2,483 results on '"TRANSPLANTATION of cell nuclei"'
Search Results
52. Sharpening the cutting edge: additional considerations for the UK debates on embryonic interventions for mitochondrial diseases.
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Haimes, Erica and Taylor, Ken
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MITOCHONDRIAL pathology , *MITOCHONDRIAL donation , *TRANSPLANTATION of cell nuclei , *BIOTECHNOLOGY , *PUBLIC health - Abstract
In October 2015 the UK enacted legislation to permit the clinical use of two cutting edge germline-altering, IVF-based embryonic techniques: pronuclear transfer and maternal spindle transfer (PNT and MST). The aim is to use these techniques to prevent the maternal transmission of serious mitochondrial diseases. Major claims have been made about the quality of the debates that preceded this legislation and the significance of those debates for UK decision-making on other biotechnologies, as well as for other countries considering similar legislation. In this article we conduct a systematic analysis of those UK debates and suggest that claims about their quality are over-stated. We identify, and analyse in detail, ten areas where greater clarity, depth and nuance would have produced sharper understandings of the contributions, limitations and wider social impacts of these mitochondrial interventions. We explore the implications of these additional considerations for (i) the protection of all parties involved, should the techniques transfer to clinical applications; (ii) the legitimacy of focussing on short-term gains for individuals over public health considerations, and (iii) the maintenance and improvement of public trust in medical biotechnologies. We conclude that a more measured evaluation of the content and quality of the UK debates is important and timely: such a critique provides a clearer understanding of the possible, but specific, contributions of these interventions, both in the UK and elsewhere; also, these additional insights can now inform the emerging processes of implementation, regulation and practice of mitochondrial interventions. [ABSTRACT FROM AUTHOR]
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- 2017
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53. Telomeres, Reproductive Aging, and Genomic Instability During Early Development.
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Keefe, David L.
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TELOMERES , *AGING , *EMBRYOLOGY , *MATERNAL age , *DNA damage , *TRANSPLANTATION of cell nuclei - Abstract
Implantation rate decreases and miscarriage rate increases with advancing maternal age. The oocyte must be the locus of reproductive aging because donation of oocytes from younger to older women abrogates the effects of aging on fecundity. Nuclear transfer experiments in a mouse model of reproductive aging show that the reproductive aging phenotype segregates with the nucleus rather than the cytoplasm. A number of factors within the nucleus have been hypothesized to mediate reproductive aging, including disruption of cohesions, reduced chiasma, aneuploidy, disrupted meiotic spindles, and DNA damage caused by chronic exposure to reactive oxygen species. We have proposed telomere attrition as a parsimonious way to explain these diverse effects of aging on oocyte function. Telomeres are repetitive sequences of DNA and associated proteins, which form a loop (t loop) at chromosome ends. Telomeres prevent the blunt end of DNA from triggering a DNA damage response. Previously, we showed that experimental telomere shortening phenocopies reproductive aging in mice. Telomere shortening causes reduced synapsis and chiasma, chromosome fusions, embryo arrest and fragmentation, and abnormal meiotic spindles. Telomere length of polar bodies predicts the fragmentation of human embryos. Telomerase, the reverse transcriptase capable of reconstituting shortened telomeres, is only minimally active in oocytes and preimplantation embryos. Intriguingly, during the first cell cycles following activation, telomeres robustly elongate via a DNA double-strand break mechanism called alternative lengthening of telomeres (ALTs). Alternative lengthening of telomere takes place even in telomerase-null mice. This mechanism of telomere elongation previously had been found only in cancer cells lacking telomerase activity. We propose that ALT elongates telomeres across generations but does so at the cost of extensive genomic instability in preimplantation embryos. [ABSTRACT FROM AUTHOR]
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- 2016
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54. The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer.
- Author
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Taweechaipaisankul, A, Jin, JX, Lee, S, Kim, GA, and Lee, BC
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TRANSPLANTATION of cell nuclei , *CANTHAXANTHIN , *MESSENGER RNA , *EMBRYO transfer , *INTRACELLULAR pathogens - Abstract
Contents The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation ( IVM) of porcine oocytes on embryonic development after parthenogenetic activation ( PA) and somatic cell nuclear transfer ( SCNT), on intracellular glutathione ( GSH) and reactive oxygen species ( ROS) levels in mature oocytes, and on gene expression in both PA- and SCNT-derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA ( p < .05), but no significant differences were observed among groups in total blastocyst cell numbers. Subsequently, oocytes were cultured in IVM medium supplemented with or without 40 μM Cx. Oocytes treated with 40 μM Cx showed significantly increased cleavage and blastocyst formation rates after SCNT compared to the control group ( p < .05). Moreover, significantly increased intracellular GSH and reduced ROS levels were observed in the Cx-treated group ( p < .05). In addition, both PA- and SCNT-derived blastocysts from the 40 μM Cx-treated group showed significantly increased mRNA expression of Bcl2 and Oct4 and decreased Caspase3 expression level ( p < .05), when compared with the control group. PA-derived blastocysts from the 40 μM Cx-treated group also exhibited significantly decreased expression of Bax ( p < .05). Our results demonstrated that treatment with 40 μM Cx during IVM improves the developmental competence of PA and SCNT embryos. Improvement of embryo development by Cx is most likely due to increased intracellular GSH synthesis, which reduces ROS levels in oocytes, and it may also positively regulate apoptosis- and development-related genes. [ABSTRACT FROM AUTHOR]
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- 2016
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55. Learning, memory and exploratory similarities in genetically identical cloned dogs.
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Chi Won Shin, Kim, Geon A., Won Jun Park, Kwan Yong Park, Jeong Min Jeon, Hyun Ju Oh, Min Jung Kim, and Byeong Chun Lee
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LEARNING in animals ,CLONING ,CLONE cells ,SOMATIC cells ,TRANSPLANTATION of cell nuclei - Abstract
Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals. [ABSTRACT FROM AUTHOR]
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- 2016
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56. Isolation of Bovine Skin-Derived Precursor Cells and Their Developmental Potential After Nuclear Transfer.
