subject:(nuclear transfer cloning)

University of Canterbury

1. Clow, Andrew Leif. Micro-scale Instruments Applied to a Bovine Nuclear Transfer System.

Degree: PhD, Electrical Engineering, 2010, University of Canterbury

► Manual handling of biological cells is routine in most laboratories. This is gradually changing with the development of robotic cell handling systems, and micro-scale lab-on-chip… (more)

▼ Manual handling of biological cells is routine in most laboratories. This is gradually changing with the development of robotic cell handling systems, and micro-scale lab-on-chip devices. Attempts were made to develop devices that automate or assist cell handling in the context of a bovine nuclear transfer (NT) system. The system, a zona-free bovine NT cloning system, formed a baseline reference for tool design and performance evaluation. Bovine NT can, as other cell handling procedures, be improved by rapid and precise cell positioning. Improvements in cell handling can increase the quantity of cells processed, and the uniformity of conditions the cells are subject to during processing. Tools were developed for two areas of cell handling: cell fusion and cell transportation. Designs suitable for implementation in microscale lab-on-chip systems were evaluated. Tool development was predominantly experimental, assisted by numerical modelling. The experimental investigation concerned device fabrication and operational performance. A number of cell handling tool designs were built and tested. Coplanar electrodes are not commonly used in bovine NT and reports on their efficacy were not available. These electrodes, which are simple to fabricate, were tested to determine fusion rates achievable in comparison with those of the baseline procedure. A novel fusion device, the micropit, was designed to assist bovine cell pairing and electrofusion. It was initially uncertain whether this device was capable of achieving cell fusion. Tests were conducted; and cell fusion and micro-positioning were demonstrated, as was an increase in biological cell processing throughput. Many miniaturised lab-on-chip systems rely on cell transportation. One illustration in the baseline procedure is the on-chip transport of cells to the cell fusion device. Potential cell transport mechanisms for a miniature cloning system were evaluated by prototype construction and testing. These mechanisms included travelling wave dielectrophoresis and capillary fluid actuation. To facilitate automation of on-chip cell transportation, a low cost electrically isolated cell detection system was developed based on a DVD pick-up unit. Various obstacles that were encountered during the course of device construction are noted, as are the fabrication methods employed.

Subjects/Keywords: Nuclear transfer; cloning; dielectrophoresis; electrofusion

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APA (6^th Edition):

Clow, A. L. (2010). Micro-scale Instruments Applied to a Bovine Nuclear Transfer System. (Doctoral Dissertation). University of Canterbury. Retrieved from http://dx.doi.org/10.26021/2780

Chicago Manual of Style (16^th Edition):

Clow, Andrew Leif. “Micro-scale Instruments Applied to a Bovine Nuclear Transfer System.” 2010. Doctoral Dissertation, University of Canterbury. Accessed April 10, 2021. http://dx.doi.org/10.26021/2780.

MLA Handbook (7^th Edition):

Clow, Andrew Leif. “Micro-scale Instruments Applied to a Bovine Nuclear Transfer System.” 2010. Web. 10 Apr 2021.

Vancouver:

Clow AL. Micro-scale Instruments Applied to a Bovine Nuclear Transfer System. [Internet] [Doctoral dissertation]. University of Canterbury; 2010. [cited 2021 Apr 10]. Available from: http://dx.doi.org/10.26021/2780.

Council of Science Editors:

Clow AL. Micro-scale Instruments Applied to a Bovine Nuclear Transfer System. [Doctoral Dissertation]. University of Canterbury; 2010. Available from: http://dx.doi.org/10.26021/2780

Colorado State University

2. De Lille, Alexandra. Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses.

Degree: PhD, Biomedical Sciences, 2012, Colorado State University

URL: http://hdl.handle.net/10217/67429

► This study was designed to measure fetal and placental characteristics in bovine day 75 nuclear transfer and control pregnancies. Responses included mRNA concentration of the… (more)

