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Production of Cloned Pigs with Targeted Attenuation of Gene Expression  [PDF]
Vilceu Bordignon, Nayla El-Beirouthi, Bernardo G. Gasperin, Marcelo S. Albornoz, Mario A. Martinez-Diaz, Carine Schneider, Denyse Laurin, David Zadworny, Luis B. Agellon
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064613
Abstract: The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45–82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.
Effects of chemical activation and season on birth efficiency of cloned pigs
YuFang Ma,Yan Li,HengXi Wei,QiuYan Li,Rui Fang,Rui Zhao,Kun Zhang,Kai Xue,YanKun Lou,YunPing Dai,LinSheng Lian,Ning Li
Science China Life Sciences , 2009, DOI: 10.1007/s11427-009-0087-3
Abstract: The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte’s molecular programming and so does not significantly improve the efficiency of producing cloned pig births.
Effects of chemical activation and season on birth efficiency of cloned pigs

MA YuFang,LI Yan,WEI HengXi,LI QiuYan,FANG Rui,ZHAO Rui,ZHANG Kun,XUE Kai,LOU YanKun,DAI YunPing,LIAN LinSheng &,LI Ning,

中国科学C辑(英文版) , 2009,
Abstract: The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte’s molecular programming and so does not significantly improve the efficiency of producing cloned pig births. Supported by National Key Basic Research and Development Program (China of Grant No. G200000161).
Cloned pigs derived from somatic cell nuclear transferembryos cultured in vitro atlow oxygen tension
Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

PAN Dengke,ZHANG Yunhai,SUN Xiuzhu,ZHANG Jian,LI Xuyang,LI Yan,GU Zhiliang,DAI Yunping,WU Changxin,LI Ning,
PAN
,Dengke,ZHANG,Yunhai,SUN,Xiuzhu,ZHANG,Jian,LI,Xuyang,LI,Yan,GU,Zhiliang,DA,Yunping,WU,Changxin,LI,Ning

科学通报(英文版) , 2006,
Abstract: 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 em- bryos 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.
Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension
Dengke Pan,Yunhai Zhang,Xiuzhu Sun,Jian Zhang,Xuyang Li,Yan Li,Zhiliang Gu,Yunping Dai,Changxin Wu,Ning Li
Chinese Science Bulletin , 2006, DOI: 10.1007/s11434-006-0839-4
Abstract: 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.
Comparison of Gene Expression and Genome-Wide DNA Methylation Profiling between Phenotypically Normal Cloned Pigs and Conventionally Bred Controls  [PDF]
Fei Gao, Yonglun Luo, Shengting Li, Jian Li, Lin Lin, Anders Lade Nielsen, Charlotte Brandt S?rensen, Gábor Vajta, Jun Wang, Xiuqing Zhang, Yutao Du, Huanming Yang, Lars Bolund
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025901
Abstract: Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.
The roots of the Eurozone sovereign debt crisis: PIGS vs non-PIGS  [PDF]
Fernandes Costa Abel L.,Mota Paulo R.
Panoeconomicus , 2011, DOI: 10.2298/pan1105631f
Abstract: The main purposes of this paper are twofold: a) to determine if there are significant differences on the determinants of public expenditures and tax revenues between the so-called PIGS and the remaining Eurozone member states; b) to uncover possible explanations for the different situations in which these countries find themselves nowadays. The paper focus on the effects of the cyclical state of the economy on those fiscal variables, and on the actual adherence to the fiscal rules imposed by the Maastricht Treaty. Based on the estimated results we conclude that the anti-cyclical reaction with respect to the unemployment rate is much stronger among non-PIGS. We also find that fiscal rules have, in general, not been followed by those two groups of countries. Moreover, PIGS, in spite of their economic frailties, have tried to emulate the fiscal behavior of their more prosperous Eurozone partners instead of executing more rigorous policies.
The Correlation between Thermal and Noxious Gas Environments, Pig Productivity and Behavioral Responses of Growing Pigs  [PDF]
Hong Lim Choi,Sang Hwa Han,Louis D. Albright,Won Kyung Chang
International Journal of Environmental Research and Public Health , 2011, DOI: 10.3390/ijerph8093514
Abstract: Correlations between environmental parameters (thermal range and noxious gas levels) and the status (productivity, physiological, and behavioral) of growing pigs were examined for the benefit of pig welfare and precision farming. The livestock experiment was conducted at a Seoul National University station in South Korea. Many variations were applied and the physiological and behavioral responses of the growing pigs were closely observed. Thermal and gas environment parameters were different during the summer and winter seasons, and the environments in the treatments were controlled in different manners. In the end, this study finds that factors such as Average Daily Gain (ADG), Adrenocorticotropic Hormone (ACTH), stress, posture, and eating habits were all affected by the controlled environmental parameters and that appropriate control of the foregoing could contribute to the improvement of precision farming and pig welfare.
Of cloned pigs and PERV
William Wells
Genome Biology , 2000, DOI: 10.1186/gb-spotlight-20000818-01
Abstract:
Entanglement and Mixed ness of Locally Cloned Non - Maximal W - State  [PDF]
Indranil Chakrabarty,Sovik Roy,Nirman Ganguly,Binayak S. Choudhury
Physics , 2008, DOI: 10.1007/s10773-009-9953-9
Abstract: In this work we describe a protocol by which two of three parties generate two bipartite entangled state among themselves without involving third party, from a non maximal W state or W - type state $|X>=\alpha|001>_{123}+\beta|010>_{123}+\gamma|100>_{123}, \alpha^{2} + \beta^{2} + \gamma^{2} = 1$ shared by three distant partners. Also we have considered the case $\beta=\gamma$, to obtain a range for $\alpha^2$, for which the local output states are separable and non local output states are inseparable. We also find out the dependence of the mixed ness of inseparable states with their amount of inseparability, for that range of $\alpha^2$.
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