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Xiao, Jiajia, Li, Qiaoqiao, Qu, Pengxiang, Zhang, Zihan, Pan, Shaohui, Wang, Yongsheng, and Zhang, Yong
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MOLECULAR cloning , *TRANSPLANTATION of cell nuclei , *PLURIPOTENT stem cells , *LABORATORY swine , *TRANSGENIC animals - Abstract
Nuclei from less differentiated stem cells yield high cloning efficiency. However, pluripotent stem cells are rather difficult to obtain from bovines. Skin-derived precursor (SKPs) cells exhibit a certain degree of pluripotency, which has been shown to enhance the efficiency of nuclear transfer (NT) in pigs. In this study, bovine SKPs were isolated and characterized. Results showed that bovine SKPs expressed nestin, fibronectin, vimentin, pluripotency-related genes, and characteristic neural crest markers, such as NGFR, PAX3, SOX9, SNAI2, and OCT4. Bovine SKPs and fibroblasts were used as NT donor cells to examine and compare the preimplantation developmental potential of reconstructed embryos after somatic cell nuclear transfer (SCNT). Bovine SKP-cloned embryos displayed higher developmental competence in terms of blastocyst formation rate and total cell number in blastocysts compared with the bovine embryonic fibroblast-cloned embryos. This study revealed that bovine SKPs may be considered excellent candidate nuclear donors for SCNT and may provide a promising platform for transgenic cattle generation. [ABSTRACT FROM AUTHOR]
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- 2016
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57. Effect of Long-Term Exposure of Donor Nuclei to the Oocyte Cytoplasm on Production of Cloned Mice Using Serial Nuclear Transfer.
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Wakayama, Sayaka, Tanabe, Yoshiaki, Nagatomo, Hiroaki, Mizutani, Eiji, Kishigami, Satoshi, and Wakayama, Teruhiko
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ANIMAL cloning , *OVUM physiology , *CYTOPLASM , *TRANSPLANTATION of cell nuclei , *LABORATORY mice - Abstract
Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT. [ABSTRACT FROM AUTHOR]
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- 2016
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58. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.
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Olivera, Ramiro, Moro, Lucia Natalia, Jordan, Roberto, Luzzani, Carlos, Miriuka, Santiago, Radrizzani, Martin, Donadeu, F. Xavier, and Vichera, Gabriel
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FIBROBLASTS , *HORSE embryos , *STROMAL cells , *MOLECULAR cloning , *TRANSPLANTATION of cell nuclei , *IN vitro studies , *IN vivo studies ,DISEASES in adults - Abstract
The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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59. 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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da Silva, Carolina Gonzales, Frederico Martins, Carlos, Cristina Cardoso, Tereza, da Cunha, Elisa Ribeiro, Christina Bessler, Heidi, Margaret McManus, Concepta, Pivato, Ivo, and Nair Báo, Sônia
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MESENCHYMAL stem cells , *TRANSPLANTATION of cell nuclei , *FIBROBLASTS , *MICROMANIPULATORS , *BLASTOCYST , *UMBILICAL cord , *CLONING , *GENETICS - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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60. THE PERILS OF CLONING.
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Park, Alice
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GENETIC engineering ,DOLLY (Sheep) ,CLONING ,DNA ,TRANSPLANTATION of cell nuclei ,METHYLATION ,MAMMALOGICAL research - Abstract
The author reports that, a decade after the cloning of the lamb, Dolly and after the cloning of dozens of animals since, it has become clear that all are in one way or another defective. Nuclear transfer, the process by which mammalian cloning is achieved, is compared to a lottery. The most common defect in clones is called large-offspring syndrome. Most of cloning errors can be traced to a process known as DNA methylation. INSETS: Dolly's creator;How to Clone A Tasty T-Bone.
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- 2006
61. Cell fate conversion-from the viewpoint of small molecules and lineage specifiers.
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Zhao, T., Li, Y., and Deng, H.
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SMALL molecules , *ECTOPIC pregnancy , *FERTILIZATION (Biology) , *SOMATIC cells , *TRANSPLANTATION of cell nuclei - Abstract
Mammalian development was generally considered a naturally unidirectional and irreversible process. However, pioneering work of recent decades has highlighted the plasticity of mammalian cells and implied the possibilities of manipulating cell fate in vitro. Pluripotent stem cells, which hold great potential for regenerative medicine, have been shown to be reprogrammed from differentiated cells either by somatic cell nuclear transfer or by ectopic expression of pluripotency factors. Nevertheless, it remained unknown whether the reprogramming could be accomplished without pluripotency genes. Recent studies show that lineage specifiers play an important role in orchestrating the process of restoring pluripotency by replacing pluripotency-associated transcription factors. Moreover, a combination of small molecules enables the acquisition of pluripotency from somatic cells without any transgenes, offering a tractable platform to precisely dissect the induction and maintenance of cell identity. Here, we will discuss recent scientific advances regarding the cell fate conversion mediated by small molecules or lineage specifiers, especially in the chemically induced somatic cell reprogramming process, and will provide new insights into the intermediate plastic state and 'seesaw model' established by chemical approaches during reprogramming. [ABSTRACT FROM AUTHOR]
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- 2016
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62. Classical swine fever virus replicated poorly in cells from MxA transgenic pigs.