▼ This study was designed to measure fetal and placental characteristics in bovine day 75 nuclear transfer and control pregnancies. Responses included mRNA concentration of the insulin-like growth factor (IGF) system [IGF-1, IGF-2, IGF1R, IGF2R, IGFBBP-1, -2, -3] and the vascular endothelial growth factor (VEGF) system [VEGF, PlGF, VEGF1R, and VEGF2R]. Fetal attrition of the cloned pregnancies up to day 75 was high (89%, 63 out of 71 frozen embryos transferred; 8 of 16 cloned conceptuses present on day 30 survived to day 75, as did 5 of 5 controls). No significant differences in mean weights of large and medium placentomes were observed between 8 clones and 5 controls. However, the variance of mean weight of large placentomes was greater in clones than in controls; one gestation had placentomes six standard deviations larger than controls. Interestingly, the mean umbilical cord weight/length ratio was significantly greater for clones (P < 0.05). Mean fetal length, fetal weight, fetal weight/length index and mean weights for heart, brain, liver, kidneys and the mean brain/liver index did not differ between cloned and control day 75 conceptuses, but numbers per group were limited. Northern blot analysis, revealed the presence of three transcripts of 3.7kb, 2.2kb and 1.7kb for VEGF and one 1.7 kb transcript for PlGF mRNA in the cotyledons and allantochorion of day 45 cloned and control gestations. All three VEGF bands were present in both cloned and control day 75 cotyledons and caruncles, but the PlGF transcript was barely detectable, except for the cotyledons of one clone. mRNA for all of genes studied could be detected with real time PCR in day 75 cotyledons and caruncles, and fetal livers contained mRNA for all IGF's and IGFBP's evaluated. In all placentomal tissues, PlGF mRNA concentration was 100-fold less than VEGF mRNA, which seems to be the driving force for placentomal vascularization at day 75. There was a trend for a reduction by half of the PlGF mRNA concentration in caruncle of clones vs. controls (P = 0.06). VEGF2R (KDR) mRNA was abundant, but VEGF1R (Flt-1), was only present in very low concentrations; our primer set did not distinguish between soluble versus membrane bound receptor mRNA for VEGF1R. Four of the cloned conceptuses contained substantially less cotyledonary IGF1R mRNA than the other clones and controls. IGFBP-3 mRNA concentrations were very high in placentomes; IGFBP-1 and -2 mRNA concentration on the other hand was very low for clones and controls. mRNA for IGFBP-1, -2, -3, however, was abundant in day 75 fetal livers, while IGF-1 mRNA was scarce in this tissue. Fetal livers from cloned pregnancies contained 4-fold more IGF-2 mRNA than controls (P<0.01). We observed that liver IGF-2 mRNA concentration and liver weight increased with weight of the largest placentome; in clones these increases were associated with a decrease in cotyledonary IGF-2 mRNA, while the opposite occurred with controls. Interestingly, there was a trend to lower IGF2R mRNA concentrations (P = 0.09), and… Advisors/Committee Members: Seidel, George (advisor), Anthony, Russell (committee member), Clay, Colin (committee member), Garry, Franklyn (committee member).

Subjects/Keywords: nuclear transfer cloning; insulin like growth factor; placenta; angiogenesis; bovine

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APA (6^th Edition):

De Lille, A. (2012). Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/67429

Chicago Manual of Style (16^th Edition):

De Lille, Alexandra. “Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses.” 2012. Doctoral Dissertation, Colorado State University. Accessed April 10, 2021. http://hdl.handle.net/10217/67429.

MLA Handbook (7^th Edition):

De Lille, Alexandra. “Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses.” 2012. Web. 10 Apr 2021.

Vancouver:

De Lille A. Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses. [Internet] [Doctoral dissertation]. Colorado State University; 2012. [cited 2021 Apr 10]. Available from: http://hdl.handle.net/10217/67429.

