Consortium for the Advanced Simulation of Light Water Reactors, or CASL, is focused on the deployment of advanced modeling and simulation capabilities to enable the nuclear industry to reduce uncertainties in the prediction of multi-physics phenomena and continue to improve the performance of todays light water reactors and their fuel. An important part of the CASL mission is the development of a next generation thermal hydraulics simulation capability, integrating the history of engineering models based on based on experimental experience with the computing technology of the future. (Author)

  12. Analysis of interactions of mechanical deformations and mass transfer on heat transfer from an underground nuclear-waste repository. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Bloom, S.G.; Hulbert, L.E.

    1979-10-01

    A review of existing models identified several effects that may need consideration in further model development. Most of these effects involved coupling equations through variable property values rather than through omission of any significant mechanism. However, it was also shown that more than one mechanism may adequately simulate a given set of experimental data and additional experimental data are needed to establish which (if any) of the possible mechanisms would actually control conditions in a nuclear waste repository. In particular, it is believed that mathematical modeling of major thermomechanical effects can be accomplished with finite element analysis computer programs, provided that adequate thermomechanical property data of salt and overburden are attained. An attempt was made to develop a general set of differential equations for simulating momentum, mass, and energy flows in geologic formations in order to illustrate the possible mechanisms and point out those included and not included in existing models. Most of the mechanisms are included in some manner in existing models although some approximations may not be adequate. More experimental data are required to assess the importance of most omitted mechanisms. Analysis of some data on brine migration in salt indicated that two mechanisms, acting simultaneously, could adequately explain the flow. These are Darcy flow and a combination of ordinary and thermal diffusion enhanced by temperature-dependent solubility. Equations based on this simultaneous action correlated the data very well and indicated the possible need to include both (and, maybe other) mechanisms in future models. A program is recommended for further study of brine mobility. An expected result of this program includes recommendations for further experimental work.

  13. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    Science.gov (United States)

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  14. Otimização do sistema de produção de clones por transferência nuclear de célula somática (NTSC)

    OpenAIRE

    Forell, Fabiana; Feltrin,Cristiano; Santos, Lucila Carboneiro dos; Costa, Ubirajara Maciel da; Vieira, Arnaldo Diniz; Hölker, Michael; Rodríguez, José Luis

    2008-01-01

    A técnica de transferência nuclear é uma ferramenta que possibilita a produção de embriões clones que podem ser utilizados tanto na clonagem reprodutiva como no modelo para o estudo de diversos mecanismos fisiológicos durante o desenvolvimento embrionário. Neste sentido, embriões clones bovinos foram produzidos por transferência nuclear, com o objetivo de estabelecer a técnica de clonagem, bem como otimizála para as condições do Laboratório de Embriologia e Biotécnicas de Reprodução da UFRGS....

  15. Patologia de neonatos bovinos originados por meio da técnica de transferência nuclear de células somáticas – clonagem

    OpenAIRE

    2010-01-01

    Os mecanismos de morte em animais clonados a partir de células somáticas são poucos elucidados. Malformações de órgãos, alterações de tamanho e peso desses animais foram anomalias já descritas, porém em casos isolados. Desse modo, estudos nos níveis anatomo e histopatológicos são de suma importância para ajudar a compreensão dos fatores responsáveis pelos altos índices de insucessos com a utilização da TNCS (transferência nuclear de células somáticas). Assim, o presente trabalho foi realizado...

  16. Review paper: a review of the pathology of abnormal placentae of somatic cell nuclear transfer clone pregnancies in cattle, sheep, and mice.

    Science.gov (United States)

    Palmieri, C; Loi, P; Ptak, G; Della Salda, L

    2008-11-01

    Cloning of cattle, sheep, and mice by somatic cell nuclear transfer (SCNT) can result in apparently healthy offspring, but the probability of a successful and complete pregnancy is less than 5%. Failures of SCNT pregnancy are associated with placental abnormalities, such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium, and altered basement membrane. The pathogenesis of these changes is poorly understood, but current evidence implicates aberrant reprogramming of donor nuclei by the recipient oocyte cytoplast, resulting in epigenetic modifications of key regulatory genes essential for normal placental development. The purpose of this review is to provide an overview of the anatomic pathology of abnormal placentae of SCNT clones and to summarize current knowledge concerning underlying pathogenetic mechanisms.

  17. An integrated high-performance beam optics-nuclear processes framework with hybrid transfer map-Monte Carlo particle transport and optimization

    Energy Technology Data Exchange (ETDEWEB)

    Bandura, L., E-mail: bandura@msu.ed [Argonne National Laboratory, Argonne, IL 60439 (United States); Erdelyi, B. [Argonne National Laboratory, Argonne, IL 60439 (United States); Northern Illinois University, DeKalb, IL 60115 (United States); Nolen, J. [Argonne National Laboratory, Argonne, IL 60439 (United States)

    2010-12-01

    An integrated beam optics-nuclear processes framework is essential for accurate simulation of fragment separator beam dynamics. The code COSY INFINITY provides powerful differential algebraic methods for modeling and beam dynamics simulations in absence of beam-material interactions. However, these interactions are key for accurately simulating the dynamics of heavy ion fragmentation and fission. We have developed an extended version of the code that includes these interactions, and a set of new tools that allow efficient and accurate particle transport: by transfer map in vacuum and by Monte Carlo methods in materials. The new framework is presented, along with several examples from a preliminary layout of a fragment separator for a facility for rare isotope beams.

  18. Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

    Institute of Scientific and Technical Information of China (English)

    FAN Yong; YAO RuQiang; YU Yang; LI ZanDong; WANG Liu; ALICE Jouneau; ZHOU Qi; TONG Man; ZHAO ChunLi; DING ChenHui; HAO Jie; LV Zhuo; DAI XiangPeng; HAI Tang; LI XueMei

    2008-01-01

    Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated.In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer-ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.

  19. Inter-organisational knowledge transfer: building and sustaining the sources of innovation in nuclear safety and security

    Energy Technology Data Exchange (ETDEWEB)

    Staude, Fabio, E-mail: fstaude@cnen.gov.br [Comissao Nacional de Energia Nuclear (CNEN-RJ), Rio de Janeiro, RJ (Brazil); Ramirez, Matias, E-mail: matias.ramirez@sussex.ac.uk [University of Sussex (SPRU/US), Brighton (United Kingdom). Science and Technology Policy Research

    2013-07-01

    The current complexity of innovation processes has led to an understanding that the models of innovation have changed from linear model to a model characterised by multiple interactions and complex networks. Within this more multifaceted environment, has emerged a new set of actors, generally termed as intermediaries, performing a variety of tasks in the innovation process. The innovation literature has recognised various important supporting activities performed by intermediaries, by linking and facilitating the movement of information and knowledge between actors within an innovation system, in order to fill information gaps. Complementary, we make the assumption that the intermediary can assume a more central role in the innovation process, performing activities beyond to filling information gaps, since they intervene to create, prioritise, and articulate meaning to practices. Under this argument, this paper explores how intermediaries work in making innovation happen in the Brazilian nuclear safety and security area, demonstrating the influence of intermediary organisations in improving nuclear regulatory activities. We make sense of these processes by analyzing intermediary roles in the recent regulatory activities improvements, specifically those related to the practices involving radiation sources in medicine. Thus, through an empirical case study, this paper examines the issue of intermediation in a wide sense, including strategic activities preformed by intermediaries, associated with accessing, diffusing, coordinating and enabling knowledge activities. (author)

  20. Enzootic bovine leukosis and Bovine leukemia virus

    OpenAIRE

    Amauri Alcindo Alfieri; Alice Fernandes Alfieri; Luis Álvaro Leuzzi Junior

    2004-01-01

    All over de World the Enzootic Bovine Leukosis is a important viral infection in cattle herds. This revision points out topics relative to the etiological agent, clinical signals, diagnosis methods, control and prophylaxis of the infection.A Leucose Enzoótica Bovina é uma infecção viral amplamente disseminada em rebanhos bovinos de todo o mundo. Esta revisão tem por objetivo apresentar tópicos relacionados ao agente etiológico, à doença clínica e aos métodos de diagnóstico, controle e profila...

  1. Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses

    Science.gov (United States)

    Czernik, Marta; Toschi, Paola; Zacchini, Federica; Iuso, Domenico; Ptak, Grażyna Ewa

    2017-01-01

    In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria. PMID:28076382

  2. Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses.

    Science.gov (United States)

    Czernik, Marta; Toschi, Paola; Zacchini, Federica; Iuso, Domenico; Ptak, Grażyna Ewa

    2017-01-01

    In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.

  3. Transfer of conservative and non-conservative radionuclides from the Sellafield nuclear fuel reprocessing plant to the coastal waters of Ireland

    Energy Technology Data Exchange (ETDEWEB)

    Mcmahon, C.A.; Fegan, M.; Wong, J.; Long, S.C.; Mckittrick, L.; Thomas, K.; Rafferty, B. [Radiological Protection Institute of Ireland, Dublin (Ireland)

    2004-07-01

    The Radiological Protection Institute of Ireland has monitored levels of anthropogenic radionuclides in the Irish marine environment for over 20 years. While the primary objective of the monitoring programme is to assess the exposure of the Irish population resulting from the presence of these radionuclides in the marine environment, the programme also aims to assess the geographical distribution and temporal variations of the radionuclides. The programme involves the routine sampling of and testing for radioactivity in fish, shellfish, seaweed, sediments and seawater. The data generated in the course of this programme, as well as in a separate study of changing plutonium isotopic ratios in Fucus vesiculosus from the west coast of Ireland, are used in this paper to estimate transport times from the Sellafield nuclear fuel reprocessing plant to the western Irish Sea and from the Irish Sea to the west coast of Ireland. The results obtained are discussed in the paper and the transfer times estimated for particle-reactive radionuclides (plutonium isotopes) compared with those obtained for more conservative radionuclides ({sup 137}Cs and {sup 99}Tc). Transfer factors (calculated as the ratio between observed concentrations in the environment and an average discharge rate {tau} years earlier, where {tau} is the transport time) are also presented. (author)

  4. 8. Innovative Technologies: Two-Phase Heat Transfer in Water-Based Nanofluids for Nuclear Applications Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Buongiorno, Jacopo; Hu, Lin-wen

    2009-07-31

    Nanofluids are colloidal dispersions of nanoparticles in water. Many studies have reported very significant enhancement (up to 200%) of the Critical Heat Flux (CHF) in pool boiling of nanofluids (You et al. 2003, Vassallo et al. 2004, Bang and Chang 2005, Kim et al. 2006, Kim et al. 2007). These observations have generated considerable interest in nanofluids as potential coolants for more compact and efficient thermal management systems. Potential Light Water Reactor applications include the primary coolant, safety systems and severe accident management strategies, as reported in other papers (Buongiorno et al. 2008 and 2009). However, the situation of interest in reactor applications is often flow boiling, for which no nanofluid data have been reported so far. In this project we investigated the potential of nanofluids to enhance CHF in flow boiling. Subcooled flow boiling heat transfer and CHF experiments were performed with low concentrations of alumina, zinc oxide, and diamond nanoparticles in water (≤ 0.1 % by volume) at atmospheric pressure. It was found that for comparable test conditions the values of the nanofluid and water heat transfer coefficient (HTC) are similar (within ±20%). The HTC increased with mass flux and heat flux for water and nanofluids alike, as expected in flow boiling. The CHF tests were conducted at 0.1 MPa and at three different mass fluxes (1500, 2000, 2500 kg/m2s) under subcooled conditions. The maximum CHF enhancement was 53%, 53% and 38% for alumina, zinc oxide and diamond, respectively, always obtained at the highest mass flux. A post-mortem analysis of the boiling surface reveals that its morphology is altered by deposition of the particles during nanofluids boiling. A confocal-microscopy-based examination of the test section revealed that nanoparticles deposition not only changes the number of micro-cavities on the surface, but also the surface wettability. A simple model was used to estimate the ensuing nucleation site

  5. Bovine milk glycome.

    Science.gov (United States)

    Tao, N; DePeters, E J; Freeman, S; German, J B; Grimm, R; Lebrilla, C B

    2008-10-01

    Bovine milk oligosaccharides have several potentially important biological activities including the prevention of pathogen binding to the intestinal epithelial and as nutrients for beneficial bacteria. It has been suggested that milk oligosaccharides are an important source of complex carbohydrates as supplements for the food and the pharmaceutical industries. However, only a small number of structures of bovine milk oligosaccharides (bMO) are known. There have been no systematic studies on bMO. High-performance mass spectrometry and separation methods are used to evaluate bMO, and nearly 40 oligosaccharides are present in bovine milk. Bovine milk oligosaccharides are composed of shorter oligomeric chains than are those in human milk. They are significantly more anionic with nearly 70%, measured abundances, being sialylated. Additionally, bMO are built not only on the lactose core (as are nearly all human milk oligosaccharides), but also on lactose amines. Sialic acid residues include both N-acetyl and N-glycolylneuraminic acid, although the former is significantly more abundant.

  6. Bovine milk exosome proteome

    Science.gov (United States)

    Exosomes are 40-100 nm membrane vesicles of endocytic origin and are found in blood, urine, amniotic fluid, bronchoalveolar lavage (BAL) fluid, as well as human and bovine milk. Exosomes are extracellular organelles important in intracellular communication/signaling, immune function, and biomarkers ...

  7. Intervet Symposium: bovine neosporosis

    NARCIS (Netherlands)

    Schetters, T.; Dubey, J.P.; Adrianarivo, A.; Frankena, K.; Romero, J.J.; Pérez, E.; Heuer, C.; Nicholson, C.; Russell, D.; Weston, J.

    2004-01-01

    This article summarises the most relevant data of presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in New Orleans, LA, USA, from 10 to 14 August 2003) in a symposium session on bovine neosporosis. The sy

  8. Bovine Spongiform Encephalopathy

    Science.gov (United States)

    Bovine spongiform encephalopathy (BSE) is caused by a novel contagion, known to as a prion. Prions are proteins capable of converting a normal cellular protein into a prion, thereby propagating an infection. BSE is the first known prion zoonotic. As such it has attracted broad scientific and, to a r...

  9. Sporadic Bovine Leukosis: A Description of Eight Calves Received at Animal Diseases Research Institute from 1974-1980

    OpenAIRE

    Bundza, A; Greig, A. S.; CHANDER, S.; Dukes, T. W.