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Yicheng Zhao, Tiedong Wang, Li Yao, Bo Liu, Chunbo Teng, and Hongsheng Ouyang
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VIRAL replication , *CLASSICAL swine fever virus , *TRANSGENIC animals , *VIRUS diseases in swine , *TRANSPLANTATION of cell nuclei - Abstract
Background: In addition to their value as livestock, pigs are susceptible to classical swine fever virus (CSFV) and can serve as reservoirs for CSFV, allowing it to develop into an epizootic. CSFV, a pestivirus of the Flaviviridae family, has a single-stranded RNA genome. Recent research has indicated that the human MxA protein inhibits the life cycles of certain RNA viruses, such as members of the Bunyaviridae family, the Flaviviridae family and others. Results: To produce pigs with antiviral protection against CSFV, transgenic pigs expressing human MxA were generated by nuclear transplantation. Cells from three MxA transgenic piglets were used to investigate in vitro antiviral activity of MxA aganist CSFV, and the results of in vitro indirect immunofluorescence assays, virus titration and real-time PCR indicated that the MxA transgenic pig has an antiviral capacity against CSFV. Conclusions: Transgene with human MxA on pigs is feasible. High levels of MxA expression do inhibit CSFV in vitro at early time points post-infection at 60-96dpi. [ABSTRACT FROM AUTHOR]
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- 2016
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63. Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs.
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Park, Bola, Lee, Hanna, Lee, Yongjin, Elahi, Fazle, Lee, Joohyeong, Lee, Seung Tae, Park, Choon-Keun, Hyun, Sang-Hwan, and Lee, Eunsong
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SWINE physiology , *FORSKOLIN , *PREIMPLANTATION genetic diagnosis , *TRANSPLANTATION of cell nuclei , *MEIOTIC drive , *GAP junctions (Cell biology) ,EMBRYONIC motility - Abstract
This study was conducted to evaluate the effects of treatment with the cAMP modulators cilostamide and/or forskolin during pre-IVM culture on meiotic progression, gap junction communication, intraoocyte cAMP level and glutathione content, embryonic development after parthenogenesis, and somatic cell nuclear transfer in pigs. Cumulus–oocyte complexes were cultured for 24 hours in unsupplemented medium or media containing 20 μM cilostamide and/or 50 μM forskolin. After pre-IVM, oocytes were cultured for 41 to 44 hours in a standard IVM medium to induce oocyte maturation. When the nuclear status of oocytes was examined after pre-IVM for 24 hours, a higher (P < 0.01) proportion of oocytes treated with forskolin (85.5%) and cilostamide + forskolin (92.6%) remained at the germinal vesicle stage compared with untreated (20.6%) and cilostamide-treated oocytes (54.7%). cAMP level in pre-IVM oocytes was significantly increased by combined treatment with cilostamide + forskolin (21.38 fmol/oocyte) relative to the no pre-IVM control, no treatment, cilostamide, and forskolin groups (2.85, 1.88, 1.74, and 8.95 fmol/oocyte, respectively). Forskolin with or without cilostamide significantly maintained open-gap junction communication relative to no treatment. Blastocyst formation in parthenogenesis was significantly (P < 0.01) improved by forskolin (65.3%) relative to other treatments (28.3% to 48.1%). Supplementation of pre-IVM with dibutyryl cAMP showed similar blastocyst formation as forskolin treatment (61.1% and 61.0%, respectively). In somatic cell nuclear transfer, simultaneous treatment with cilostamide + forskolin significantly (P < 0.05) increased embryonic development to the blastocyst stage (42.9%) relative to the no pre-IVM, control, and cilostamide groups (32.3, 28.6, and 32.8%, respectively). The glutathione contents in pre-IVM oocytes were increased by no treatment, forskolin, and cilostamide + forskolin (1.38, 1.39, and 1.27 pixels/oocyte, respectively) compared with no pre-IVM and cilostamide (1.00 and 0.99 pixels/oocyte, respectively; P < 0.05). Our results reported that the meiotic progression of immature pig oocytes could be reversibly attenuated by cAMP, whereas treatment with cilostamide and forskolin during pre-IVM had positive effects on developmental competence of oocytes in pigs, probably by improving cytoplasmic maturation. [ABSTRACT FROM AUTHOR]
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- 2016
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64. Efficient TALEN-mediated myostatin gene editing in goats.
- Author
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Baoli Yu, Rui Lu, Yuguo Yuan, Ting Zhang, Shaozheng Song, Zhengqiang Qi, Bin Shao, Mengmin Zhu, Fei Mi, and Yong Cheng
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MYOSTATIN genetics , *GENOME editing , *GOAT genetics , *SKELETAL muscle , *TRANSPLANTATION of cell nuclei - Abstract
Background: Myostatin (MSTN) encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. In this study, we aimed to test whether targeted MSTN editing, mediated by transcription activator-like effector nucleases (TALENs), is a viable approach to create myostatin-modified goats (Capra hircus). Results: We obtained a pair of TALENs (MTAL-2) that could recognize and cut the targeted MSTN site in the goat genome. Fibroblasts from pedigreed goats were co-transfected with MTAL-2, and 272 monoclonal cell strains were confirmed to have mono- or bi-allelic mutations in MSTN. Ten cell strains with different genotypes were used as donor cells for somatic cell nuclear transfer, which produced three cloned kids (K179/MSTN-/-, K52-2/MSTN+/-, and K52-1/MSTN+/+). Conclusions: The results suggested that the MTAL-2 could disrupt MSTN efficiently in the goat genome. The mutated somatic cells could be used to produce MSTN-site mutated goats without developmental disruption. Thus, TALENs is an effective method for accurate genome editing to produce site-modified goats. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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65. Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos.
- Author
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Sepulveda-Rincon, Lessly P., Solanas, Edgar del Llano, Serrano-Revuelta, Elisa, Ruddick, Lydia, Maalouf, Walid E., and Beaujean, Nathalie
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CATTLE embryology , *TRANSPLANTATION of cell nuclei , *COMPARATIVE studies , *CATTLE fertility , *CATTLE embryos - Abstract
Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased—thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization—and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation—the parthenote—and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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66. Production of Water Buffalo Clone Embryos Using Ear Skin Fibroblasts as Donor Cell in Nuclear Transfer.