Council of Science Editors:

De Lille A. Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses. [Doctoral Dissertation]. Colorado State University; 2012. Available from: http://hdl.handle.net/10217/67429

Louisiana State University

3. Sansinena, Marina Julia. Somatic cell interspecies nuclear transfer.

Degree: PhD, Animal Sciences, 2003, Louisiana State University

URL: etd-01202004-174310 ; https://digitalcommons.lsu.edu/gradschool_dissertations/642

► The low efficiency of the nuclear transfer (NT) procedure requires large number of oocytes to produce embryos and live offspring. A series of experiments were… (more)

▼ The low efficiency of the nuclear transfer (NT) procedure requires large number of oocytes to produce embryos and live offspring. A series of experiments were conducted to evaluate the ability of the bovine cytoplast to reprogram nuclei from horses and llamas. In a preliminary study, equine oocytes from small (<20mm diameter) follicles were either pretreated with roscovitine or placed in maturation (IVM only) prior to NT. Roscovitine pretreatment did not improve nuclear maturation rates (roscovitine pretreatment 57% vs. IVM only 66%) and no fusion was obtained from roscovitine-pretreated oocytes after NT. Another preliminary study was conducted with the objective to produce llama NT embryos and to compare their development in two in vitro culture conditions (G1.2® vs. CR1aa). No difference was found in the number of embryos cleaved after 2 d of culture. This resulted in the first scientific report of somatic cell NT, in vitro culture and transfer of NT embryos in the llama. In the next experiment, adult horse and llama fibroblasts were injected into enucleated cow oocytes. The results showed the cow cytoplasm is capable of partially reprogramming nuclei from other species and support mitotic divisions. However, this study also showed a consistent embryonic developmental arrest at the 8- to 16- cell stage when horse or llama donor cells were used as donor nuclei. When a more closely related species of donor cell (banteng) and recipient oocyte (domestic cattle) were used for NT, no embryonic developmental arrest was found. Embryos progressed to achieve high blastocyst rates (banteng male cell line 28% vs. banteng female cell line 15%). Two banteng interspecies NT pregnancies were established and subsequently lost from the banteng male cell line. In the final study, the effect of a mixed mitochondrial population (heteroplasmy) on early embryonic development was investigated. Ooplasmic transfer performed in combination with NT procedure indicated presence of foreign mitochondria clustered in a small portion of the cytoplasm in early stages of embryo development. When goat ooplasm was transferred into interspecies (cow oocyte-goat donor cell) NT embryos, fusion and cleave rates were reduced suggesting an increased level of heteroplasmy or nuclear-ooplasmic incompatibilities.

Subjects/Keywords: cloning; nuclear transfer; horse; llama; banteng; mitochondria

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APA (6^th Edition):

Sansinena, M. J. (2003). Somatic cell interspecies nuclear transfer. (Doctoral Dissertation). Louisiana State University. Retrieved from etd-01202004-174310 ; https://digitalcommons.lsu.edu/gradschool_dissertations/642

Chicago Manual of Style (16^th Edition):

Sansinena, Marina Julia. “Somatic cell interspecies nuclear transfer.” 2003. Doctoral Dissertation, Louisiana State University. Accessed April 10, 2021. etd-01202004-174310 ; https://digitalcommons.lsu.edu/gradschool_dissertations/642.

MLA Handbook (7^th Edition):

Sansinena, Marina Julia. “Somatic cell interspecies nuclear transfer.” 2003. Web. 10 Apr 2021.

Vancouver:

Sansinena MJ. Somatic cell interspecies nuclear transfer. [Internet] [Doctoral dissertation]. Louisiana State University; 2003. [cited 2021 Apr 10]. Available from: etd-01202004-174310 ; https://digitalcommons.lsu.edu/gradschool_dissertations/642.

Council of Science Editors:

Sansinena MJ. Somatic cell interspecies nuclear transfer. [Doctoral Dissertation]. Louisiana State University; 2003. Available from: etd-01202004-174310 ; https://digitalcommons.lsu.edu/gradschool_dissertations/642

4. Dindot, Scott Victor. The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses.

Degree: PhD, Genetics, 2004, Texas A&M University

URL: http://hdl.handle.net/1969.1/1161

► The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that… (more)

▼ The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer. Advisors/Committee Members: Derr, James (advisor), Piedrahita, Jorge (advisor), Davis, George (committee member), Womack, James (committee member), Skow, Loren (committee member).