    1980-01-01

    Eight calves with sporadic bovine leukosis are described. The common features were generalized lymphadenopathy, visceral involvement and raised total leukocyte and lymphocyte counts. Agar gel immunodiffusion tests for bovine leukemia virus antibodies were negative in eight animals and in all animals from three herds of origin. Lymphocytic nuclear pockets were found in the tissues of one calf but attempts to isolate bovine leukemia virus from two animals were unsuccessful.

  10. Functional analysis of bovine Nramp1 and production of transgenic cloned embryos in vitro.

    Science.gov (United States)

    Cheng, Xiang; Yu, Xiaoli; Liu, Yajun; Deng, Jie; Ma, Xiaoling; Wang, Huayan

    2015-02-01

    Natural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study, Nramp1 cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth of Salmonella abortusovis and Brucella abortus in macrophages. Calf fibroblasts stably transfected with pSP-NRAMP1-HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturated in vitro and the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produce Nramp1 transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

  11. Conjugation of Chitooligosaccharide-5-fluorouracil with Bovine Serum Albumin

    Institute of Scientific and Technical Information of China (English)

    Rong Min WANG; Jing Feng SONG; Yu Feng HE; Juan Juan MAO; Yan LI

    2006-01-01

    The interaction between chitooligosaccharide-5-fluorouracil (COS-5FU) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy. It was found that an energy transfer between COS-5FU and BSA had been occurred. The binding constants were calculated,between the donor and acceptor, the distance between BSA and COS-5FU was determined.

  12. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

    Directory of Open Access Journals (Sweden)

    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  13. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  14. Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin

    Institute of Scientific and Technical Information of China (English)

    FAN Ji-cai; CHEN Xiang; WANG Yun; FAN Cheng-ping; SHANG Zhi-cai

    2006-01-01

    The binding of pefloxacin mesylate (PFLX) to bovine lactoferrin (BLf) and human serum albumin (HSA) in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Forster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.

  15. 31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices.

    Science.gov (United States)

    Morris, P G; Feeney, J; Cox, D W; Bachelard, H S

    1985-05-01

    The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.

  16. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Directory of Open Access Journals (Sweden)

    Dawei Yu

    Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  17. Transcriptome profile of bovine elongated conceptus obtained from SCNT and IVP pregnancies.

    Science.gov (United States)

    Betsha, Simret; Hoelker, Michael; Salilew-Wondim, Dessie; Held, Eva; Rings, Franka; Grosse-Brinkhause, Christine; Cinar, Mehmet Ulas; Havlicek, Vitea; Besenfelder, Urban; Tholen, Ernst; Looft, Christian; Schellander, Karl; Tesfaye, Dawit

    2013-04-01

    In the present study we analyzed the gene expression changes induced by somatic cell nuclear transfer (SCNT) and in vitro production (IVP) in bovine elongated embryos using Affymetrix bovine genome array. For this, Day-16 bovine embryos from SCNT, IVP, and artificial insemination (AI) were recovered from recipients and used for transcriptome analysis. Despite comparable in vivo development rates, considerable reduction in elongation size was observed in SCNT compared to non-cloned embryos (93.3 mm for SCNT vs. 186.6 mm and 196.3 mm for IVP and AI embryos, respectively). Gene expression analysis revealed that the transcript levels of 477 genes, which are involved in various pathways including arginine and proline or glycerolipid and fatty acid metabolism, were significantly altered in SCNT compared to AI embryos. Similarly, 365 genes were differentially expressed in IVP embryos compared to AI. Thus, several pathways including TNRF-1 signaling and tight junction pathways were affected. To predict whether the altered transcripts were associated with culture condition or errors in transcriptional reprogramming, unique or common differentially expressed genes were analyzed in SCNT and IVP embryos compared to AI or fibroblast donor cells. Accordingly, 71 transcripts were found to be not transcriptionally reprogrammed, as their expression resembled the donor cells more than AI embryos; the remaining transcripts were either partially or incompletely reprogrammed. In conclusion, the present study identified deviations in elongation size, gene expression, and the corresponding molecular pathways in Day-16 SCNT and IVP conceptuses compared to their AI counterparts, which may subsequently be associated with the outcome of fetal development.

  18. Effects of embryo-derived exosomes on the development of bovine cloned embryos

    Science.gov (United States)

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Wang, Yongsheng

    2017-01-01

    The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation. PMID:28350875

  19. Distribution and transfer of radiocesium and radioiodine in the environment following the Fukushima nuclear accident - Distribution and transfer of radiocesium and radioiodine in the environment of Fukushima Prefecture following the nuclear accident

    Energy Technology Data Exchange (ETDEWEB)

    Muramatsu, Yasuyuki; Ohno, Takeshi; Sugiyama, Midori [Gakushuin University, Toshima-ku, Tokyo, 171-8588 (Japan); Sato, Mamoru [Fukushima Agricultural Technology Centre, Koriyama, Fukushima 963-0531 (Japan); Matsuzaki, Hiroyuki [The University of Tokyo, Bunkyo-ku, Tokyo 113-8656 (Japan)

    2014-07-01

    Large quantities of radioiodine and radiocesium were released from the accident at the Fukushima Daiichi Nuclear Power Plant (FDNPP) in March 2011. We have carried out intensive studies on the distribution and behaviour of these nuclides in the environment following the accident. Two topics obtained from our studies are presented. (1) Retrospective estimation of I-131 deposition through the analysis of I-129 in soil: It is necessary to obtain deposition data of radioiodine in Fukushima Prefecture for the assessment of thyroid doses due to the accident. However, short half-life of I-131 (8 days) made it impossible to obtain adequate sample coverage that would permit direct determination of the regional deposition patterns of I-131 within the prefecture and surrounding areas. On the other hand, I-129 released simultaneously during the accident still remains in soil, due to its long half-life of 1.57x10{sup 7} years. In order to reconstruct the I-131 deposition, we have determined I-129 concentrations by accelerator mass spectrometry (AMS). A good correlation was found between the measured concentrations of I-131 and I-129 in soils collected in the vicinity of FDNPP. We have analyzed I-129 in more than 500 soil samples collected systematically from Fukushima Prefecture. Using the obtained results, the I-131 deposition was calculated in different areas and the deposition map for I-131 was constructed. We also studied the vertical distribution of I-129 in soil. (2) Peculiar accumulation of radiocesium to some plants and mushrooms The radioactivity levels in agricultural crops decreased markedly in some months following the accident and their concentrations became lower than the Japanese guideline for foodstuffs (500 Bq/kg in 2011, and 100 Bq/kg after 2012). However, some agricultural products such as tea leaves and citrus fruits showed relatively higher values. Our analytical results obtained for the distribution of radiocesium in tea trees show that the root uptake

  20. Scriptaid Treatment Decreases DNA Methyltransferase 1 Expression by Induction of MicroRNA-152 Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

    Directory of Open Access Journals (Sweden)

    Shuang Liang

    Full Text Available Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152. Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos.

  1. Effect of epigallocatechin-3-gallate on the in vitro developmental potential of porcine oocytes and embryos obtained parthenogenetically and by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Yunsheng Li

    2014-03-01

    Full Text Available The present study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG on the in vitro development of porcine oocytes, parthenogenetic activation embryos (PA, and somatic cell nuclear transfer (SCNT embryos. In Experiment 1, 0 (control, 10, 30, and 50 μg/mL EGCG were added to in vitro maturation (IVM medium to explore the effect of EGCG on IVM of pig oocytes. The matured oocytes were then used to produce PA and SCNT embryos. Either for nuclear maturation of oocytes or for the rates of cleavage and blastocyst of PA and SCNT embryos, no significant difference was found among all groups. However, the total cell number per cloned blastocyst was significantly lower in blastocysts derived from oocytes matured in 50 μg/mL EGCG (P<0.05 as compared with the other groups. In Experiment 2, we cultured pig SCNT and PA embryos in medium containing various concentrations of EGCG to examine the effect of EGCG on preimplantation development. The cleavage and blastocyst rates and the total cell number per blastocyst did not significantly differ between PA and SCNT embryos among all groups. However, the reactive oxygen species level was significantly lower in the PA embryos cultured in 10 μg/mL EGCG than the other groups (P<0.05. Our results suggest that high doses of EGCG in IVM are harmful to the oocytes as evidenced by the decreased quality of SCNT embryos, and EGCG has no beneficial effects on in vitro development of pig cloned embryos.

  2. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer

    Directory of Open Access Journals (Sweden)

    Madhusudana Girija Sanal

    2014-09-01

    Full Text Available Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC, we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient’s somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is

  3. Testing nuclear forces by polarization transfer coefficients in d(p,p)d and d(p,d)p reactions at Ep=22.7 MeV

    Energy Technology Data Exchange (ETDEWEB)

    Evgeny Epelbaum

    2005-05-01

    The proton to proton polarization transfer coefficients K{sub x}{sup x'}, K{sub y}{sup y'}, K{sub z}{sup x'} and the proton to deuteron polarization transfer coefficients K{sub x}{sup x'}, K{sub y}{sup y'}, K{sub z}{sup x'}, K{sub x}{sup y'z'}, K{sub y}{sup z'z'}, K{sub z}{sup y'z'}, K{sub y}{sup x'z'} and K{sub y}{sup x'x'-y'y'} have been measured in d({rvec p}, {rvec p})d and d({rvec p}, {rvec d})p reactions at E{sub p}{sup lab} = 22.7 MeV, respectively. The data have been compared to predictions of modern nuclear forces obtained by solving the three-nucleon Faddeev equations in momentum space. Realistic (semi) phenomenological nucleon-nucleon potentials combined with model three-nucleon forces and modern chiral nuclear forces have been used. The AV18, CD Bonn, Nijm I and II nucleon-nucleon interactions have been applied alone or combined with the Tucson-Melbourne 99 three-nucleon force, adjusted separately for each potential to reproduce the triton binding energy. For the AV18 potential also the Urbana IX three-nucleon force have been used. In addition chiral NN potentials in the next-to-leading-order and chiral two- and three-nucleon forces in the next-to-next-to-leading-order have been applied. Only when three-nucleon forces are included a satisfactory description of all data results. For the chiral approach the restriction to the forces in the next-to-leading order is insufficient. Only when going over to the next-to-next-to-leading order one gets a satisfactory description of the data, similar to the one obtained with the (semi) phenomenological forces.

  4. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    BOU; ShorGan

    2009-01-01

    In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  5. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GUO XuDong; YANG DongShan; Ao XuDong; WU Xia; LI GuangPeng; WANG LingLing; BAO MingTao; XUE Lian; BOU ShorGan

    2009-01-01

    In the present study, cashmere goat fetal flbroblasta were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1(IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasta cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h.Parthenogenetic ooctyes were used as a model to Investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% va 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05).After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysta were randomly selected to identify transgene by polymeraee chain reaction. Both were positive. These results showed that: (i) RFP and Neo genes were correctly expressed indicating that transgenlc somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  6. Mycotic bovine nasal granuloma.

    Science.gov (United States)

    Conti Díaz, Ismael Alejandro; Vargas, Roberto; Apolo, Ada; Moraña, José Antonio; Pedrana, Graciela; Cardozo, Elena; Almeida, Edgardo

    2003-01-01

    A case of mycotic bovine nasal granuloma in a 10 year-old Jersey cow, produced by Drechslera halodes is presented. Histopathological sections showed abundant hyaline and pigmented extra and intracellular fungal structures together with a polymorphic cellular granuloma formed by neutrophils, lymphocytes, plasmocytes, histiocytes and giant cells of the Langhans type. It is the first case of mycotic bovine nasal granuloma recognized in Uruguay although this disease seems to be frequent according to the opinion of veterinarian specialists. Another similar clinical case also in a Jersey cow from the same dairy house with an intense cellular infiltrate rich in eosinophils without granulomatous image, together with extracellular hyaline and fuliginous fungal forms, is also referred for comparative purposes. Geotrichum sp. was isolated. The need of an early diagnosis and treatment of the disease is stressed.

  7. Selenium in bovine spermatozoa.

    Science.gov (United States)

    Niemi, S M; Kuzan, F B; Senger, P L

    1981-05-01

    This study investigated the association of selenium with ejaculated bovine spermatozoa. Over 75% of the radioactive spermatozoa. Over 75% of the radioactive selenium-75 was released after 30 min of incubation in 2 X 10(-3) dithiothreitol. Of the selenium-75 released by dithiothreitol, 85% was associated with spermatozoal protein. Protein containing selenium-75 was found predominantly in a single band after polyacrylamide gel electrophoresis. Molecular weight was approximately 21,500 daltons.

  8. Infectious bovine keratoconjunctivitis (pinkeye)

    OpenAIRE

    Angelos, JA

    2015-01-01

    Copyright © 2015 Elsevier Inc. All rights reserved. As is the case for controlling other infectious livestock diseases, the most successful efforts to control infectious bovine keratoconjunctivitis (IBK) will include consideration of the host, the environment, herd management, and ongoing surveillance even after the immediate crisis has passed. Research over many years has led to the discovery of a variety of antibiotic treatments and antibiotic regimens that can be effective against IBK. The...

  9. The orphan nuclear receptor SF-1 is involved in the effect of PCBs, DDT, and DDE on the secretion of steroid hormones and oxytocin from bovine luteal cells during the estrous cycle in vitro.

    Science.gov (United States)

    Mlynarczuk, J; Wrobel, M H; Kotwica, J

    2014-04-15

    The orphan receptor steroidogenic factor-1 (SF-1) is involved in the regulation of ovarian steroidogenesis in cows. It is hypothesized that estrogen-like chlorinated compounds might affect SF-1, and thus impair the function of the ovary. Bovine luteal cells from the estrous cycle (Days: 1-5, 6-10, 11-15, and 16-19) were treated for 50 hours with DDT, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene, 3,3'4,4'-tetrachlorobiphenyl or 2'2'4,4',5,5'-hexachlorobiphenyl (each at a dose of 10 ng/mL). Luteal cells were also treated with 4-(heptyloxy)phenol (1 × 10(-7) M), an SF-1 agonist, and F0160 (1 × 10(-6) M), an SF-1 blocker, jointly or separately. The secretion of progesterone and oxytocin and the expression of oxytocin precursor (NP-I/OT) messenger RNA were increased (P 0.05) SF-1 messenger RNA expression. In summary, SF-1 is involved in the adverse effect of chlorinated xenobiotics on the regulation of the bovine CL.