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Atabay, Edwin C. and Atabay, Eufrocina P.
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FIBROBLASTS , *CLONING , *WATER buffalo , *TRANSPLANTATION of cell nuclei , *EMBRYOS , *BLASTOCYST - Abstract
The present study investigated the possibility of producing water buffalo clone embryos using ear skin fibroblasts from ear skin tissues tested for viability postcollection and cryopreservation. Water buffalo ear skin samples were collected and maintained in 4ºC at different time points: 12, 24, 48, 72, 96, 120 and 168 hr post-collection. After storage at specified time, tissue samples were processed for primary culture. Fibroblasts obtained from ear skin tissues were cryopreserved using different freezing procedures and were then used as donor nuclei for nuclear transfer. The maximum time lapse when ear skin fibroblast proliferation did not decrease significantly was at 120-hr post-collection (hpc). The post-thaw viability of the fibroblasts cryopreserved with the alternative rapid freezing procedures was comparable with that of the control group. The fibroblasts survived the alternative cryopreservation procedure and reached cell confluency at sub-culture. When used further for nuclear transfer, no significant differences were observed from the control group in terms of rates of cleavage and development to blastocyst, and cell numbers of the blastocyst formed. The present findings indicate that ear skin fibroblasts derived from tissues stored up to 120 hpc can be used as donor nuclei without compromising the developmental competence of the reconstructed embryos. [ABSTRACT FROM AUTHOR]
- Published
- 2016
67. Tumorigenesis of nuclear transfer-derived embryonic stem cells is reduced through differentiation and enrichment following transplantation in the infarcted rat heart.
- Author
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QIANG FU, DECHUN SU, KE WANG, and YINGJUN ZHAO
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EMBRYONIC stem cells , *LABORATORY rats , *TRANSPLANTATION of cell nuclei , *NEOPLASTIC cell transformation , *CELL differentiation , *HEART transplantation , *MAMMALS - Abstract
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 (P<0.05) between the nt-mESC-CMs and nt-mESCs. The mean tumor volumes were 82.72±6.52, 83.17±3.58 and 50.40±5.98 mm³, respectively (P>0.05 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. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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68. Nuclear transfer nTreg model reveals fate-determining TCR-β and novel peripheral nTreg precursors.
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Manching Ku, Shih-En Chang, Hernandez, Julio, Abadejos, Justin R., Sabouri-Ghomi, Mohsen, Muenchmeier, Niklas J., Schwarz, Anna, Valencia, Anna M., and Kirak, Oktay
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SOMATIC cell nuclear transfer , *TRANSPLANTATION of cell nuclei , *T cells , *SCURFIN (Protein) , *SOMATIC cells - Abstract
To study the development and function of "natural-arising" T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3+ CD4+ Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) β-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR β-chain was able to provide stronger TCR signals. This TCR-β-driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3- CD4+ T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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69. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming.
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Bai, Lin, Li, Mengqi, Sun, Junli, Yang, Xiaogan, Lu, Yangqing, Lu, Shengsheng, and Lu, Kehuan
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RNA sequencing , *OVUM physiology , *SOMATIC cell nuclear transfer , *EMBRYOLOGY , *BLASTOCYST , *TRANSPLANTATION of cell nuclei - Abstract
The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
70. Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.
- Author
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Hara, Hiromasa, Goto, Teppei, Takizawa, Akiko, Sanbo, Makoto, Jacob, Howard J., Kobayashi, Toshihiro, Nakauchi, Hiromitsu, Hochi, Shinichi, and Hirabayashi, Masumi
- Subjects
- *
BLASTOCYST , *CELL enucleation , *CYCLOHEXIMIDE , *EMBRYONIC stem cells , *TRANSPLANTATION of cell nuclei , *CELL culture - Abstract
Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B- tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes ( n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
71. Production of Bovine Embryos and Calves Cloned by Nuclear Transfer Using Mesenchymal Stem Cells from Amniotic Fluid and Adipose Tissue.
- Author
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da Silva, Carolina Gonzales, Martins, Carlos Frederico, Cardoso, Tereza Cristina, da Cunha, Elisa Ribeiro, Bessler, Heidi Christina, Martins, George Henrique Lima, Pivato, Ivo, and Báo, Sônia Nair
- Subjects
- *
TRANSPLANTATION of cell nuclei , *CATTLE embryology , *MESENCHYMAL stem cells , *AMNIOTIC liquid , *ADIPOSE tissues - Abstract
The less differentiated the donor cells are used in nuclear transfer (NT), the more easily are they reprogrammed by the recipient cytoplasm. In this context, mesenchymal stem cells (MSCs) appear as an alternative to donor nuclei for NT. The amniotic fluid and adipose tissue are sources of MSCs that have not been tested for the production of cloned embryos in cattle. The objective of this study was to isolate, characterize, and use MSCs derived from amniotic fluid (MSC-AF) and adipose tissue (MSC-AT) to produce cloned calves. Isolation of MSC-AF was performed using in vivo ultrasound-guided transvaginal amniocentesis, and MSC-AT were isolated by explant culture. Cellular phenotypic and genotypic characterization by flow cytometry, immunohistochemistry, and RT-PCR were performed, as well as induction in different cell lineages. The NT was performed using MSC-AF and MSC-AT as nuclear donors. The mesenchymal markers of MSC were expressed in bovine MSC-AF and MSC-AT cultures, as evidenced by flow cytometry, immunohistochemistry, and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes, and adipocytes. Embryo production was similar between the cell types, and two calves were born. The calf from MSC-AT was born healthy, and this fact opens a new possibility of using this type of cell to produce cloned cattle by NT. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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72. Role of linker histone H1c during the reprogramming of Chinese swamp buffalo (Bubalus Bubalis) embryos produced by somatic cell nuclear transfer.