Subjects/Keywords: imprinting; epigenetics; bovine; cloning; nuclear transfer

…associated with nuclear transfer Presently, cloning by somatic cell nuclear transfer is an… …59 III EPIGENETIC AND GENOMIC IMPRINTING ANALYSIS IN NUCLEAR TRANSFER DERIVED Bos gaurus… …16 1.2 Genomic imprinting in normal and nuclear transfer derived bovine embryos......23… …3.3 Bos gaurus/Bos taurus day 72 donor fetus and placenta and day 40 nuclear transfer… …and nuclear transfer derived fetus and placenta…

10 more images…

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APA (6^th Edition):

Dindot, S. V. (2004). The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/1161

Chicago Manual of Style (16^th Edition):

Dindot, Scott Victor. “The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses.” 2004. Doctoral Dissertation, Texas A&M University. Accessed April 10, 2021. http://hdl.handle.net/1969.1/1161.

MLA Handbook (7^th Edition):

Dindot, Scott Victor. “The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses.” 2004. Web. 10 Apr 2021.

Vancouver:

Dindot SV. The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses. [Internet] [Doctoral dissertation]. Texas A&M University; 2004. [cited 2021 Apr 10]. Available from: http://hdl.handle.net/1969.1/1161.

Council of Science Editors:

Dindot SV. The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses. [Doctoral Dissertation]. Texas A&M University; 2004. Available from: http://hdl.handle.net/1969.1/1161

University of Georgia

5. Adams, Allison Marie. The knockdown of Dnmt1 using small inhibitory RNA.

Degree: 2014, University of Georgia

URL: http://hdl.handle.net/10724/22045

► Increasing evidence has implicated the incomplete or aberrant reprogramming of donor nuclei as a contributing factor to the observed inefficiencies and outcomes inherent with the… (more)

Subjects/Keywords: Nuclear transfer; livestock cloning; reprogramming; genomic imprinting; DNA methylation; Dnmt1; RNA interference; RNAi; small inhibitory RNA; siRNA

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APA (6^th Edition):

Adams, A. M. (2014). The knockdown of Dnmt1 using small inhibitory RNA. (Thesis). University of Georgia. Retrieved from http://hdl.handle.net/10724/22045

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Adams, Allison Marie. “The knockdown of Dnmt1 using small inhibitory RNA.” 2014. Thesis, University of Georgia. Accessed April 10, 2021. http://hdl.handle.net/10724/22045.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Adams, Allison Marie. “The knockdown of Dnmt1 using small inhibitory RNA.” 2014. Web. 10 Apr 2021.

Vancouver:

Adams AM. The knockdown of Dnmt1 using small inhibitory RNA. [Internet] [Thesis]. University of Georgia; 2014. [cited 2021 Apr 10]. Available from: http://hdl.handle.net/10724/22045.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Adams AM. The knockdown of Dnmt1 using small inhibitory RNA. [Thesis]. University of Georgia; 2014. Available from: http://hdl.handle.net/10724/22045

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Brigham Young University

6. McCarrey, Sariah Cottrell. Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology.

Degree: MS, 2013, Brigham Young University

URL: https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5169&context=etd

► Within many communities and religions, including the LDS community, there is some controversy surrounding the use of stem cells – particularly embryonic stem cells… (more)

Subjects/Keywords: embryonic stem cells (ESC); personhood; spirit; body; in vitro fertilization (IVF); deontology; moral status; cloning; extinct; endangered; conservation; ethics; utilitarianism; somatic cell nuclear transfer; Biology

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APA (6^th Edition):

McCarrey, S. C. (2013). Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology. (Masters Thesis). Brigham Young University. Retrieved from https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5169&context=etd

Chicago Manual of Style (16^th Edition):

McCarrey, Sariah Cottrell. “Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology.” 2013. Masters Thesis, Brigham Young University. Accessed April 10, 2021. https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5169&context=etd.

MLA Handbook (7^th Edition):

McCarrey, Sariah Cottrell. “Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology.” 2013. Web. 10 Apr 2021.

Vancouver:

McCarrey SC. Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology. [Internet] [Masters thesis]. Brigham Young University; 2013. [cited 2021 Apr 10]. Available from: https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5169&context=etd.