  10. EPR detected polarization transfer between Gd3+ and protons at low temperature and 3.3 T: The first step of dynamic nuclear polarization

    Science.gov (United States)

    Nagarajan, Vijayasarathi; Hovav, Yonatan; Feintuch, Akiva; Vega, Shimon; Goldfarb, Daniella

    2010-06-01

    Electron-electron double resonance pulsed electron paramagnetic resonance (EPR) at 95 GHz (3.3 T) is used to follow the dynamics of the electron spin polarization during the first stages of dynamic nuclear polarization in solids. The experiments were performed on a frozen solution of Gd+3 (S =7/2) in water/glycerol. Focusing on the central |-1/2⟩→|+1/2⟩ transition we measured the polarization transfer from the Gd3+ electron spin to the adjacent H1 protons. The dependence of the echo detected EPR signal on the length of the microwave irradiation at the EPR "forbidden" transition corresponding to an electron and a proton spin flip is measured for different powers, showing dynamics on the microsecond to millisecond time scales. A theoretical model based on the spin density matrix formalism is suggested to account for this dynamics. The central transition of the Gd3+ ion is considered as an effective S =1/2 system and is coupled to H1 (I =1/2) nuclei. Simulations based on a single electron-single nucleus four level system are shown to deviate from the experimental results and an alternative approach taking into account the more realistic multinuclei picture is shown to agree qualitatively with the experiments.

  11. Effect of calf death loss on cloned cattle herd derived from somatic cell nuclear transfer: clones with congenital defects would be removed by the death loss.

    Science.gov (United States)

    Watanabe, Shinya

    2013-09-01

    To increase public understanding on cloned cattle derived from somatic cell nuclear transfer (SCNT), the present review describes the effect of calf death loss on an SCNT cattle herd. The incidence of death loss in SCNT cattle surviving more than 200 days reached the same level as that in conventionally bred cattle. This process could be considered as removal of SCNT cattle with congenital defects caused by calf death loss. As a result of comparative studies of SCNT cattle and conventionally bred cattle, the substantial equivalences in animal health status, milk and meat productive performance have been confirmed. Both sexes of SCNT cattle surviving to adulthood were fertile and their reproductive performance, including efficiency of progeny production, was the same as that in conventionally bred cattle. The presence of substantial equivalence between their progeny and conventionally bred cattle also existed. Despite these scientific findings, the commercial use of food products derived from SCNT cattle and their progeny has not been allowed by governments for reasons including the lack of public acceptance of these products and the low efficiency of animal SCNT. To overcome this situation, communication of the low risk of SCNT technology and research to improve SCNT efficiency are required.

  12. TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos

    Science.gov (United States)

    Ding, Biao; Zuo, Xiaoyuan; Li, Hui; Ding, Jianping; Li, Yunsheng; Huang, Weiping; Zhang, Yunhai

    2017-01-01

    The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression. PMID:28114389

  13. Paddy-field contamination with 134Cs and 137Cs due to Fukushima Dai-ichi Nuclear Power Plant accident and soil-to-rice transfer coefficients.

    Science.gov (United States)

    Endo, Satoru; Kajimoto, Tsuyoshi; Shizuma, Kiyoshi

    2013-02-01

    The transfer coefficient (TF) from soil to rice plants of (134)Cs and (137)Cs in the form of radioactive deposition from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident in March 2011 was investigated in three rice paddy fields in Minami-Soma City. Rice crops were planted in the following May and harvested at the end of September. Soil cores of 30-cm depth were sampled from rice-planted paddy fields to measure (134)Cs and (137)Cs radioactivity at 5-cm intervals. (134)Cs and (137)Cs radioactivity was also measured in rice ears (rice with chaff), straws and roots. The rice ears were subdivided into chaff, brown rice, polished rice and rice bran, and the (134)Cs and (137)Cs radioactivity concentration of each plant part was measured to calculate the respective TF from the soil. The TF of roots was highest at 0.48 ± 0.10 in the field where the (40)K concentration in the soil core was relatively low, in comparison with TF values of 0.31 and 0.38 in other fields. Similar trends could be found for the TF of whole rice plants, excluding roots. The TF of rice ears was relatively low at 0.019-0.026. The TF of chaff, rice bran, brown rice and polished rice was estimated to be 0.049, 0.10-0.16, 0.013-0.017 and 0.005-0.013, respectively.

  14. Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Miyoshi, Kazuchika; Mori, Hironori; Mizobe, Yamato; Akasaka, Eri; Ozawa, Akio; Yoshida, Mitsutoshi; Sato, Masahiro

    2009-12-01

    The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.

  15. Diagnostic imaging in bovine orthopedics.

    Science.gov (United States)

    Kofler, Johann; Geissbühler, Urs; Steiner, Adrian

    2014-03-01

    Although a radiographic unit is not standard equipment for bovine practitioners in hospital or field situations, ultrasound machines with 7.5-MHz linear transducers have been used in bovine reproduction for many years, and are eminently suitable for evaluation of orthopedic disorders. The goal of this article is to encourage veterinarians to use radiology and ultrasonography for the evaluation of bovine orthopedic disorders. These diagnostic imaging techniques improve the likelihood of a definitive diagnosis in every bovine patient but especially in highly valuable cattle, whose owners demand increasingly more diagnostic and surgical interventions that require high-level specialized techniques.

  16. 4 years of successful knowledge transfer - the nuclear technology training center of the TUeV Nord Group; 4 Jahre erfolgreicher Wissenstransfer - das Ausbildungszentrum fuer Kerntechnik der TUeV Nord Gruppe

    Energy Technology Data Exchange (ETDEWEB)

    Willenbockel, I. [TUeV NORD EnSys Hannover GmbH und Co. KG, Abt. ETE, Hannover (Germany); Tietze, U. [TUeV NORD SysTec, Ausbildungszentrum fuer Kerntechnik, Hamburg (Germany)

    2007-02-15

    In connection with the 2002 amendment to the German Atomic Energy Act, the topics of generational change and maintenance of competence grew in importance and necessitated new solution approaches. To this end, various activities were launched, with the aim of conducting conceptual analyses of these topics. Examples include the 'National Competence Network for Nuclear Technology' (Nationaler Kompetenzverbund fuer Kerntechnik), various networks established by colleges and universities, the 'Knowledge Management for the Maintenance and Transfer of Competence in Reactor Safety' (Wissensmanagement zum Kompetenzerhalt und -transfer in der Reaktorsicherheit) workshop held in 2001 in Garching near Munich (Germany) and the 'Ad-hoc Workgroup on the Maintenance of Competence' (Ad-hoc-Arbeitskreis Kompetenzerhalt) of the VdTUeV. The nuclear technology departments of the TUeV Nord Group were aware of te challenges associated with the generational change early on. By establishing the 'Nuclear Technology Training Center' (Ausbildungszentrum fuer Kerntechnik, AfK), the TUeV Nord Group intended to ensure the required knowledge transfer during the generational change as well as maintain the renowned high qualification as regards the subject of nuclear technology and thus continue to provide - in the sense of social responsibility - crucial contribution to the long-term safety of nuclear plants. Four years have passed since the training center held the first courses in the fall of 2002. Up to now, more than 350 participants have been trained in the courses conducted by the AfK. In the opinion of the TUeV Nord Group, the activities of the AfK have laid the foundation for a successful change of generations within the group's nuclear technology organizations. (orig.)

  17. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    Science.gov (United States)

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  18. Structure and Function of Bovine and Camel Chymosin

    DEFF Research Database (Denmark)

    Jensen, Jesper Langholm

    The central step in cheese making is the separation of milk into curd and whey. This can be done enzymatically by hydrolysis of the Phe105-Met106 bond or nearby bonds in bovine κ-casein, which releases its hydrophilic C-terminal leading to coagulation of the milk. The preferred enzyme...... for this action is bovine chymosin from the calf's stomach, as it has a high activity towards the Phe105-Met106 bond, and a low activity towards other bonds in the milk proteins, as the latter can lead to loss of protein in the whey and release of peptides with a bitter taste. Chymosin was isolated from camel...... this difference through the study of the structures of bovine and camel chymosin, and preparation of catalytically inactive enzymes in complex with substrate. Their milk-clotting activities was determined using the traditional assay on skimmed milk, and a fluorescence resonance energy transfer (FRET) assay...

  19. Splitting and biopsy for bovine embryo sexing under field conditions.

    Science.gov (United States)

    Lopes, R F; Forell, F; Oliveira, A T; Rodrigues, J L

    2001-12-01

    Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.

  20. Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes%体细胞核移植生产绵羊转hALR基因囊胚

    Institute of Scientific and Technical Information of China (English)

    马玉珍; 任宇; 周雪原; 刘东军; 旭日干

    2011-01-01

    扩增人肝细胞再生增强因子(human augmenter of liver regeneration,ALR)基因,利用质粒pIRES2-EGFP 构建新霉素(Neo)、增强绿色荧光蛋白(enhanced green fluorescence protein,EGFP)双标记基因且EGFP和ALR基因为双顺反子的真核表达载体.LipofectAMINETM介导其转染体外培养的绵羊胎儿成纤维细胞(sheep fetal fibroblast cells,sFFCs);经G418筛选转基因细胞;激光共聚焦显微镜挑选绿色荧光单克隆细胞.PCR、RT-PCR和免疫组织化学方法进一步检测ALR基因及其表达;稳定表达外源基因的sFFCs作供体,移入去核的绵羊卵母细胞中,进行体细胞核移植.通过激光共聚焦显微镜和ALR抗体检测EGFP、ALR基因在胚胎水平上的表达,其结果表明:由IRES连接的EGFP和ALR基因可在绵羊胎儿成纤维细胞内同时表达,由此细胞核移植产生的转基因胚胎在发育的各阶段均可见绿色荧光;囊胚中所有细胞表达EGFP基因;发绿色荧光的胚胎中ALR基因同时存在.因此,由IRES连接标记基因和目的基因,以标记基因指示目的基因的表达,可简化检测目的基因的繁琐手段;用筛选的转基因早期胚胎进行移植,可提高制备转基因动物的效率.%Human ALR gene sequence was amplified by PCR from human total DNA and inserted into PIRES2-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/AZJ? By the induction of lipofectAMINE?. The positive cell clones were selected with medium containing G418 (800 ug/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and

  1. Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy.

    Science.gov (United States)

    Slonina, Dominika; Kowalik, Magdalena K; Kotwica, Jan

    2012-01-01

    The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.

  2. Bovine coronavirus hemagglutinin protein.

    Science.gov (United States)

    King, B; Potts, B J; Brian, D A

    1985-02-01

    Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.

  3. Infectious bovine keratoconjunctivitis (pinkeye).

    Science.gov (United States)

    Angelos, John A

    2015-03-01

    As is the case for controlling other infectious livestock diseases, the most successful efforts to control infectious bovine keratoconjunctivitis (IBK) will include consideration of the host, the environment, herd management, and ongoing surveillance even after the immediate crisis has passed. Research over many years has led to the discovery of a variety of antibiotic treatments and antibiotic regimens that can be effective against IBK. The discoveries of Mor bovoculi and reports of IBK associated with Mycoplasma spp without concurrent Mor bovis or Mor bovoculi have raised new questions into the roles that other organisms may play in IBK pathogenesis.

  4. Experimental investigations of heat transfer and temperature fields in models simulating fuel assemblies used in the core of a nuclear reactor with a liquid heavy-metal coolant

    Science.gov (United States)

    Belyaev, I. A.; Genin, L. G.; Krylov, S. G.; Novikov, A. O.; Razuvanov, N. G.; Sviridov, V. G.

    2015-09-01

    The aim of this experimental investigation is to obtain information on the temperature fields and heat transfer coefficients during flow of liquid-metal coolant in models simulating an elementary cell in the core of a liquid heavy metal cooled fast-neutron reactor. Two design versions for spacing fuel rods in the reactor core were considered. In the first version, the fuel rods were spaced apart from one another using helical wire wound on the fuel rod external surface, and in the second version spacer grids were used for the same purpose. The experiments were carried out on the mercury loop available at the Moscow Power Engineering Institute National Research University's Chair of Engineering Thermal Physics. Two experimental sections simulating an elementary cell for each of the fuel rod spacing versions were fabricated. The temperature fields were investigated using a dedicated hinged probe that allows temperature to be measured at any point of the studied channel cross section. The heat-transfer coefficients were determined using the wall temperature values obtained at the moment when the probe thermocouple tail end touched the channel wall. Such method of determining the wall temperature makes it possible to alleviate errors that are unavoidable in case of measuring the wall temperature using thermocouples placed in slots milled in the wall. In carrying out the experiments, an automated system of scientific research was applied, which allows a large body of data to be obtained within a short period of time. The experimental investigations in the first test section were carried out at Re = 8700, and in the second one, at five values of Reynolds number. Information about temperature fields was obtained by statistically processing the array of sampled probe thermocouple indications at 300 points in the experimental channel cross section. Reach material has been obtained for verifying the codes used for calculating velocity and temperature fields in channels with

  5. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

    Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  6. Potential Synthetic Biolubricant as an Alternative to Bovine Serum

    Directory of Open Access Journals (Sweden)

    Susan C. Scholes

    2016-11-01

    Full Text Available It is crucial that orthopaedic implant materials are tested in conditions that replicate the natural body’s environment as closely as possible. Bovine serum is currently recommended for use by the International Organisation for Standardisation (ISO for the wear testing of these implant materials, however, the rheological properties of bovine serum do not match fully those of the body’s natural lubricant, synovial fluid. This study looks at a potential alternative to bovine serum for the testing of orthopaedic implant materials; 0.5% gellan gum fluid gel. Wear tests using multidirectional motion were conducted on ultra-high molecular weight polyethylene (UHMWPE pins rubbing against stainless steel plates. Roughness measurements were performed during testing along with particle analysis of the testing lubricant. At two million cycles (equivalent to 121.3 km of sliding, the mean wear factor for the four UHMWPE pins was 0.25 (standard deviation (SD 0.03 × 10−6 mm3/Nm and there was no evidence of any transfer film on the plate surfaces. The wear factor produced by 0.5% gellan gum fluid gel was lower than that measured in previous studies using bovine serum as the lubricant but greater than the wear factor shown in published work using a similar alternative lubricant (sodium alginate mixed with gellan gum. Work on the development of a suitable alternative lubricant to bovine serum will continue.