- Author
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Huang, Gao-Bo, Quan, Li, Zeng, Yong-Lian, Yang, Jian, Lu, Ke-Huan, and Lu, Sheng-Sheng
- Subjects
- *
WATER buffalo , *HISTONE genetics , *SOMATIC cell nuclear transfer , *TRANSPLANTATION of cell nuclei , *ANIMAL genetics research , *ANIMAL reproduction - Abstract
During reprogramming, there is exchange of histone H1c and the oocyte-specific linker histone, and H1c may play a critically important role in the reprogramming process of somatic cell nuclear transfer (SCNT). The aim of the present study was to investigate the role of the H1c gene in SCNT reprogramming in Chinese swamp buffalo (Bubalus bubalis) using RNA interference (RNAi). Chinese swamp buffalo H1c gene sequences were obtained and H1c-RNAi vectors were designed, synthesised and then transfected into a buffalo fetal skin fibroblast cell line. Expression of H1c was determined by real-time polymerase chain reaction to examine the efficiency of vector interference. These cells were then used as a nuclear donor for SCNT so as to observe the further development of SCNT embryos. Inhibition of H1c gene expression in donor cells significantly improved the developmental speed of embryos from the 1-cell to 8-cell stage. Furthermore, compared with the control group, inhibition of H1c gene expression significantly reduced the blastocyst formation rate. It is concluded that linker histone H1c is very important in SCNT reprogramming in Chinese swamp buffalo. Correct expression of the H1c gene plays a significant role in preimplantation embryonic development in B. bubalis. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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73. Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer.
- Author
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Kyung-Mee PARK, Kamal Hany HUSSEIN, Heung-Myong WOO, Seok-Ho HONG, Se-Ran YANG, Joohyeong LEE, and Eunsong LEE
- Subjects
PLURIPOTENT stem cells ,TRANSPLANTATION of cell nuclei ,TRANSGENIC animals - Abstract
Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for the development of genetically engineered pig models, because they can be expanded without senescence and have the potential for multiple gene manipulation. They are also useful cell sources for disease modeling and treatment. However, the generation of transgenic porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has not yet been reported. We report here the generation of liver-specific oncogenes (TGF-a/c- Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They expressed stem cell characteristics and were differentiated into hepatocyte-like cells that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells successfully developed blastocysts in vitro. As an initial approach toward porcine transgenic iPS cell generation and their developmental competence after NT, this study provides foundations for the efficient generation of genetically modified porcine iPS cells and animal models. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
74. The Complete Genome Sequences, Unique Mutational Spectra, and Developmental Potency of Adult Neurons Revealed by Cloning.
- Author
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Hazen, Jennifer L., Faust, Gregory G., Rodriguez, Alberto R., Ferguson, William C., Shumilina, Svetlana, Clark, Royden A., Boland, Michael J., Martin, Greg, Chubukov, Pavel, Tsunemoto, Rachel K., Torkamani, Ali, Kupriyanov, Sergey, Hall, Ira M., and Baldwin, Kristin K.
- Subjects
- *
NEURON development , *SOMATIC mutation , *NUCLEOTIDE sequencing , *CLONE cells , *TRANSPLANTATION of cell nuclei , *LABORATORY mice - Abstract
Summary Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor ∼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0–2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
75. Nuclear Transfer using Human CD59 and IL-18BP Double Transgenic Fetal Fibroblasts in Miniature Pigs.
- Author
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Junghyun Ryu, Minjeong Kim, Jin Seop Ahn, Kwang Sung Ahn, and Hosup Shim
- Subjects
- *
TRANSPLANTATION of cell nuclei , *CD59 antigen , *INTERLEUKIN-18 , *TRANSGENIC animals , *FIBROBLASTS , *MINIATURE pigs - Abstract
Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of IFN-γ. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
76. Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies.
- Author
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Brouwer, Marinka, Zhou, Huiqing, and Nadif Kasri, Nael
- Subjects
- *
INDUCED pluripotent stem cells , *SOMATIC cells , *REGENERATIVE medicine , *TRANSPLANTATION of cell nuclei , *NEOPLASTIC cell transformation , *NEUROSCIENCES - Abstract
The ability to generate human induced pluripotent stem cells (iPSCs) from somatic cells provides tremendous promises for regenerative medicine and its use has widely increased over recent years. However, reprogramming efficiencies remain low and chromosomal instability and tumorigenic potential are concerns in the use of iPSCs, especially in clinical settings. Therefore, reprogramming methods have been under development to generate safer iPSCs with higher efficiency and better quality. Developments have mainly focused on the somatic cell source, the cocktail of reprogramming factors, the delivery method used to introduce reprogramming factors and culture conditions to maintain the generated iPSCs. This review discusses the developments on these topics and briefly discusses pros and cons of iPSCs in comparison with human embryonic stem cells generated from somatic cell nuclear transfer. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
77. Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally.
- Author
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Wasson, Jadiel A., Simon, Ashley K., Myrick, Dexter A., Wolf, Gernot, Driscoll, Shawn, Pfaff, Samuel L., Macfarlan, Todd S., and Katz, David J.
- Subjects
- *
DNA methylation , *METHYLCYTOSINE , *SOMATIC cells , *TRANSPLANTATION of cell nuclei , *EMBRYOLOGY - Abstract
Somatic cell nuclear transfer has established that the oocyte contains maternal factors with epigenetic reprogramming capacity. Yet the identity and function of these maternal factors during the gamete to embryo transition remains poorly understood. In C. elegans, LSD1/KDM1A enables this transition by removing H3K4me2 and preventing the transgenerational inheritance of transcription patterns. Here we show that loss of maternal LSD1/KDM1A in mice results in embryonic arrest at the 1-2 cell stage, with arrested embryos failing to undergo the maternal-to-zygotic transition. This suggests that LSD1/KDM1A maternal reprogramming is conserved. Moreover, partial loss of maternal LSD1/KDM1A results in striking phenotypes weeks after fertilization; including perinatal lethality and abnormal behavior in surviving adults. These maternal effect hypomorphic phenotypes are associated with alterations in DNA methylation and expression at imprinted genes. These results establish a novel mammalian paradigm where defects in early epigenetic reprogramming can lead to defects that manifest later in development. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
78. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.
