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      In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

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          Abstract

          The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals.

          Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

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          Most cited references 32

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          Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.

          Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.
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            Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos.

            Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.
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              Identification of apoptotic cells by formamide-induced dna denaturation in condensed chromatin.

              In this article we describe a novel effect of formamide on DNA of apoptotic nuclei and present a method for specific detection of apoptotic cells based on this effect. Our observations show that formamide induces DNA denaturation in apoptotic nuclei but has no such effect on DNA of non-apoptotic cells. Formamide-induced DNA denaturation combined with detection of denatured DNA with a monoclonal antibody (MAb) against single-stranded DNA made it possible to specifically identify the apoptotic cells. This procedure produced intense staining of the condensed chromatin in the apoptotic nuclei. In contrast, necrotic cells from cultures treated with sodium azide, saponin, or hyperthermia did not bind this antibody, demonstrating the specificity of the formamide-MAb assay for the apoptotic cells. However, TUNEL stained 90-100% of necrotic cells in all three models of necrosis. Because the MAb did not stain cells with single- or double-stranded DNA breaks in the absence of apoptosis, we conclude that staining of the apoptotic nuclei is not influenced by DNA breaks and is induced by specific changes in condensed chromatin, such as damage to the DNA-histone interactions. Importantly, the formamide-MAb technique identified apoptotic cells in frozen sections and in histological sections of formalin-fixed, paraffin-embedded tissues.
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                Author and article information

                Affiliations
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                Author notes
                xurg@cae.cn
                Contributors
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China
                Journal
                Front Agric Sci Eng
                FASE
                CN10-1204/S
                Frontiers of Agricultural Science and Engineering
                Higher Education Press (4 Huixin Dongjie, Chaoyang District, Beijing 100029, China )
                2095-7505
                2014
                : 1
                : 1
                : 28-36
                10.15302/J-FASE-2014012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Categories
                RESEARCH ARTICLE

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