  7. Quality and safety of bovine clones and their products.

    Science.gov (United States)

    Heyman, Y; Chavatte-Palmer, P; Fromentin, G; Berthelot, V; Jurie, C; Bas, P; Dubarry, M; Mialot, J P; Remy, D; Richard, C; Martignat, L; Vignon, X; Renard, J P

    2007-08-01

    cattle derived from somatic nuclear transfer than in cattle born from conventional reproduction. Our results confirm that the quality and safety of products (milk and meat) from adult and clinically healthy cloned cattle is globally similar to normal animals. However, from a strictly biological point of view, the slightly delayed maturation we observed in the muscle of clones together with some marginal differences identified in FA composition of both muscle and milk, point to the need for more refined analysis to totally exclude any risks from the consumption of those products.

  8. Tumorigenesis of nuclear transfer-derived embryonic stem cells is reduced through differentiation and enrichment following transplantation in the infarcted rat heart.

    Science.gov (United States)

    Fu, Qiang; Su, Dechun; Wang, Ke; Zhao, Yingjun

    2016-06-01

    The aim of the present study was to evaluate the tumorigenic potential of nuclear transfer-derived (nt) mouse embryonic stem cells (mESCs) transplanted into infarcted rat hearts. The nt‑mESCs were cultured using a bioreactor system to develop embryoid bodies, which were induced with 1% ascorbic acid to differentiate into cardiomyocytes. The nt‑mESC‑derived cardiomyocytes (nt‑mESCs‑CMs) were enriched using Percoll density gradient separation to generate nt‑mESCs‑percoll‑enriched (PE)‑CMs. Ischemia was induced by ligating the left anterior descending coronary artery in female Sprague‑Dawley rats. Immunosuppressed rats (daily intraperitoneal injections of cyclosporine A and methylprednisolone) were randomly assigned to receive an injection containing 5x106 mESCs, nt‑mESCs, nt‑mESC‑CMs or nt‑mESC‑PE‑CMs. Analysis performed 8 weeks following transplantation revealed teratoma formation in 80, 86.67 and 33.33% of the rats administered with the mESCs, nt‑mESCs and nt‑mESC‑CMs, respectively, indicating no significant difference between the mESCs and nt‑mESCs; but significance (P0.05 mESCs, vs. nt‑mESCs; P<0.05 nt‑mESC‑CMs, vs. nt‑mESCs). By contrast, no teratoma formation was detected in the rats, which received nt‑mESC‑PE‑CMs. Octamer‑binding transcription factor‑4, a specific marker of undifferentiated mESCs, was detected using polymerase chain reaction in the rats, which received nt‑mESCs and nt‑mESC‑CMs, but not in rats administered with nt‑mESC‑PE‑CMs. In conclusion, nt‑mESCs exhibited the same pluripotency as mESCs, and teratoma formation following nt‑mESC transplantation was reduced by cell differentiation and enrichment.

  9. 一例体细胞克隆水牛脂肪及皮肤的组织结构观察%Histological Structure Observation of the Fat and Skin in Somatic Nuclear Transfered Buffalo

    Institute of Scientific and Technical Information of China (English)

    王晓丽; 周石琼; 李波; 魏晓瑞; 崔奎青; 石德顺

    2012-01-01

    应用光镜及HE染色等技术对体细胞克隆水牛的脂肪和皮肤组织及其细胞类型和形态进行了详细的观察,旨在为以后体细胞克隆牛的研究提供一定的理论依据,结果表明,脂肪组织外周有疏松结缔组织包裹,脂肪小叶不明显;皮肤由表皮和真皮构成,借助皮下组织与深部的肌肉相连,局部表皮有脱落,皮肤土有毛囊分布.%To provide a certain basis for the study of somatic nuclear transfer buffalo, the histological structure and its cell morphology of adipose and skin tissue in somatic nuclear transferred buffalo were observed by anatomy, HE staining techniques. The results showed that the adipose tissue of somatic nuclear transfer buffalo were encapsulated by loose connective tissue, and the fat lobule was not obvious; The skin was composed of epidermis and dermis, and connected with muscle by hypodermis. Part of epidermis was abscission. There were hair follicles.

  10. Transfer of financial obligations for the disposal of nuclear waste and decommissioning of German NPP's. Legal aspects of a trust model; Sicherstellung der finanziellen Entsorgungsvorsorge fuer die Stilllegungs- und Rueckbaukosten der deutschen Kernkraftwerke. Rechtliche Randbedingungen eines Stiftungsmodells

    Energy Technology Data Exchange (ETDEWEB)

    Schewe, Markus; Wiesendahl, Stefan [Kuemmerlein Rechtsanwaelte und Notare, Essen (Germany)

    2015-04-15

    The nuclear power plant operators have to bear the costs associated with the closure and the decommissioning of the German nuclear power plants as well as the costs for the disposal of nuclear waste. For that purpose, the operators have to build up sufficient reserves for the decommissioning phase. These reserves at the end of 2013 amounted to approximately 36 billion Euro. Changing this system is discussed very so often. Last in May 2014, a public debate started dealing with the so called trust model (''Stiftungsmodell''). The press published deliberations of several operators to transfer their entire nuclear business to the Federal Republic of Germany. Under this deliberation the current nuclear power plant operations, as well as closure obligations would be contributed to trust. Further, also the reserves should be ''transferred'' to the trust. RAG-Foundation (RAG-Stiftung) - which will assume the financial obligations in connection with Germany's closure of underground coal mining activities - sometimes is cited as a role model. The article covers elements of German trust law and atomic energy law regarding such deliberations. In trust law e.g. it can be debated whether the trust should be established under public or - as in the case of RAG-Foundation - under private law. In this context we will set out the major differences between those two options. In the public law part we will notably address issues arising from individual licensing requirements for nuclear power plants and focus on questions concerning reliability, requisite qualification and organizational structures.

  11. Human metabolism and ecological transfer of radioactive caesium: Comparative studies of Chernobyl debris and nuclear weapons fallout, in Southern Sweden and in Bryansk, Russia

    Science.gov (United States)

    Raaf, Christopher Leopold

    The whole-body content of radiocaesium was measured in a South-Swedish urban group of people residing in the city of Lund (55.7°N, 13.2°E) between 1960 and 1994. The results from the survey have been analysed in order to estimate the ecological half-time, Teff,eco, of fallout radiocaesium and the aggregate transfer from ground deposition to man in the region. After 1987, the biological half-times, Ts, of 137Cs and 40K in man were also determined in the reference group through whole-body content measurements in combination with 24-hour urine sampling. Relationships between 24-hour urinary excretion and body burden of 137Cs in the group together with data from the literature were then applied to urine samples collected in 1994 and 1995 from adult subjects living in the highly contaminated region of Bryansk, Russia, in order to estimate their average body burden of 137Cs. The equivalent biological half-time for 137Cs in females of the Lund reference group was, on average (+/-1 WSE), 66 +/- 3 d, which agrees with other findings, whereas the value for the males, 81 +/- 4 d, was, on average, significantly lower than what is found in the literature. This is partly explained by the elevated mean age and relatively low mean body muscle mass of the males investigated in the group during the post-Chernobyl study period. The effective ecological half-time for 137Cs from Chernobyl was found to be 1.8 +/- 0.2 y. The aggregate transfer of 137Cs from deposition to mean activity concentration in man was estimated to be 1.7 Bq kg-1/kBq m-2. These vales may be compared with an effective ecological half-time of 1.3 years found in the reference group in the 1960s, and an aggregate transfer factor of 9.8 Bq kg-1/kBq m-2. The average committed effective dose from ingested 137Cs Chernobyl fallout in the study group was estimated to be 0.02 mSv and from the nuclear weapons fallout to 0.20 mSv. The estimates of whole-body content of 137Cs in the Russian subjects obtained through

  12. Viral infections and bovine mastitis: a review

    NARCIS (Netherlands)

    Wellenberg, G.J.; Poel, van der W.H.M.; Oirschot, van J.T.

    2002-01-01

    This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or para

  13. New insight into the stereoselective interactions of quinine and quinidine, with bovine serum albumin.

    Science.gov (United States)

    Liu, Yan; Chen, Mingmao; Wang, Shuaihua; Lin, Jingjing; Cai, Lizhen; Song, Ling

    2014-05-01

    Quinine (QN) and quinidine (QD), the chief quinoline alkaloids of various species of cinchona bark, are stereoisomers to each other. In this study, a series of appropriate and efficient methods have been applied to compare the binding modes of QN and QD with bovine serum albumin (BSA). The isothermal titration calorimetry and room temperature phosphorescence results show that both QN and QD can interact with BSA at one binding site to form drug-protein complexes, mainly through enthalpic driving force with the binding affinity order: QN > QD. The fluorescence resonance energy transfer and time-resolved fluorescence spectroscopy exhibits that QN has a larger energy transfer and more intensified binding capacity for BSA than QD. Data of dynamic light scattering reveal that the aggregate state of BSA is changed during this binding process, and the particle size distribution of QN-BSA bioconjugate is larger than that of QD. Nuclear magnetic resonance analysis indicates that aromatic protons make more contribution during ligand-protein complexation than that of aliphatic protons. The circular dichroism spectra exhibit different degrees of changes in BSA secondary structures in the presence of QN and QD, respectively.

  14. ¿Es posible la recuperación de especies silvestres extintas a través de la transferencia somática nuclear? Is the recovery of extinct wild species through somatic nuclear transfer possible?

    Directory of Open Access Journals (Sweden)

    Janet López-Saucedo

    2010-08-01

    technology approaches. However, considering that Mexico is one of the megadiverse countries with a great number of species of threatened mammals, biotechnological tools are urgently needed for conservation research of Mexican species. Furthermore, these tools need to be utilized and developed by our own scientific Latin-American community. In this note, we discuss the scientific advances that have proposed the use of somatic nuclear transfer as a conservation tool, its real possibilities, the needs and suggestions for its development in threatened Mexican / Latin-American mammal species that should allow modern biotechnology to turn into one more tool in the battle of biological conservation.

  15. Human metabolism and ecological transfer of radioactive caesium. Comparative studies of Chernobyl debris and nuclear weapons fallout, in southern Sweden and in Bryansk, Russia

    Energy Technology Data Exchange (ETDEWEB)

    Raeaef, C.L

    2000-05-01

    The whole-body content of radiocaesium was measured in a South Swedish urban group of people residing in the city of Lund between 1960 and 1994. The results from the survey have been analysed in order to estimate the ecological half time, T{sub eff,eco} of fallout radiocaesium, and the aggregate transfer from ground deposition to man in the region. After 1987, the biological half times, T{sub e} of {sup 137}Cs and {sup 40}K in man were also determined in the reference group through whole-body content measurements in combination with 24-hour urine sampling. Relationships between 24-hour urinary excretion and body burden of {sup 137}Cs in the group together with data from the literature were then applied to urine samples collected in 1994 and 1995 from adult subjects living in the highly contaminated region of Bryansk, Russia, in order to estimate their average body burden of {sup 137}Cs. The equivalent biological half-time for {sup 137}Cs in females of the Lund reference group was, on average 66{+-}3 d, which agrees with other findings, whereas the value for the males, 81{+-}4 d, was, on average, significantly lower than what is found in the literature. This is partly explained by the elevated mean age and relatively low mean body muscle mass of the males investigated. The {sup 137}Cs from nuclear weapons tests in the 1950s and 1960s still gave a significant contribution to the total {sup 137}Cs levels in man during the post-Chernobyl study period (1987-1994). About 10% of the peak post-Chernobyl concentration level of {sup 137}Cs (3.5-4 Bq/kg) in 1987, was attributed to pre-Chernobyl {sup 137}Cs. The effective ecological half-time for {sup 137}Cs from Chernobyl was found to be 1.8{+-}0.2 y. The time-integrated aggregate transfer of {sup 137}Cs from ground deposition to mean activity concentration in man was estimated to be 0.4 Bq/kg/kBq/m{sup 2}. These values may be compared with an effective ecological half-time of 1.3 years found in the Lund reference group in

  16. Review of the safety concept for fusion reactor concepts and transferability of the nuclear fission regulation to potential fusion power plants

    Energy Technology Data Exchange (ETDEWEB)

    Raeder, Juergen; Weller, Arthur; Wolf, Robert [Max-Planck-Institut fuer Plasmaphysik (IPP), Garching (Germany); Jin, Xue Zhou; Boccaccini, Lorenzo V.; Stieglitz, Robert; Carloni, Dario [Karlsruher Institute fuer Technologie (KIT), Eggenstein-Leopoldshafen (Germany); Pistner, Christoph [Oeko-Institut e.V., Darmstadt (Germany); Herb, Joachim [Gesellschaft fuer Anlagen- und Reaktorsicherheit, Koeln (Germany)

    2016-01-15

    This paper summarizes the current state of the art in science and technology of the safety concept for future fusion power plants (FPPs) and examines the transferability of the current nuclear fission regulation to the concepts of future fusion power plants. At the moment there exist only conceptual designs of future fusion power plants. The most detailed concepts with regards to safety aspects were found in the European Power Plant Conceptual Study (PPCS). The plant concepts discussed in the PPCS are based on magnetic confinement of the plasma. The safety concept of fusion power plants, which has been developed during the last decades, is based on the safety concepts of installations with radioactive inventories, especially nuclear fission power plants. It applies the concept of defence in depth. However, there are specific differences between the implementations of the safety concepts due to the physical and technological characteristics of fusion and fission. It is analysed whether for fusion a safety concept is required comparable to the one of fission. For this the consequences of a purely hypothetical release of large amounts of the radioactive inventory of a fusion power plant and a fission power plant are compared. In such an event the evacuation criterion outside the plant is exceeded by several orders of magnitude for a fission power plant. For a fusion power plant the expected radiological consequences are of the order of the evacuation criterion. Therefore, a safety concept is also necessary for fusion to guarantee the confinement of the radioactive inventory. The comparison between the safety concepts for fusion and fission shows that the fundamental safety function ''confinement of the radioactive materials'' can be transferred directly in a methodical way. For a fusion power plant this fundamental safety function is based on both, physical barriers as well as on active retention functions. After the termination of the fusion

  17. Review of the safety concept for fusion reactor concepts and transferability of the nuclear fission regulation to potential fusion power plants