- Author
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Hoin KANG, Jong Im PARK, and Sangho ROH
- Subjects
SOMATIC cell nuclear transfer ,TRANSPLANTATION of cell nuclei ,GENES - Abstract
In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
79. Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig.
- Author
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Song, Yuning, Lai, Liangxue, Li, Li, Huang, Yongye, Wang, Anfeng, Tang, Xiaochun, Pang, Daxin, Li, Zhanjun, and Ouyang, Hongsheng
- Subjects
GERM cells ,GENE expression ,RECOMBINASES ,PROMOTERS (Genetics) ,TRANSPLANTATION of cell nuclei ,IMMUNOHISTOCHEMISTRY ,LABORATORY swine - Abstract
The Cre- loxP system is a powerful tool for genetic analysis of distinct cell lineages and tissue-specific gene knockout in animal models. VASA is specifically expressed in reproductive tissues, and is known to play important roles in spermatogenesis and germ-cell growth. In this study, Cre recombinase transgenic pigs under the control of the VASA promoter were generated by somatic cell nuclear transfer. Germ cell-specific expression of Cre recombinase in VASA-Cre transgenic pigs was shown by western blotting and immunohistochemistry. VASA-Cre transgenic pigs will be a useful tool for germ cell-specific gene knockout and a disease model for disorders of the reproductive system. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
80. Genome-Wide Dynamic Profiling of Histone Methylation during Nuclear Transfer-Mediated Porcine Somatic Cell Reprogramming.
- Author
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Cao, Zubing, Li, Yunsheng, Chen, Zhen, Wang, Heng, Zhang, Meiling, Zhou, Naru, Wu, Ronghua, Ling, Yinghui, Fang, Fugui, Li, Ning, and Zhang, Yunhai
- Subjects
- *
HISTONE methylation , *TRANSPLANTATION of cell nuclei , *SOMATIC cells , *EPIGENETICS , *GENETIC programming - Abstract
The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
81. Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.
- Author
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Akira Tsujii, Yoichi Miyamoto, Tetsuji Moriyama, Yuko Tsuchiya, Chikashi Obuse, Kenji Mizuguchi, Masahiro Oka, and Yoshihiro Yoneda
- Subjects
- *
RETINOBLASTOMA protein , *TRANSPLANTATION of cell nuclei , *CELLULAR aging , *CYTOPLASM , *EUKARYOTIC cells - Abstract
Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
82. SCIENCE.
- Author
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PRENTICE, DAVID A.
- Subjects
- *
CELL differentiation , *PLURIPOTENT stem cells , *REGENERATIVE medicine , *SMALL molecules , *GENETIC transformation , *TRANSPLANTATION of cell nuclei , *GENETIC engineering - Abstract
The article examines the efficacy of using the direct cell conversion method than creating induced pluripotent stem cells to derive targeted differentiated cells for the laboratory or regenerative medicine. The author finds that the method can be solely used to achieve complete nuclear genetic reprogramming. Other topics discussed include the initiation of cell conversion using small molecules or through gene transfer and the chemical induction of direct cell conversion.
- Published
- 2015
83. Somatic cell nuclear transfer: origins, the present position and future opportunities.
- Author
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Wilmut, Ian, Yu Bai, and Taylor, Jane
- Subjects
- *
TRANSPLANTATION of cell nuclei , *SOMATIC cells , *CELL transplantation , *HUMAN cell cycle , *GENETIC transcription , *PLURIPOTENT stem cells , *TRANSPLANTATION of organs, tissues, etc. - Abstract
Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce 'induced pluripotent stem cells' is providing revolutionary new opportunities in research and cell therapy. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
84. From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?
- Author
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Yamada, Mitsutoshi, Byrne, James, and Egli, Dieter
- Subjects
- *
STEM cells , *PLURIPOTENT stem cells , *SOMATIC cells , *TRANSPLANTATION of cell nuclei , *OVUM , *CYTOLOGY - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
85. Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.
- Author
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Moro, LN, Jarazo, J, Buemo, C, Hiriart, MI, Sestelo, A, and Salamone, DF
- Subjects
- *
CAT reproduction , *EMBRYOS , *TRANSPLANTATION of cell nuclei , *BLASTOCYST , *MOLECULAR cloning - Abstract
Contents The aim of this study was to evaluate three different cloning strategies in the domestic cat ( Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning ( iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger ( Panthera tigris) donor cells. In experiment 1, zona-free ( ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
86. Establishment, Differentiation, Electroporation and Nuclear Transfer of Porcine Mesenchymal Stem Cells.
- Author
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Song, Z, Cong, P, Ji, Q, Chen, L, Nie, Y, Zhao, H, He, Z, and Chen, Y
- Subjects
- *
TRANSPLANTATION of cell nuclei , *CELL differentiation , *MESENCHYMAL stem cells , *SWINE genetics , *SOMATIC cells , *EPIGENETICS - Abstract
Contents The limited success of somatic cell nuclear transfer ( SCNT) is largely attributed to defects in epigenetic reprogramming of the donor genome. Donor cell types with distinct potential competence may offer different epigenetic flexibility for subsequent genome reprogramming in SCNT. Stem cells possibly enable their genomes to be more readily reprogrammed than differentiated cells. To improve the efficiency of cloning, porcine mesenchymal stem cells ( pMSCs) were isolated and well identified by 6-channel flow cytometry and differentiation assays and were used as donors in SCNT. Compared with porcine embryonic fibroblasts ( pEFs), our results showed that pMSCs markedly enhanced cloned embryo development in terms of cleavage and blastocyst formation (p < 0.05). To enhance the epigenetic flexibility of pMSCs, classical reprogramming factors ( RFs) were transfected by electroporation, and we achieved optimization with ectopic expression of RFs in pMSCs. Our results suggest that the epigenetic status of donor cells has an improvement on genome reprogramming, and multipotent pMSCs favoured subsequent embryonic development. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
87. Breakup mechanisms for 7Li + 197Au, 204Pb systems at sub-barrier energies.
- Author
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Luong, D. H., Dasgupta, M., Hinde, D. J., du Rietz, R., Rafiei, R., Evers, M., Lin, C. J., Wakhle, A., Ramachandran, K., Carter, I. P., and Diaz-Torres, A.