Council of Science Editors:

McCarrey SC. Personhood and Cloning: Modern Applications and Ethics of Stem Cell and Cloning Technology. [Masters Thesis]. Brigham Young University; 2013. Available from: https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=5169&context=etd

University of Edinburgh

7. Thakrar, Sanjay. Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells.

Degree: PhD, 2009, University of Edinburgh

URL: http://hdl.handle.net/1842/4510

► In normal embryonic development, cells generated from a fertilised oocyte lose their pluripotent status and become restricted to a particular differentiation pathway. This production of… (more)

▼ In normal embryonic development, cells generated from a fertilised oocyte lose their pluripotent status and become restricted to a particular differentiation pathway. This production of functionally distinct cell lineages is thought to be mediated by epigenetic processes that help control gene expression both temporally and spatially without any changes to the DNA sequence. These epigenetic changes consist of posttranslational modifications of the N-terminal tails of histones and differential DNA methylation. Together these act by altering local chromatin structure, which in turn directs gene transcription by regulating the accessibility of the underlying DNA. To examine the potential developmental roles of these modifications, we determined the global cellular patterns of DNA methylation, as well as histone H3 lysine 9 (H3K9) and histone H4 lysine 20 (H4K20) methylation in the developing zebrafish embryo. These modifications are seen as hallmarks of heterochromatin, which consists of DNA that is tightly packaged, gene-poor and transcriptionally silent. Thus using immunostaining techniques, we confirmed the occurrence of genome-wide DNA methylation changes during zebrafish embryogenesis, as well as observing the unique localisation of this mark around the nuclear periphery in conjunction with pericentric heterochromatin. For mono-, di- and tri-methylated H3K9, it was observed by both immunostaining and immunoblotting that these marks became apparent after the onset of zygotic transcription. Ultimately their levels increased as development progressed, in a fashion similar to that of DNA methylation, consistent with a link between these epigenetic marks. Using the same methodology, the three methylation states of H4K20 were seen to vary differentially during zebrafish development, where in particular the levels of H4K20me1 decreased in concert with a potentially sumoylated form. In contrast, the levels of H4K20me2 increased progressively during embryogenesis, while those of H4K20me3 decreased rapidly after the mid-blastula transition. Together, these findings demonstrate that both DNA and histone lysine methylation take place in a highly dynamic manner, further supporting their roles in augmenting chromatin structure and directing cellular differentiation, while also providing a valuable comparison to the developmental epigenetics of other model organisms characterised to date. Preparatory work for somatic cell nuclear transfer in zebrafish was also undertaken. In future studies, the dynamics of these marks could be compared with those of cloned embryos, so that the specific epigenetic profiles necessary for development can be elucidated. Epigenetically, a homologous process occurs within pluripotent embryonic stem cells (ESCs), which can differentiate into any cell type or undergo indefinite self-renewal. Advantageously, we were able to derive zebrafish ESC-like clusters which were morphologically similar to those derived from mice. These clusters were alkaline phosphatase-positive and expressed key ESC markers as…

Subjects/Keywords: 591.35; zebrafish; epigenetics; H3K9; H4K20; stem cells; cloning; nuclear transfer

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APA (6^th Edition):

Thakrar, S. (2009). Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells. (Doctoral Dissertation). University of Edinburgh. Retrieved from http://hdl.handle.net/1842/4510

Chicago Manual of Style (16^th Edition):

Thakrar, Sanjay. “Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells.” 2009. Doctoral Dissertation, University of Edinburgh. Accessed April 10, 2021. http://hdl.handle.net/1842/4510.

MLA Handbook (7^th Edition):

Thakrar, Sanjay. “Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells.” 2009. Web. 10 Apr 2021.

Vancouver:

Thakrar S. Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells. [Internet] [Doctoral dissertation]. University of Edinburgh; 2009. [cited 2021 Apr 10]. Available from: http://hdl.handle.net/1842/4510.