    Energy Technology Data Exchange (ETDEWEB)

    Raeder, Juergen; Weller, Arthur; Wolf, Robert [Max-Planck-Institut fuer Plasmaphysik (IPP), Garching (Germany); Jin, Xue Zhou; Boccaccini, Lorenzo V.; Stieglitz, Robert; Carloni, Dario [Karlsruher Institute fuer Technologie (KIT), Eggenstein-Leopoldshafen (Germany); Pistner, Christoph [Oeko-Institut e.V., Darmstadt (Germany); Herb, Joachim [Gesellschaft fuer Anlagen- und Reaktorsicherheit, Koeln (Germany)

    2016-01-15

    This paper summarizes the current state of the art in science and technology of the safety concept for future fusion power plants (FPPs) and examines the transferability of the current nuclear fission regulation to the concepts of future fusion power plants. At the moment there exist only conceptual designs of future fusion power plants. The most detailed concepts with regards to safety aspects were found in the European Power Plant Conceptual Study (PPCS). The plant concepts discussed in the PPCS are based on magnetic confinement of the plasma. The safety concept of fusion power plants, which has been developed during the last decades, is based on the safety concepts of installations with radioactive inventories, especially nuclear fission power plants. It applies the concept of defence in depth. However, there are specific differences between the implementations of the safety concepts due to the physical and technological characteristics of fusion and fission. It is analysed whether for fusion a safety concept is required comparable to the one of fission. For this the consequences of a purely hypothetical release of large amounts of the radioactive inventory of a fusion power plant and a fission power plant are compared. In such an event the evacuation criterion outside the plant is exceeded by several orders of magnitude for a fission power plant. For a fusion power plant the expected radiological consequences are of the order of the evacuation criterion. Therefore, a safety concept is also necessary for fusion to guarantee the confinement of the radioactive inventory. The comparison between the safety concepts for fusion and fission shows that the fundamental safety function ''confinement of the radioactive materials'' can be transferred directly in a methodical way. For a fusion power plant this fundamental safety function is based on both, physical barriers as well as on active retention functions. After the termination of the fusion

  18. Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis

    OpenAIRE

    2007-01-01

    International audience; Bovine herpesvirus 1 (BoHV-1), classified as an alphaherpesvirus, is a major pathogen of cattle. Primary infection is accompanied by various clinical manifestations such as infectious bovine rhinotracheitis, abortion, infectious pustular vulvovaginitis, and systemic infection in neonates. When animals survive, a life-long latent infection is established in nervous sensory ganglia. Several reactivation stimuli can lead to viral re-excretion, which is responsible for the...

  19. Intervet symposium: bovine neosporosis.

    Science.gov (United States)

    Schetters, T; Dubey, J P; Adrianarivo, A; Frankena, K; Romero, J J; Pérez, E; Heuer, C; Nicholson, C; Russell, D; Weston, J

    2004-10-28

    This article summarises the most relevant data of presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)held in New Orleans, LA, USA, from 10 to 14 August 2003) in a symposium session on bovine neosporosis. The symposium was organised by Juan Muñoz-Bielsa,Wicher Holland, Enzo Foccoliand Theo Schetters (chairman). The focus was on the present state of knowledge of the biology, epidemiology(presented by J.P. Dubey) and immunology of Neospora infection (presented by A. Adrianarivo),with special emphasis on the prospects of vaccination of cattle against Neospora-induced abortion (presentations of K. Frankena (Costa Rican trial) and C. Heuer (New Zealand trial)).

  20. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  1. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Jun Liu

    Full Text Available The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3 in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli, Staphylococcus aureus (S. aureus and Streptococcus agalactiae (S. agalactiae that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05. The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3 and 95.4×10(3 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC in infected glands reached up to 260.4×10(5 and 622.2×10(5 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  2. Binding of several benzodiazepines to bovine serum albumin: Fluorescence study

    Science.gov (United States)

    Machicote, Roberta G.; Pacheco, María E.; Bruzzone, Liliana

    2010-10-01

    The interactions of lorazepam, oxazepam and bromazepam with bovine serum albumin (BSA) were studied by fluorescence spectrometry. The Stern-Volmer quenching constants and corresponding thermodynamic parameters Δ H, Δ G and Δ S were calculated. The binding constants and the number of binding sites were also investigated. The distances between the donor (BSA) and the acceptors (benzodiazepines) were obtained according to fluorescence resonance energy transfer and conformational changes of BSA were observed from synchronous fluorescence spectra.

  3. Density content of nuclear symmetry energy from nuclear observables

    Indian Academy of Sciences (India)

    B K Agrawal

    2014-11-01

    The nuclear symmetry energy at a given density measures the energy transferred in converting symmetric nuclear matter into the pure neutron matter. The density content of nuclear symmetry energy remains poorly constrained. Our recent results for the density content of the nuclear symmetry energy, around the saturation density, extracted using experimental data for accurately known nuclear masses, giant resonances and neutron-skin thickness in heavy nuclei are summarized.

  4. The host defense proteome of human and bovine milk.

    Directory of Open Access Journals (Sweden)

    Kasper Hettinga

    Full Text Available Milk is the single source of nutrients for the newborn mammal. The composition of milk of different mammals has been adapted during evolution of the species to fulfill the needs of the offspring. Milk not only provides nutrients, but it also serves as a medium for transfer of host defense components to the offspring. The host defense proteins in the milk of different mammalian species are expected to reveal signatures of evolution. The aim of this study is therefore to study the difference in the host defense proteome of human and bovine milk. We analyzed human and bovine milk using a shot-gun proteomics approach focusing on host defense-related proteins. In total, 268 proteins in human milk and 269 proteins in bovine milk were identified. Of these, 44 from human milk and 51 from bovine milk are related to the host defense system. Of these proteins, 33 were found in both species but with significantly different quantities. High concentrations of proteins involved in the mucosal immune system, immunoglobulin A, CD14, lactoferrin, and lysozyme, were present in human milk. The human newborn is known to be deficient for at least two of these proteins (immunoglobulin A and CD14. On the other hand, antimicrobial proteins (5 cathelicidins and lactoperoxidase were abundant in bovine milk. The high concentration of lactoperoxidase is probably linked to the high amount of thiocyanate in the plant-based diet of cows. This first detailed analysis of host defense proteins in human and bovine milk is an important step in understanding the function of milk in the development of the immune system of these two mammals.

  5. A dynamic model to calculate cadmium concentrations in bovine tissues from basic soil characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Waegeneers, Nadia, E-mail: nadia.waegeneers@var.fgov.be; Ruttens, Ann; De Temmerman, Ludwig

    2011-06-15

    A chain model was developed to calculate the flow of cadmium from soil, drinking water and feed towards bovine tissues. The data used for model development were tissue Cd concentrations of 57 bovines and Cd concentrations in soil, feed and drinking water, sampled at the farms were the bovines were reared. Validation of the model occurred with a second set of measured tissue Cd concentrations of 93 bovines of which age and farm location were known. The exposure part of the chain model consists of two parts: (1) a soil-plant transfer model, deriving cadmium concentrations in feed from basic soil characteristics (pH and organic matter content) and soil Cd concentrations, and (2) bovine intake calculations, based on typical feed and water consumption patterns for cattle and Cd concentrations in feed and drinking water. The output of the exposure model is an animal-specific average daily Cd intake, which is then taken forward to a kinetic uptake model in which time-dependent Cd concentrations in bovine tissues are calculated. The chain model was able to account for 65%, 42% and 32% of the variation in observed kidney, liver and meat Cd concentrations in the validation study. - Research highlights: {yields} Cadmium transfer from soil, drinking water and feed to bovine tissues was modeled. {yields} The model was based on 57 bovines and corresponding feed and soil Cd concentrations. {yields} The model was validated with an independent data set of 93 bovines. {yields} The model explained 65% of variation in kidney Cd in the validation study.

  6. Successful vitrification of bovine blastocysts on paper container.

    Science.gov (United States)

    Kim, Y M; Uhm, S J; Gupta, M K; Yang, J S; Lim, J-G; Das, Z C; Heo, Y T; Chung, H-J; Kong, I-K; Kim, N-H; Lee, H T; Ko, D H

    2012-09-15

    Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P 0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos.

  7. Bovine respiratory disease model based on dual infections with infection with bovine viral diarrhea virus and bovine corona virus

    Science.gov (United States)

    Bovine respiratory disease complex (BRDC) is the leading cause of economic loss in the U.S. cattle industry. BRDC likely results from simultaneous or sequential infections with multiple pathogens including both viruses and bacteria. Bovine viral diarrhea virus (BVDV) and bovine corona virus (BoCV...

  8. Cold nuclear fusion

    Directory of Open Access Journals (Sweden)

    Huang Zhenqiang Huang Yuxiang

    2013-10-01

    Full Text Available In normal temperature condition, the nuclear force constraint inertial guidance method, realize the combination of deuterium and tritium, helium and lithium... And with a magnetic moment of light nuclei controlled cold nuclear collide fusion, belongs to the nuclear energy research and development in the field of applied technology "cold nuclear collide fusion". According to the similarity of the nuclear force constraint inertial guidance system, the different velocity and energy of the ion beam mixing control, developed ion speed dc transformer, it is cold nuclear fusion collide, issue of motivation and the nuclear power plant start-up fusion and power transfer system of the important equipment, so the merger to apply for a patent

  9. Application of a nuclear localization signal gene in transgene mice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Efficient gene transfer by cytoplasm co-injec- tion will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine (?-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.

  10. Relationship between Beta-Lactoglobulin and Bovine Submaxillary Mucin: Structure and Tribology Studies

    DEFF Research Database (Denmark)

    Celebioglu, Hilal Yilmaz; Guðjónsdóttir, María; Chronakis, Ioannis S.;

    ), the major whey protein, and bovine submaxillary mucin (BSM), a (model) major salivary component, when mixed (1:1) at different pHs (pH 3.0, 5.0 and 7.4) in order to broaden our understanding of food oral processing on the molecular level. High and low field Nuclear Magnetic Resonance (NMR), Dynamic Light...

  11. Sex determination of bovine preimplantation embryos by oligonucleotide microarray.

    Science.gov (United States)

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin

    2013-06-01

    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  12. Material Properties of Inorganic Bovine Cancellous Bovine: Nukbone

    Science.gov (United States)

    Piña, Cristina; Palma, Benito; Munguía, Nadia

    2006-09-01

    In this work, inorganic cancellous bovine bone implants prepared in the Instituto de Investigaciones en Materiales — UNAM were characterized. Elementary chemical analysis was made, toxic elements concentration were measured and the content of organic matter also. These implants fulfill all the requirements of the ASTM standards, and therefore it is possible their use in medical applications.

  13. Comparative Genomic Analysis of the ICESa2603 Family ICEs and Spread of erm(B- and tet(O-Carrying Transferable 89K-Subtype ICEs in Swine and Bovine Isolates in China

    Directory of Open Access Journals (Sweden)

    Jinhu eHuang

    2016-02-01

    Full Text Available Integrative and conjugative elements (ICEs of the ICESa2603 family have been isolated from several species of Streptococcus spp.; however, the comparative genomic and evolutionary analyses of these particular ICEs are currently only at their initial stages. By investigating 13 ICEs of the ICESa2603 family and two ICESa2603 family-like ICEs derived from diverse hosts and locations, we have determined that ICEs comprised a backbone of 30 identical syntenic core genes and accessory genes that were restricted to the intergenic sites or the 3'-end of the non-conserved domain of core genes to maintain its function. ICESa2603 family integrase IntICESa2603 specifically recognized a 15-bp att sequence (TTATTTAAGAGTAAC at the 3'-end of rplL, which was highly conserved in genus Streptococcus. Phylogenetic analyses suggest that extensive recombination/insertion and the occurrence of a hybrid/mosaic in the ICESa2603 family were responsible for the significant increase in ICE diversity, thereby broadening its host range. Approximately 42.5% and 38.1% of the tested Streptococcus suis and Streptococcus agalactiae clinical isolates respectively contained ICESa2603 family Type Ⅳ secretion system (T4SS genes, and 80.5% and 62.5% of which also respectively carried intICESa2603, indicating that ICESa2603 family is widely distributed across these bacteria. Sequencing and conjugation transfer of a novel sequence type ST303 clinical S. suis isolate HB1011 demonstrated that the 89K-subtype ICESsuHB1011 retained its transferrable function, thereby conferring tetracycline and macrolide resistance.

  14. Indução do parto de bovinos aos 270 dias de gestação com a utilização prévia de glicocorticoide de longa ação e a transferência de imunidade passiva = Inducing bovine parturition at 270 pregnancy days with early use of a long-acting glucocorticoid and passive immunity transfer

    Directory of Open Access Journals (Sweden)

    Alfredo Quites Antoniazzi

    2009-01-01

    Full Text Available O trabalho teve por objetivo avaliar a taxa de transferência de imunidade passiva e de retenção de placenta com a utilização prévia de um glicocorticoide de longa ação na indução do parto bovino aos 270 dias de gestação, bem como verificar a eficácia dos protocolos. Foram utilizadas 42 vacas prenhes, de corte e de leite, leatoriamente separadas em três grupos: Grupo-controle (C que não recebeu aplicação hormonal, mas o mesmo manejo; Grupo longa ação + indução (LA que recebeu 30 mg de acetato de metilprednisolona, via subcutânea, no dia 255 de gestação e 20 mg de dexametasona associada a 0,5 mg de cloprostenol sódico, via intramuscular, no dia 270 de gestação; e Grupo indução (I que recebeu 20 mg de dexametasona associada a 0,5 mg de cloprostenol sódico no dia 270 de gestação. A utilização prévia do glicocorticoide de longa ação não mostrou diferença na taxa de transferência de imunidade passiva e nos índices de retenção de placenta. As fêmeas dos Grupos LA e I pariram em 41,36 ± 1,89h após a aplicação hormonal de dexametasona associada ao cloprostenol, independente de aplicação prévia de glicocorticoide de longa ação no Grupo LA. A indução da parição com dexametasonaassociada ao cloprostenol, independente da aplicação prévia de acetato de metilprednisolona, mostrou-se eficaz em todos dos animais.This experiment had the objective of evaluating passive immunity transfer and placental retention rates with the early use of a long-acting glucocorticoid to induce bovine parturition at 270 pregnancy days, as well as comparing protocol efficacy regarding the timing of parturition induction. Fortytwo pregnant cows were selected from a beef and dairy herd and randomly assigned into one of three groups: control (C without drug injection, but with the same management; long-acting + induction group (LA, with one injection of 30 mg prednisolone methylacetate, subcutaneous, on pregnancy day 255 and

  15. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.;

    2005-01-01

    The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos...... were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...... embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas...