- Subjects
- *
TRANSPLANTATION of cell nuclei , *BINARY control systems , *FUSION (Phase transformation) , *INTERCHANGEABLE mechanisms , *AUTOMATIC control systems - Abstract
Coincidence measurements of breakup fragments were carried out for the 7Li + 197Au and 204Pb systems at sub-barrier energies. The mechanisms triggering breakup, and time-scales of each process, were identified through the reaction Q-values and the relative energy of the breakup fragments. Binary breakup of 7Li were found to be predominantly triggered by nucleon transfer, with p-pickup leading to 8Be → α + α decay being the preferred breakup mode. From the time-scales of each process, the coincidence yields were separated into prompt and delayed components, allowing the identification of breakup process important in the suppression of complete fusion of 7Li at above-barrier energies. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
88. Mitochondrial replacement therapy: born in the USA: the untold story of a conceptual breakthrough.
- Author
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Adashi, Eli Y. and Cohen, I. Glenn
- Subjects
MITOCHONDRIAL pathology ,MITOCHONDRIAL DNA abnormalities ,TRANSPLANTATION of cell nuclei ,MELAS syndrome ,CELL fusion ,THERAPEUTICS - Published
- 2017
- Full Text
- View/download PDF
89. '3-parent baby' success.
- Author
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Hamzelou, Jessica
- Subjects
- *
DNA , *INFANT boys , *CHILDBIRTH , *TRANSPLANTATION of cell nuclei - Abstract
The article discusses the birth of a baby boy using a technique that incorporates DNA from three people. Topics include a description of the technique and the mother's genes with Leigh syndrome, an explanation of the pronuclear transfer as well as the spindle nuclear transfer, and safety for the baby as a concern. Also mentioned are John Zhang of New Hope Fertility Center and the American Society for Reproductive Medicine's Scientific Congress.
- Published
- 2016
- Full Text
- View/download PDF
90. Lymphoid lineage differentiation potential of mouse nuclear transfer embryonic stem cells.
- Author
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Eslami-Arshaghi, Tarlan, Salehi, Mohammad, Soleimani, Masoud, Gholipourmalekabadi, Mazaher, Mossahebi-Mohammadi, Majid, Ardeshirylajimi, Abdolreza, and Rajabi, Hoda
- Subjects
- *
TRANSPLANTATION of cell nuclei , *LYMPHOID tissue , *CELL differentiation , *EMBRYONIC stem cells , *STEM cell treatment , *LABORATORY mice - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
91. Transgenic cattle produced by nuclear transfer of fetal fibroblasts carrying Ipr1 gene at a specific locus.
- Author
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Wang, Yong Sheng, He, Xiaoning, Du, Yue, Su, Jianmin, Gao, Mingqing, Ma, Yefei, Hua, Song, Quan, Fusheng, Liu, Jun, and Zhang, Yong
- Subjects
- *
TRANSGENE expression , *CATTLE breeding , *BACTERIAL diseases in animals , *MYCOBACTERIUM bovis , *TRANSPLANTATION of cell nuclei , *FIBROBLASTS , *GENETIC vectors , *POLYMERASE chain reaction , *PREVENTION - Abstract
This study aimed to assess the effects of the intracellular pathogen resistance 1 (Ipr1) transgene on preventing infection of Mycobacterium bovis in cattle. A specific expression vector for the Ipr1 gene was constructed and inserted in the genome between surfactant protein A and methionine adenosyltransferase I of bovine fetal fibroblasts. After SCNT, cleavage (86.9% vs. 87.4%, P > 0.05) and blastocyst developmental rates (34.6% vs. 33.5%, P > 0.05) were similar between transgenic and nontransgenic bovine fetal fibroblasts. Four surviving and one dead Ipr1 -transgenic female cattle were produced by transfer of the SCNT blastocysts. Polymerase chain reaction and Southern blot analyses confirmed that the Ipr1 transgene of the cattle was located at the expected site. Inserting Ipr1 gene did not affect the expression of the surrounding genes. Main death modality of M bovis –infected peripheral blood mononuclear cells (PBMCs) derived from Ipr1 -transgenic cattle was apoptosis, whereas that of PBMCs from control cattle was necrosis. In addition, the number of colony-forming units in PBMCs of Ipr1 -transgenic cattle was significantly lower than that of the control cattle (P < 0.05). The finding that expression of Ipr1 transgene in PBMCs significantly increased anti– M bovis activity suggested breeding anti– M bovis cattle population by the transgenic SCNT technique could be a feasible strategy. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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92. Scriptaid Treatment Decreases DNA Methyltransferase 1 Expression by Induction of MicroRNA-152 Expression in Porcine Somatic Cell Nuclear Transfer Embryos.
- Author
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Liang, Shuang, Zhao, Ming-Hui, Choi, Jeong-woo, Kim, Nam-Hyung, and Cui, Xiang-Shun
- Subjects
- *
DNA methyltransferases , *GENE expression , *MICRORNA , *SOMATIC cells , *TRANSPLANTATION of cell nuclei , *DNA methylation - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
93. Nuclear transport factors: global regulation of mitosis.
- Author
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Forbes, Douglass J, Travesa, Anna, Nord, Matthew S, and Bernis, Cyril
- Subjects
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MITOSIS , *TRANSPLANTATION of cell nuclei , *SPINDLE apparatus , *KARYOPHERINS , *MICROTUBULES - Abstract
The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin , a nuclear import receptor, and Crm1/Xpo1 , an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator – the γ-TuRC complex – and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
94. Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.