  16. Superovulatory response and embryonic progressive in Iranian Qezel ewes treated with two different concentrations of bovine somatotropin

    Directory of Open Access Journals (Sweden)

    Amir Hossein Asgari Safdar

    2016-05-01

    Conclusions: The treatment 50 mg bovine somatotropins enhance the ratio and growth of the transferable embryos. Embryos of bST-50 treatment indicated an improved embryonic development but bST did not affect the pregnancy rates of transferred embryos.

  17. 核电项目建造阶段中的焊接工艺评定转移%Welding procedure qualification transference to different project of nuclear power item during the construction period

    Institute of Scientific and Technical Information of China (English)

    马新朝

    2011-01-01

    论述了核电站在建造阶段焊接工艺评定报告由已完成建造任务的核电项目至正在建设中的核电项目中的焊接工艺评定报告的转移情况,分析了工艺评定转移的可行性,转移后施工单位、监理单位、政府监督单位所应关注的重点,突出了质量保证及质量控制,更关注了政府监督单位(核安全局及地区核与辐射监督站)的监督职能.焊接工艺评定的有效转移,一方面节约了重复评定时的人力和物力投入,降低了建造成本;另一方面有力的推动了工程的顺利建设,促进焊接技术及经验的持续发展,从而提离了管理水平.%The paper shows us the Welding producer report transference to different project of nuclear power item during the construction period,analyses the practicability to the producer report transference. After transference,the paper discusses the focal point to welding in construction enterprises,surveillance management corporation as well as the responsible for supervision departments of the Government,give prominence to the Quality assurance and Quality control. The Nuclear Safety Bureau and the Area Departments Responsible for Supervision on radiation should give more attention to the surveillance on transference.The effective transfer of PQR can reduce expenses ,material and manpower in duplicate welding procedure qualification,reduce construction costs ;the other side,which can strongly promotes the project construction,accelerate the welding technology and experience continue development,and improve the management standards.

  18. Bovine spongiform encephalopathy in sheep?

    NARCIS (Netherlands)

    Schreuder, B.E.C.; Somerville, R.A.

    2003-01-01

    Bovine spongiform encephalopathy (BSE) in sheep has not been identified under natural conditions at the time of writing and remains a hypothetical issue. However, rumours about the possible finding of a BSE-like isolate in sheep have led to great unrest within the sheep industry, among the general p

  19. Nuclear power industry

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-04-28

    This press dossier presented in Shanghai (China) in April 1999, describes first the activities of the Framatome group in the people`s republic of China with a short presentation of the Daya Bay power plant and of the future Ling Ao project, and with a description of the technological cooperation with China in the nuclear domain (technology transfers, nuclear fuels) and in other industrial domains (mechanics, oil and gas, connectors, food and agriculture, paper industry etc..). The general activities of the Framatome group in the domain of energy (nuclear realizations in France, EPR project, export activities, nuclear services, nuclear fuels, nuclear equipments, industrial equipments) and of connectors engineering are presented in a second and third part with the 1998 performances. (J.S.)

  20. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos

    NARCIS (Netherlands)

    Wagtendonk-de Leeuw, van A.M.; den Daas, J.H.; Kruip, T.A.; Rail, W.F.

    1995-01-01

    Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii) conv

  1. 77 FR 15847 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-03-16

    ... been fed ruminant protein, other than milk protein, during their lifetime; The bovines from which the... from animals that are not known to have been fed ruminant protein, other than milk protein, during... community is that the agent is an abnormal form of a normal protein known as cellular prion protein. The...

  2. The criterion for blanking-off heat-transfer tubes in the steam generators at VVER-based nuclear power plants based on the results of eddy-current examination

    Science.gov (United States)

    Lunin, V. P.; Zhdanov, A. G.; Chegodaev, V. V.; Stolyarov, A. A.

    2015-05-01

    The problem of defining the criterion for blanking off heat-transfer tubes in the steam generators at nuclear power plants on the basis of signals obtained from the standard multifrequency eddy-current examination is considered. The decision about blanking off one or another tube is presently made with reference to one parameter of the relevant signal at the working frequency, namely, with reference to its phase, which directly depends on the depth of the flaw being detected, i.e., a crack in the tube. The crack depth equal to 60% of the tube wall thickness is regarded to be the critical one, at which a decision about withdrawing such a tube out from operation (blanking off) must be taken. However, since mechanical tensile rupture tests of heat-transfer tubes show the possibility of their further use with such flaws, the secondary parameter of the signal, namely, its amplitude, must be used for determining the blanking-off criterion. The signals produced by the standard flow-type transducers in response to flaws in the form of a longitudinal crack having the depth and length within the limits permitted by the relevant regulations were calculated using 3D finite-element modeling. Based on the obtained results, the values of the eddy-current signal amplitude were determined, which, together with the signal phase value, form a new amplitude-phase criterion for blanking off heat-transfer tubes. For confirming the effectiveness of this technique, the algorithm for revealing the signal indications satisfying the proposed amplitude-phase criterion was tested on real signals obtained from operational eddy-current examination of the state of steam generator heat-transfer tubes carried out within the framework of planned preventive repair.

  3. Fusion: a tale of recombination in an asexual fungus: The role of nuclear dynamics and hyphal fusion in horizontal chromosome transfer in Fusarium oxysporum

    OpenAIRE

    Shahi, S.

    2016-01-01

    Recent studies have shown that not only meiotic recombination is responsible for the fast evolution of fungal pathogens. In the asexual fungus F. oxysporum (Fo) the "fast" evolving part of the genome is organized into small chromosomes and one such chromosome houses all effector genes and is referred to as the "pathogenicity" chromosome. This pathogenicity chromosome can be horizontally transferred to a non-pathogenic strain, conferring pathogenicity. Here we use Fo as a model organism to add...

  4. The transfer of radiocesium from the bark to the stemflow of chestnut trees (Castanea crenata) contaminated by radionuclides from the Fukushima Dai-ichi nuclear power plant accident.

    Science.gov (United States)

    Sasaki, Yoshito; Abe, Hironobu; Mitachi, Katsuaki; Watanabe, Takayoshi; Ishii, Yasuo; Niizato, Tadafumi

    2016-09-01

    We report on the behavior of radiocesium in tree bark and its transfer into the stemflows of chestnut trees in a forest in the Fukushima Prefecture, Japan. In stems that were present at the time of the accident, the radiocesium concentration of the bark was found to be approximately 10 times that of the wood. The average (137)Cs concentration of the dissolved fraction (0.45 μm) of the stemflow were strongly adsorbed radiocesium.

  5. Nuclear Confidence

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    The Fukushima nuclear accident provides valuable lessons for China national nuclear Corp.as it continues to expand its operations AS Japan’s Fukushima nuclear crisis sparks a global debate over nuclear safety,China National Nuclear Corp. (CNNC),the country’s largest nuclear plant operator, comes under the spotlight.

  6. Oversight framework over oocyte procurement for somatic cell nuclear transfer: comparative analysis of the Hwang Woo Suk case under South Korean bioethics law and U.S. guidelines for human embryonic stem cell research.

    Science.gov (United States)

    Kim, Mi-Kyung

    2009-01-01

    We examine whether the current regulatory regime instituted in South Korea and the United States would have prevented Hwang's potential transgressions in oocyte procurement for somatic cell nuclear transfer, we compare the general aspects and oversight framework of the Bioethics and Biosafety Act in South Korea and the US National Academies' Guidelines for Human Embryonic Stem Cell Research, and apply the relevant provisions and recommendations to each transgression. We conclude that the Act would institute centralized oversight under governmental auspices while the Guidelines recommend politically-independent, decentralized oversight bodies including a special review body for human embryonic stem cell research at an institutional level and that the Guidelines would have provided more vigorous protection for the women who had undergone oocyte procurement for Hwang's research than the Act. We also suggest additional regulations to protect those who provide oocytes for research in South Korea.

  7. Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear

    OpenAIRE

    2010-01-01

    Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2) com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante se...

  8. A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.

    Science.gov (United States)

    Zhang, Hui; Wang, LiJun; Li, WenZhe; Mao, QingFu; Wang, YongSheng; Li, Qian; Hua, Song; Zhang, Yong

    2015-10-01

    RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used.

  9. Corrosion of aluminum clad spent nuclear fuel in the 70 ton cask during transfer from L area to H-canyon

    Energy Technology Data Exchange (ETDEWEB)

    Mickalonis, J. I. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2015-08-01

    Aluminum-clad spent nuclear fuel will be transported for processing in the 70-ton nuclear fuel element cask from L Basin to H-canyon. During transport these fuels would be expected to experience high temperature aqueous corrosion from the residual L Basin water that will be present in the cask. Cladding corrosion losses during transport were calculated for material test reactor (MTR) and high flux isotope reactors (HFIR) fuels using literature and site information on aqueous corrosion at a range of time/temperature conditions. Calculations of the cladding corrosion loss were based on Arrhenius relationships developed for aluminum alloys typical of cladding material with the primary assumption that an adherent passive film does not form to retard the initial corrosion rate. For MTR fuels a cladding thickness loss of 33% was found after 1 year in the cask with a maximum temperature of 263 °C. HFIR fuels showed a thickness loss of only 6% after 1 year at a maximum temperature of 180 °C. These losses are not expected to impact the overall confinement function of the aluminum cladding.

  10. Corrosion of aluminum clad spent nuclear fuel in the 70 ton cask during transfer from L area to H-canyon

    Energy Technology Data Exchange (ETDEWEB)

    Mickalonis, J. I. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2015-08-31

    Aluminum-clad spent nuclear fuel will be transported for processing in the 70-ton nuclear fuel element cask from L Basin to H-canyon. During transport these fuels would be expected to experience high temperature aqueous corrosion from the residual L Basin water that will be present in the cask. Cladding corrosion losses during transport were calculated for material test reactor (MTR) and high flux isotope reactors (HFIR) fuels using literature and site information on aqueous corrosion at a range of time/temperature conditions. Calculations of the cladding corrosion loss were based on Arrhenius relationships developed for aluminum alloys typical of cladding material with the primary assumption that an adherent passive film does not form to retard the initial corrosion rate. For MTR fuels a cladding thickness loss of 33 % was found after 1 year in the cask with a maximum temperature of 263 °C. HFIR fuels showed a thickness loss of only 6% after 1 year at a maximum temperature of 180 °C. These losses are not expected to impact the overall confinement function of the aluminum cladding.

  11. CORROSION OF ALUMINUM CLAD SPENT NUCLEAR FUEL IN THE 70 TON CASK DURING TRANSFER FROM L AREA TO H-CANYON

    Energy Technology Data Exchange (ETDEWEB)

    Mickalonis, J.

    2014-06-01

    Aluminum-clad spent nuclear fuel will be transported for processing in the 70-ton nuclear fuel element cask from L Basin to H-canyon. During transport these fuels would be expected to experience high temperature aqueous corrosion from the residual L Basin water that will be present in the cask. Cladding corrosion losses during transport were calculated for material test reactor (MTR) and high flux isotope reactors (HFIR) fuels using literature and site information on aqueous corrosion at a range of time/temperature conditions. Calculations of the cladding corrosion loss were based on Arrhenius relationships developed for aluminum alloys typical of cladding material with the primary assumption that an adherent passive film does not form to retard the initial corrosion rate. For MTR fuels a cladding thickness loss of 33 % was found after 1 year in the cask with a maximum temperature of 260 {degrees}C. HFIR fuels showed a thickness loss of only 6% after 1 year at a maximum temperature of 180 {degrees}C. These losses are not expected to impact the overall confinement function of the aluminum cladding.

  12. Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer%体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究

    Institute of Scientific and Technical Information of China (English)

    刘凤军; 张玉玲; 杨自军; 陈兴启; 孙达权; 王国华; 张涌

    2008-01-01

    [Objective] The aim of this study is to understand the effects of donor cell type, embryo stage, number and transfer position on the efficiency of goat transgenic clone. [Method] Using somatic cell nuclear transfer technology, the single goat fetal fibroblasts (GFF) and mammary gland epithelial cells (GMGE) harboring human lactoferrin (hLF) gene were transferred to the enucleated oocyte. Reconstructed karyoplast-cytoplast couplets were fused, activated, and cultured in vitro. Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients, and blastocysts were transferred into uterine horn. [Result] The pregnancy rate was similar between GFF and GMGE (oviduct transfer:26.47% vs. 20.00%), and between oviduct transfer and uterine horn transfer (26.47% vs. 25.00%) for GFF group; pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group (40.00% vs. 26.67% and 21.43%). [Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type, embryo stage and transfer position but be done by the number of embryo transferred per recipient. In addition, the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.

  13. Bovine cysticercosis situation in Brazil

    Directory of Open Access Journals (Sweden)

    Gabriel Augusto Marques Rossi

    2014-02-01

    Full Text Available The taeniasis-cysticercosis complex is a long known zoonotic parasitosis characteristic of underdeveloped countries. In addition to its public health significance, this parasitosis is cause of economic losses to the beef production chain, and synonymous of technical inadequacy in relation to the adoption of Good Agricultural Practices. The occurrences of both human teniasis and bovine cysticercosis could and should be controlled with basic sanitary measures. However, there is much variation in the occurrence of the disease in cattle, characterizing a low rate of technical development as well as problems related to the adoption of basic sanitation measures. This review describes, in details, the causative agent and its epidemiological chain, besides raising current information about the occurrence of bovine cysticercosis in different regions of Brazil, aiming at the adoption of prophylactic measures by different segments responsible.

  14. Estimation of soil-to-plant transfer factors of radiocesium in 99 wild plant species grown in arable lands 1 year after the Fukushima 1 Nuclear Power Plant accident.

    Science.gov (United States)

    Yamashita, Jun; Enomoto, Takashi; Yamada, Masao; Ono, Toshiro; Hanafusa, Tadashi; Nagamatsu, Tomohiro; Sonoda, Shoji; Yamamoto, Yoko

    2014-01-01

    One year after the deposition of radionuclides from the Fukushima 1 Nuclear Power Plant (A formal name is Fukushima Daiichi Nuclear Power Station) in March 2011, radiocesium (¹³⁴Cs, ¹³⁷Cs) concentrations ([Cs]) were comprehensively investigated in the wild plants of 99 species most of which were annual or summer green perennial herbs and started to grow from April 2012 at the heavily contaminated fields of paddy (three study sites) and upland (one study site) in Fukushima Prefecture. The survey was conducted three times (April, July and October) in the year. In each site, soils (soil cores of 5-cm depth) and plants (aerial shoots) were collected for determination of [Cs] on a dry weight basis, and then the transfer factor (TF) of radiocesium from soil to plant ([Cs]plant/[Cs]soil) was estimated in each species. The [Cs] values of both soils and plants largely varied. However, some species exhibited relatively high TF values (more than 0.4) (e.g., Athyrium yokoscense, Dryopteris tokyoensis, and Cyperus brevifolius), while others exhibited almost negligible values (less than 0.01) (e.g., Salix miyabeana, Humulus scandens, and Elymus tsukushiensis). In addition, judging from the 11 species grown in both paddy and upland fields, TF values were generally higher in the paddy fields. The estimation of phytoextraction efficiency of soil radiocesium by weed communities in the paddy fields suggests that the weed community is not a practical candidate for phytoremediation technique.

  15. Transcriptomic and genomic evidence for Streptococcus agalactiae adaptation to the bovine environment

    Science.gov (United States)

    2013-01-01

    Background Streptococcus agalactiae is a major cause of bovine mastitis, which is the dominant health disorder affecting milk production within the dairy industry and is responsible for substantial financial losses to the industry worldwide. However, there is considerable evidence for host adaptation (ecotypes) within S. agalactiae, with both bovine and human sourced isolates showing a high degree of distinctiveness, suggesting differing ability to cause mastitis. Here, we (i) generate RNAseq data from three S. agalactiae isolates (two putative bovine adapted and one human) and (ii) compare publicly available whole genome shotgun sequence data from an additional 202 isolates, obtained from six host species, to elucidate possible genetic factors/adaptations likely important for S. agalactiae growth and survival in the bovine mammary gland. Results Tests for differential expression showed distinct expression profiles for the three isolates when grown in bovine milk. A key finding for the two putatively bovine adapted isolates was the up regulation of a lactose metabolism operon (Lac.2) that was strongly correlated with the bovine environment (all 36 bovine sourced isolates on GenBank possessed the operon, in contrast to only 8/151 human sourced isolates). Multi locus sequence typing of all genome sequences and phylogenetic analysis using conserved operon genes from 44 S. agalactiae isolates and 16 additional Streptococcus species provided strong evidence for acquisition of the operon via multiple lateral gene transfer events, with all Streptococcus species known to be major causes of mastitis, identified as possible donors. Furthermore, lactose fermentation tests were only positive for isolates possessing Lac.2. Combined, these findings suggest that lactose metabolism is likely an important adaptation to the bovine environment. Additional up regulation in the bovine adapted isolates included genes involved in copper homeostasis, metabolism of purine, pyrimidine

  16. Infectious bovine keratoconjunctivitis: a review.

    Science.gov (United States)

    Brown, M H; Brightman, A H; Fenwick, B W; Rider, M A

    1998-01-01

    The economic impact of infectious bovine keratoconjunctivitis (IBK) warrants continued investigation of the mechanisms by which Moraxella bovis survives on and colonizes the corneal surface. Virulent strains of M bovis produce hemolysin and exhibit different plasmid profiles than nonvirulent strains. Interactions among host, environment, vector, season, and concurrent infection influence the prevalence of IBK. Mycoplasma sp. or infectious bovine rhinotracheitis virus may enhance or hasten the disease process. The manifestations of IBK may range from mild conjunctivitis to severe ulceration, corneal perforation, and blindness. Treatment of IBK is dictated by economic considerations, intended animal use, and feasibility of administration. Antibiotic therapy is aimed at achieving drug concentrations in tears to meet or exceed the minimum inhibitory concentration for prolonged periods. At present, IBK is not a preventable disease. Affected animals must be separated from the herd and vector control vigorously instituted. Carrier animals must be identified and removed from the herd. Vaccination trials have been unsuccessful because of pili antigen cross-reactivity, variable strains, and uncontrolled environmental factors. Recent investigations have determined that M bovis may utilize host iron sources via iron-repressible outer membrane proteins and siderophores for growth. Elucidation of normal defense mechanisms of the bovine eye may lead to new strategies to enhance the immune response against M bovis.

  17. Accumulation and transfer of 137Cs and 90Sr in the plants of the forest ecosystem near the Ignalina Nuclear Power Plant.

    Science.gov (United States)

    Lukšienė, B; Marčiulionienė, D; Gudelienė, I; Schönhofer, F

    2013-02-01

    The radioecological state of the forest ecosystem in the vicinity of the Ignalina Power Plant prior to decommissioning was analysed with specific emphasis on (137)Cs and (90)Sr activity concentrations in plant species growing in two reference sampling sites (Tilze and Grikiniskes). In the period of 1996-2008 the mean contamination of plants with (137)Cs was from 45 to 119 Bq/kg and with (90)Sr - from 3 to 42 Bq/kg. Measured (137)Cs TF values for soil-root transfer mainly ranged between 1.0-1.4, except for Calamagrostis arundinacea which had a TF value of 0.1. On average, the (137)Cs TF value from root to shoot was 1.7 fold higher than for soil to root transfer. (90)Sr TF values (soil-root) were in the range of 1.2-1.8 but for Calluna vulgaris it was 0.2. The mean root to shoot TF value for (90)Sr was 7.7 fold higher. These results indicate the higher (90)Sr bioavailability than that of (137)Cs in the forested area. The Grikiniskes reference site is located nearby the Ignalina NPP, specifically the heated water outlet channel, which results in altered microclimatic conditions. These specific microclimatic conditions result in relationships between (137)Cs TF (soil-root) values and pH, moisture and organic matter content in the soil at Grikiniskes which appear to be different to those at the Tilze reference sampling site.

  18. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Energy Technology Data Exchange (ETDEWEB)

    Souza, D.K. de [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Faculdade da Ceilândia, Universidade de Brasília, Brasília, DF (Brazil); Salles, L.P. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Departamento de Biologia Molecular, Instituto de Biologia, Universidade de Brasília, Brasília, DF (Brazil); Rosa e Silva, A.A.M. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil)

    2015-01-23

    Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  19. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Directory of Open Access Journals (Sweden)

    D.K. de Souza

    2015-03-01

    Full Text Available Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  20. Bovine Chymosin: A Computational Study of Recognition and Binding of Bovine κ-Casein

    DEFF Research Database (Denmark)

    Palmer, David S.; Christensen, Anders Uhrenholt; Sørensen, Jesper

    2010-01-01

    Bovine chymosin is an aspartic protease that selectively cleaves the milk protein κ-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine κ-casein complexed with bovine chymosin, using ligand docking, conformational...

  1. Charge transfer in Li/CFx-silver vanadium oxide hybrid cathode batteries revealed by solid state 7Li and 19F nuclear magnetic resonance spectroscopy

    Science.gov (United States)

    Sideris, Paul J.; Yew, Rowena; Nieves, Ian; Chen, Kaimin; Jain, Gaurav; Schmidt, Craig L.; Greenbaum, Steve G.

    2014-05-01

    Solid state 7Li and 19F magic angle spinning nuclear magnetic resonance (MAS NMR) experiments are conducted on several cathodes containing CFx-Silver vanadium oxide (CFx-Ag2V4O11) hybrid cathodes discharged to 50% depth of discharge (DoD) and stored at their open-circuit voltage for a period of one and three months. Three carbonaceous sources for the CFx phase are investigated: petroleum coke-based, fibrous, and mixed fibrous. For each hybrid cathode, a measurable increase in the relative amount of lithium fluoride is observed after a three month resting period in both the 7Li and 19F NMR spectra. These changes are attributed to lithium ion migration from the silver vanadium oxide to the CFx phase during the resting period, and help clarify the mechanism behind high power handling capability of this cathode.

  2. Nuclear moments

    CERN Document Server

    Kopferman, H; Massey, H S W

    1958-01-01

    Nuclear Moments focuses on the processes, methodologies, reactions, and transformations of molecules and atoms, including magnetic resonance and nuclear moments. The book first offers information on nuclear moments in free atoms and molecules, including theoretical foundations of hyperfine structure, isotope shift, spectra of diatomic molecules, and vector model of molecules. The manuscript then takes a look at nuclear moments in liquids and crystals. Discussions focus on nuclear paramagnetic and magnetic resonance and nuclear quadrupole resonance. The text discusses nuclear moments and nucl

  3. Nuclear safeguards; Salvaguardias nucleares

    Energy Technology Data Exchange (ETDEWEB)

    Zurron, O.

    2015-07-01

    Safeguards control at the Juzbado Plant is implemented through the joint IAEA/EURATOM partnership approach in force within the European Union for all nuclear facilities. this verification agreement is designed to minimize burden on the operators whilst ensuring that both inspectorate achieve the objectives related to their respective safeguards regimes. This paper outlines the safeguards approaches followed by the inspectorate and the particularities of the Juzbado Plants nuclear material accountancy and control system. (Authors)

  4. Influence of heat transfer on walls due to aerosol decomposition rate in the containment building of nuclear power plants during heavy incidents; Einfluss des Waermeuebergangs an Waenden auf die Aerosolabbaurate im Sicherheitsbehaelter von Kernkraftwerken bei schweren Stoerfaellen

    Energy Technology Data Exchange (ETDEWEB)

    Braun, T.

    2004-07-01

    Today, German nuclear power plants are leading in safety standards worldwide. Increasing potentials arise continuously along with improvements in technology. One of these potentials is the best-estimate simulation of fission product transport in case of a severe accident. A main part of the fission products is allocated on aerosols. Therefore, the aerosol behavior before containment leakage is important for the radioactive source term to the environment. Having a good knowledge about the main aerosol phenomena, it is possible to simulate them numerically. This enables to develop and test safety measures to limit damages before accidents occur. Within this study, the main aerosol phenomena have been ascertained and accordingly classified into formation, transport and reduction. On this basis, simulations of one- and multi-component aerosol experiments of the KAEVER series have been performed with the COCOSYS code. Due to an overprediction of the computed volume condensation rate, the results showed an overestimation of the reduction rate of insoluble aerosols. The reason was found to be the underestimation of the wall condensation rate. Based on an additional plain thermal hydraulic multi compartment experiment, these uncertainties in the wall heat transfer correlations were investigated in detail. The results show a strong dependency between the wall condensation rate and the convective heat transfer, resp. the characteristic length. In case of mainly forced convection, correct values for the characteristic length led to an underestimation of the calculated heat transfer coefficients. The analysis of the heat transfer models show an inconsistency in the coupling of free and forced convection. Therefore, an improved and consistent convection model has been developed and implemented. Both models have been tested on different experiments. Although the new model shows only minor improvements, it could be proven that the influence for forced convection is significant

  5. Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes.

    Science.gov (United States)

    Moulavi, F; Hosseini, S M; Tanhaie-Vash, N; Ostadhosseini, S; Hosseini, S H; Hajinasrollah, M; Asghari, M H; Gourabi, H; Shahverdi, A; Vosough, A D; Nasr-Esfahani, M H

    2017-03-01

    Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at -20 °C and in vitro matured domestic cat oocytes. No cells growth was detected in primary culture of skin and tendon pieces or following culture of singled cells prepared by enzymatic digestion. Furthermore, no live cells were detected following differential viable staining and almost all cells had ruptured membrane. Therefore, direct injection of donor nuclei into enucleated cat oocytes matured in vitro was carried out for SCNT experiments. Early signs of nuclear remodeling were observed as early as 2 h post-iSCNT and significantly increased at 4 h post-iSCNT. The percentages of iSCNT reconstructs that cleaved and developed to 4-16 cell and morula stages were 32.3 ± 7.3, 18.2 ± 9.8 and 5.9 ± 4.3%, respectively. However, none of the iSCNT reconstructs developed to the blastocyst stage. When domestic cat somatic and oocytes were used for control SCNT and parthenogenetic activation, the respective percentages of oocytes that cleaved (51.3 ± 13.9 and 77.3 ± 4.0%) and further developed to the blastocyst stage (11.3 ± 3.3 and 16.8 ± 3.8%) were comparable. In summary, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate non-viable nuclei of Asiatic cheetah and support its reprogramming back to the embryonic stage. To our knowledge, this is

  6. 体细胞克隆技术在企业肉羊繁殖生产中的初步应用%Application of Somatic Cell Nuclear Transfer Techniques in Sheep Farm

    Institute of Scientific and Technical Information of China (English)

    孟蝶; 白春玲; 银凤霞; 苏广华; 魏著英; 程磊; 李俊龙; 李光鹏

    2013-01-01

    Although the efficiency of somatic call nuclear transfer (SCNT),especially in farm animals is still very low,the potential applications of this technique in excellent farm animal reproduction are valued to make trials.The present study is a farm-university-research cooperation,the farm as a main body,to produce the proven excellent Dorper rams by SCNT techniques.The results show that the barn feeding recipient sheep are more suitable for cloned embryo transfer than the pastured sheep.Estrus synchronization protocols and shipment of cloned embryos affect the recipient synchronization and pregnancies.One hundred recipient ewes got pregnancy,with 13.6% pregnant rate during the last three years.Sixty-one cloned lambs are healthily survived.Most of the cloned Dorper rams are currently used in the production of sheep with local Mongolian sheep.In conclusion,this preliminary study indicates the application of SCNT techniques in sheep farm to produce the excellent sheep individuals is highly available.%虽然体细胞克隆技术(somatic cell nuclear transfer,SCNT)的总体效率不高,但其潜在的开发价值仍值得尝试.本试验与企业密切合作,并以企业为开发主体,开展优秀杜泊种公羊的克隆生产与应用研究.结果表明,舍饲条件下的受体母羊比草原放养母羊更适合于作克隆胚胎移植受体,同期发情处理方式与胚胎运输影响克隆胚胎的妊娠效率.在过去3年间,共有100只受体羊妊娠至3个月以上,妊娠率为13.6%.目前有61只健康存活.其中一部分克隆公羊作为种羊与本地蒙古羊配种,参与企业的肉羊繁殖生产.由此可以看出,以企业为主体的利用克隆技术进行优秀种羊规模化生产是可行的.