- Author
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Whitworth, Kristin M., Mao, Jiude, Lee, Kiho, Spollen, William G., Samuel, Melissa S., Walters, Eric M., Spate, Lee D., and Prather, Randall S.
- Subjects
- *
BLASTOCYST , *GENETIC transcription , *FERTILIZATION in vitro , *TRANSPLANTATION of cell nuclei , *HISTONE deacetylase inhibitors , *SOMATIC cell nuclear transfer - Abstract
Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro-fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid ( p<0.05). Two treatments, 1.0 μM ISAHA and 1.0 μM SAHA, had higher mean cell number than No HDACi treatment ( p<0.021). Embryo transfers performed with 10 μM SAHA- and 1 μM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos ( p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
95. Artificial cloning of domestic animals.
- Author
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Keefer, Carol L.
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CLONING , *DOMESTIC animals , *EMBRYOS , *TRANSPLANTATION of cell nuclei , *OVUM - Abstract
Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
96. Evolutionary perspectives on clonal reproduction in vertebrate animals.
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Avise, John C.
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CLONING , *VERTEBRATES , *REPRODUCTION , *TRANSPLANTATION of cell nuclei , *GYNOGENESIS - Abstract
A synopsis is provided of different expressions of whole-animal vertebrate clonality (asexual organismal-level reproduction), both in the laboratory and in nature. For vertebrate taxa, such clonal phenomena include the following: human-mediated cloning via artificial nuclear transfer; intergenerational clonality in nature via parthenogenesis and gynogenesis; intergenerational hem iclonality via hybridogenesis and kleptogenesis; intragenerational clonality via polyembryony; and what in effect qualifies as clonal replication via self-fertilization and intense inbreeding by simultaneous hermaphrodites. Each of these clonal or quasi-clonal mechanisms is described, and its evolutionary genetic ramifications are addressed. By affording an atypical vantage on standard vertebrate reproduction, clonality offers fresh perspectives on the evolutionary and ecological significance of recombination-derived genetic variety. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
97. Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells.
- Author
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Yuan, Yan, Zhou, Quan, Wan, Haifeng, Shen, Bin, Wang, Xuepeng, Wang, Mei, Feng, Chunjing, Xie, Mingming, Gu, Tiantian, Zhou, Tao, Fu, Rui, Huang, Xingxu, Zhou, Qi, Sha, Jiahao, and Zhao, Xiao-Yang
- Subjects
GENETIC mutation ,MALE infertility ,GAMETES ,TESTIS ,STEM cells ,TRANSPLANTATION of cell nuclei ,GENOME editing - Abstract
Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit
w /Kitwv mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kitw /Kitwv somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kitw /Kitwv infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
98. Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells.
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Hidetaka Akita, Dai Kurihara, Schmeer, Marco, Schleef, Martin, and Hideyoshi Harashima
- Subjects
- *
TRANSPLANTATION of cell nuclei , *DNA , *TRANSGENE expression , *PROTAMINES , *MICROINJECTION (Cytology) , *GENE expression - Abstract
The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
99. Regulation of NF-κB Oscillation by Nuclear Transport: Mechanisms Determining the Persistency and Frequency of Oscillation.
- Author
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Ohshima, Daisuke and Ichikawa, Kazuhisa
- Subjects
- *
NF-kappa B regulation , *NUCLEAR transport , *FREQUENCIES of oscillating systems , *TRANSPLANTATION of cell nuclei , *CYTOPLASM , *GENE expression - Abstract
The activated transcription factor NF-κB shuttles between the cytoplasm and the nucleus resulting in the oscillation of nuclear NF-κB (NF-κBn). The oscillation pattern of NF-κBn is implicated in the regulation of gene expression profiles. Using computational models, we previously reported that spatial parameters, such as the diffusion coefficient, nuclear to cytoplasmic volume ratio, transport through the nuclear envelope, and the loci of translation of IκB protein, modified the oscillation pattern of NF-κBn. In a subsequent report, we elucidated the importance of the “reset” of NF-κBn (returning of NF-κB to the original level) and of a “reservoir” of IκB in the cytoplasm. When the diffusion coefficient of IκB was large, IκB stored at a distant location from the nucleus diffused back to the nucleus and “reset” NF-κBn. Herein, we report mechanisms that regulate the persistency and frequency of NF-κBn oscillation by nuclear transport. Among the four parameters of nuclear transport tested in our spatio-temporal computational model, the export of IκB mRNA from the nucleus regulated the persistency of oscillation. The import of IκB to the nucleus regulated the frequency of oscillation. The remaining two parameters, import and export of NF-κB to and from the nucleus, had virtually no effect on the persistency or frequency. Our analyses revealed that lesser export of IκB mRNA allowed NF-κBn to transcript greater amounts of IκB mRNA, which was retained in the nucleus, and was subsequently exported to the cytoplasm, where large amounts of IκB were synthesized to “reset” NF-κBn and drove the persistent oscillation. On the other hand, import of greater amounts of IκB led to an increase in the influx and the efflux of NF-κB to and from the nucleus, resulting in an increase in the oscillation frequency. Our study revealed the importance of nuclear transport in regulating the oscillation pattern of NF-κBn. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
100. Disruption of Mitochondrion-To-Nucleus Interaction in Deceased Cloned Piglets.
- Author
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Park, Joonghoon, Lai, Liangxue, Samuel, Melissa S., Wax, David, Prather, Randall S., and Tian, Xiuchun
- Subjects
- *
PIGLET physiology , *SWINE cloning , *SOMATIC cells , *TRANSPLANTATION of cell nuclei , *MITOCHONDRIAL DNA , *OXIDATIVE phosphorylation - Abstract
Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
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