  7. Modeling and simulation of coupled nuclear heat energy deposition and transfer in the fuel assembly of the Ghana Research Reactor-1 (GHARR-1)

    Energy Technology Data Exchange (ETDEWEB)

    Ameyaw, Felix, E-mail: fafeknoc@yahoo.co.uk [Department of Nuclear Engineering and Material Sciences, School of Nuclear and Allied Sciences (SNAS), University of Ghana, P.O. Box AE 1, Atomic Energy, Accra (Ghana); Ayensu, Akwasi; Akaho, E.H.K. [Department of Nuclear Engineering and Material Sciences, School of Nuclear and Allied Sciences (SNAS), University of Ghana, P.O. Box AE 1, Atomic Energy, Accra (Ghana)

    2011-12-15

    Highlights: Black-Right-Pointing-Pointer We model heat energy distribution without exceeding thermal limits Black-Right-Pointing-Pointer We ascertain the hottest fuel rod is within design limits. Black-Right-Pointing-Pointer Axial fuel rod heat energy increases until maximum. Black-Right-Pointing-Pointer Radial energy profile suggest the hottest region in the core. Black-Right-Pointing-Pointer We model convective heat transfer processes of the core. - Abstract: Monte Carlo N-Particle (MCNP) code coupled with PLTEMP/ANL code were used to model and simulate the heat transfer problems in the fuel elements assembly of the Ghana Research Reactor-1 (GHARR-1) by solving Boltzmann transport approximation to the heat conduction equation. Coupled neutron radiation-thermal codes were used to determine the spatial variations of thermal energy in the fuel channels, the heat energy distribution in the radial and axial segments of the fuel assembly and the convective heat transfer processes in the entire core of the reactor. The thermal energy at maximum reactivity load of 4 mk, reactor power of 30 kW and inlet system pressure of 101.3 kPa were found to be 8.896 Multiplication-Sign 10{sup -16} J for a single fuel pin, and 1.104 Multiplication-Sign 10{sup -15} J and 7.376 Multiplication-Sign 10{sup -16} J, for the radial and axial sectioning of the core respectively. Using the PLTEMP/ANL V4.0 code and given that the inlet coolant temperature was 30 Degree-Sign C, the maximum outlet coolant temperature was 51 Degree-Sign C. The energy values were obtained using the following thermodynamic parameters as maximum pressure drop of 0.7 MPa and mass flow rate of 0.4 kg/s. Neutronics point kinetics model and Safety Analysis Report used to validate the results confirmed that the heat distribution in the core did not exceed 100 Degree-Sign C. The heat energy profiles based on the data suggested no nucleate boiling at the simulated energies, and since the melting point of U-Al alloy fuel

  8. Insights on bovine genetic engineering and cloning

    Directory of Open Access Journals (Sweden)

    Fabiana F. Bressan

    2013-12-01

    Full Text Available Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

  9. Bovine in vitro embryo production : An overview

    Directory of Open Access Journals (Sweden)

    V. S. Suthar

    Full Text Available Dairy industry perfected the application of the first reproductive biotechnology, i.e. artificial insemination (AI - a great success story and also remains the user of embryo transfer technology (ETT. In addition, recently the researchers taking interest to embraced the field of Transvaginal OocyteRecovery (TVOR and in vitro production (IVEP of embryos. IVF provides the starting point for the generation of reproductive material for a number of advanced reproduction techniques including sperm microinjection and nuclear transfer (cloning. In several countries commercial IVF facilities are already being employed by cattle ET operators. Various research groups have reported on modification of TVOR technique to give greater efficiency. Much research is still needed in domestic animal (Especially Indian species on mechanisms controlling embryo development and on development of totally in vitro system for embryo culture. [Vet World 2009; 2(12.000: 478-479`

  10. Interaction between Wine Phenolic Acids and Salivary Proteins by Saturation-Transfer Difference Nuclear Magnetic Resonance Spectroscopy (STD-NMR) and Molecular Dynamics Simulations.

    Science.gov (United States)

    Ferrer-Gallego, Raúl; Hernández-Hierro, José Miguel; Brás, Natércia F; Vale, Nuno; Gomes, Paula; Mateus, Nuno; de Freitas, Victor; Heredia, Francisco J; Escribano-Bailón, María Teresa

    2017-03-10

    The interaction between phenolic compounds and salivary proteins is highly related to the astringency perception. Recently, it has been proven the existence of synergisms on the perceived astringency when phenolic acids were tested as mixtures in comparison to individual compounds, maintaining constant the total amount of the stimulus. The interactions between wine phenolic acids and the peptide fragment IB712 have been studied by saturation-transfer difference (STD) NMR spectroscopy. This technique provided the dissociation constants and the percentage of interaction between both individual and mixtures of hydroxybenzoic and hydroxycinnamic acids and the model peptide. It is noteworthy that hydroxybenzoic acids showed higher affinity for the peptide than hydroxycinnamic acids. To obtain further insights into the mechanisms of interaction, molecular dynamics simulations have been performed. Results obtained not only showed the ability of these compounds to interact with salivary proteins but also may justify the synergistic effect observed in previous sensory studies.

  11. Cell Infectivity in relation to bovine leukemia virus gp51 and p24 in bovine milk exosomes.

    Directory of Open Access Journals (Sweden)

    Tetsuya Yamada

    Full Text Available Exosomes are small membranous microvesicles (40-100 nm in diameter and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env and p24 (Gag, were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells.

  12. An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

    Directory of Open Access Journals (Sweden)

    Abdelwahed Chtarto

    Full Text Available Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS. This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

  13. Radiative heat transfer

    CERN Document Server

    Modest, Michael F

    2013-01-01

    The third edition of Radiative Heat Transfer describes the basic physics of radiation heat transfer. The book provides models, methodologies, and calculations essential in solving research problems in a variety of industries, including solar and nuclear energy, nanotechnology, biomedical, and environmental. Every chapter of Radiative Heat Transfer offers uncluttered nomenclature, numerous worked examples, and a large number of problems-many based on real world situations-making it ideal for classroom use as well as for self-study. The book's 24 chapters cover the four major areas in the field: surface properties; surface transport; properties of participating media; and transfer through participating media. Within each chapter, all analytical methods are developed in substantial detail, and a number of examples show how the developed relations may be applied to practical problems. It is an extensive solution manual for adopting instructors. Features: most complete text in the field of radiative heat transfer;...

  14. 14 November 2013 - Director of Indian Institute of Technology Indore P. Mathur with members of the Indian community working at CERN; visiting the LHC tunnel at Point 2, the ALICE experimental area and SM18 with ALICE Collaboration Spokesperson, Istituto Nazionale Fisica Nucleare P. Giubellino and Technology Department, Accelerator Beam Transfer Group Leader V. Mertens

    CERN Multimedia

    Jean-Claude Gadmer

    2013-01-01

    14 November 2013 - Director of Indian Institute of Technology Indore P. Mathur with members of the Indian community working at CERN; visiting the LHC tunnel at Point 2, the ALICE experimental area and SM18 with ALICE Collaboration Spokesperson, Istituto Nazionale Fisica Nucleare P. Giubellino and Technology Department, Accelerator Beam Transfer Group Leader V. Mertens

  15. The impact of oceanic circulation and phase transfer on the dispersion of radionuclides released from the Fukushima Dai-ichi Nuclear Power Plant

    Directory of Open Access Journals (Sweden)

    Y. Choi

    2013-07-01

    Full Text Available The mechanism behind the dispersion of radionuclides released from the Fukushima Dai-ichi Nuclear Power Plant on March 2011 is investigated using a numerical model. This model is a Lagrangian particle tracking–ocean circulation coupled model that is capable of solving the movement and migration of radionuclides between seawater, particulates, and bottom sediments. Model simulations show the radionuclides dispersing rapidly into the interior of the North Pacific once they enter a meso-scale eddy. However, some radionuclides also remain near the coast, with spatial distribution depending strongly on the oceanic circulation during the first month after the release. Major adsorption to bottom sediments occurs during this first month and many of these radionuclides remain on the sea floor once they are adsorbed. Model results suggest that weak offshore advection during the first month will increase the adsorption of radionuclides to bottom sediments and decelerate the dispersion to the open ocean. If vertical mixing is weak, however, fewer radionuclides reach the sea floor and adsorb to bottom sediments. More radionuclides will then quickly disperse to the open ocean.

  16. Nuclear matter and electron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Sick, I. [Dept. fuer Physik und Astronomie, Univ. Basel (Switzerland)

    1998-06-01

    We show that inclusive electron scattering at large momentum transfer allows a measurement of short-range properties of nuclear matter. This provides a very valuable constraint in selecting the calculations appropriate for predicting nuclear matter properties at the densities of astrophysical interest. (orig.)

  17. Nuclear ventriculography

    Science.gov (United States)

    ... ventriculography (RNV); Multiple gate acquisition scan (MUGA); Nuclear cardiology; Cardiomyopathy - nuclear ventriculography ... 56. Udelson JE, Dilsizian V, Bonow RO. Nuclear cardiology. In: Bonow RO, Mann DL, Zipes DP, Libby ...

  18. Nuclear Medicine.

    Science.gov (United States)

    Badawi, Ramsey D.

    2001-01-01

    Describes the use of nuclear medicine techniques in diagnosis and therapy. Describes instrumentation in diagnostic nuclear medicine and predicts future trends in nuclear medicine imaging technology. (Author/MM)

  19. Studies on Reaction Mechanism Between Sparfloxacin and Bovine Serum Albumin

    Institute of Scientific and Technical Information of China (English)

    GUO Ming; ZOU Jian-wei; YI Ping-gui; SHANG Zhi-cai; HU Gui-xiang; YU Qing-sen

    2004-01-01

    The binding of sparfloxacin and bovine serum albumin(BSA) in aqueous solution was studied by means of fluorescence and absorbance spectra, and the interactions influenced by Fe3+ and Cu2+ were explored. Based on the Scatchard′s site binding model and fluorescence quenching, practical formulas for a small molecule ligand attaching to a bio-macromolecule are proposed. The binding parameters were measured according to the suggested models, and the binding distance, the transfer efficiency of energy between sparfloxacin and BSA were obtained in view of the Frster theory of non-radiation energy transfer. The effect of sparfloxacin on the conformation of BSA was analyzed by means of synchronous fluorescence spectroscopy.

  20. In vitro production of bovine embryos

    DEFF Research Database (Denmark)

    Stroebech, L.; Mazzoni, Gianluca; Pedersen, Hanne Skovsgaard;

    2015-01-01

    In vitro production (IVP) of bovine embryos has become a widespread technology implemented in cattle breeding and production. The implementation of genomic selection and systems biology adds great dimensions to the impact of bovine IVP. The physical procedures included in the IVP process can still...

  1. Nuclear Theory - Nuclear Power

    Science.gov (United States)

    Svenne, J. P.; Canton, L.; Kozier, K. S.

    2008-01-01

    The results from modern nuclear theory are accurate and reliable enough to be used for practical applications, in particular for scattering that involves few-nucleon systems of importance to nuclear power. Using well-established nucleon-nucleon (NN) interactions that fit well the NN scattering data, and the AGS form of the three-body theory, we have performed precise calculations of low-energy neutron-deuteron (n+d) scattering. We show that three-nucleon force effects that have impact on the low-energy vector analyzing powers have no practical effects on the angular distribution of the n+d cross-section. There appear to be problems for this scattering in the evaluated nuclear data file (ENDF) libraries, at the incident neutron energies less than 3.2 MeV. Supporting experimental data in this energy region are rather old (>25 years), sparse and often inconsistent. Our three-body results at low energies, 50 keV to 10.0 MeV, are compared to the ENDF/B-VII.0 and JENDL (Japanese Evaluated Nuclear Data Library) -3.3 evaluated angular distributions. The impact of these results on the calculated reactivity for various critical systems involving heavy water is shown.

  2. Bovine milk antibodies for health.

    Science.gov (United States)

    Korhonen, H; Marnila, P; Gill, H S

    2000-11-01

    The immunoglobulins of bovine colostrum provide the major antimicrobial protection against microbial infections and confer a passive immunity to the newborn calf until its own immune system matures. The concentration in colostrum of specific antibodies against pathogens can be raised by immunising cows with these pathogens or their antigens. Immune milk products are preparations made of such hyperimmune colostrum or antibodies enriched from it. These preparations can be used to give effective specific protection against different enteric diseases in calves and suckling pigs. Colostral immunoglobulin supplements designed for farm animals are commercially available in many countries. Also, some immune milk products containing specific antibodies against certain pathogens have been launched on the market. A number of clinical studies are currently in progress to evaluate the efficacy of immune milks in the prevention and treatment of various human infections, including those caused by antibiotic resistant bacteria. Bovine colostrum-based immune milk products have proven effective in prophylaxis against various infectious diseases in humans. Good results have been obtained with products targeted against rotavirus, Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutans, Cryptosporidium parvum and Helicobacter pylori. Some successful attempts have been made to use immune milk in balancing gastrointestinal microbial flora. Immune milk products are promising examples of health-promoting functional foods, or nutraceuticals. This review summarises the recent progress in the development of these products and evaluates their potential as dietary supplements and in clinical nutrition.

  3. [Toxinology of bovine paraplegic syndrome].

    Science.gov (United States)

    Sevcik, C; Brito, J C; D'Suze, G; Mijares, A J; Domínguez, M G

    1993-01-01

    A clinical entity named "Bovine Paraplegic Syndrome" ("Síndrome Parapléjico de los Bovinos") has spread alarmingly, in the cattle growing areas of the central and eastern plains of Venezuela. Approximately four million cattle are bread in the area were the disease occurs. The mortality index due to the disease ranges 5 to 25% of the animals at risk, mostly cows, pregnant or lactating. The principal characteristic of the bovine paraplegic syndrome is decubitus, ventral or sternal, in animals that make vane efforts to stand when stimulated. The diagnosis is established ruling out, clinically and with laboratory findings, that the animals are suffering known diseases with similar symptoms such as paralytic rabies, botulism and blood parasites such Trypanosoma sp., Babesia sp., and Anaplasma sp.. Death occurs always, usually after few days, and to this date there is no known treatment able to save the sick cows. In