Council of Science Editors:

Thakrar S. Epigenetic profiling of the developing zebrafish embryo, and technical developments towards cloning zebrafish and isolating pluripotent stem cells. [Doctoral Dissertation]. University of Edinburgh; 2009. Available from: http://hdl.handle.net/1842/4510

Université Paris-Sud – Paris XI

8. Veillard, Anne-Clémence. Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse.

Degree: Docteur es, Biologie du développement, 2013, Université Paris-Sud – Paris XI

URL: http://www.theses.fr/2013PA112267

►

Les cellules souches pluripotentes sont capables de donner naissance à tous les types cellulaires constituant un organisme, ce qui leur confère un fort intérêt thérapeutique.… (more)

▼

Les cellules souches pluripotentes sont capables de donner naissance à tous les types cellulaires constituant un organisme, ce qui leur confère un fort intérêt thérapeutique. A partir de l’embryon de souris on peut en dériver deux types : les cellules souches embryonnaires (ES) au stade blastocyste et les cellules souches d’épiblaste (EpiSC) au stade œuf cylindre. Ces deux types de cellules partagent leurs propriétés pluripotentes mais se distinguent par de nombreux aspects comme leurs conditions de culture et les gènes qu’elles expriment. Nous avons montré que la reprogrammation par clonage par transfert de noyau permet d’obtenir des EpiSC présentant un méthylome et un transcriptome similaires à ceux des EpiSC issues d’embryons après fécondation. Nous avons également caractérisé le profil de méthylation de l’ADN des EpiSC, et montré une tendance à l’hyperméthylation des promoteurs des EpiSC par-rapport aux cellules ES et à l’épiblaste. De plus, l’absence de méthylation empêche la conversion des cellules ES en EpiSC. Les EpiSC semblent donc dépendre fortement de la méthylation de l’ADN pour réguler l’expression de leurs gènes, ce qui les distingue des cellules ES.

Pluripotent stem cells are of great therapeutic interest because of their capability to give rise to all the cells composing an organism. We can derive two types of these stem cells from the mouse embryo: embryonic stem cells (ESCs) from the blastocyst and epiblast stem cells (EpiSCs) from the egg cylinder stage. These two cell types share their pluripotent properties but are distinct on several features, like their culture conditions and gene expression. We showed that reprogramming using cloning by nuclear transfer allows the obtention of EpiSCs with a methylome and a transcriptome similar to those of EpiSCs derived from embryo after fertilisation. We also characterised the DNA methylation pattern of EpiSCs and showed their tendency to present a hypermethylation at their promoters compared to ESCs and epiblast. We also observed that the absence of DNA methylation blocks the conversion of ESCs into EpiSCs. As a conclusion, it seems that EpiSCs are strongly dependant of DNA methylation to regulate gene expression, which distinguishes them from ESCs.

Advisors/Committee Members: Jouneau, Alice (thesis director).

Subjects/Keywords: Pluripotence; Cellules souches d'épiblaste; Cellules souches embryonnaires; Méthylation de l'ADN; Clonage par transfert de noyau; Pluripotency; Epiblast stem cells; Embryonic stem cells; DNA methylation; Cloning by nuclear transfer

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APA (6^th Edition):

Veillard, A. (2013). Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse. (Doctoral Dissertation). Université Paris-Sud – Paris XI. Retrieved from http://www.theses.fr/2013PA112267

Chicago Manual of Style (16^th Edition):

Veillard, Anne-Clémence. “Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse.” 2013. Doctoral Dissertation, Université Paris-Sud – Paris XI. Accessed April 10, 2021. http://www.theses.fr/2013PA112267.

MLA Handbook (7^th Edition):

Veillard, Anne-Clémence. “Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse.” 2013. Web. 10 Apr 2021.

Vancouver:

Veillard A. Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse. [Internet] [Doctoral dissertation]. Université Paris-Sud – Paris XI; 2013. [cited 2021 Apr 10]. Available from: http://www.theses.fr/2013PA112267.

Council of Science Editors:

Veillard A. Profil de méthylation de l’ADN des cellules souches d’épiblaste issues d’embryons après fécondation ou clonage et comparaison avec les cellules souches embryonnaires chez la souris : DNA methylation profil of epiblast stem cells from embryos after fertilisation or cloning and comparison with embryonic stem cells in the mouse. [Doctoral Dissertation]. Université Paris-Sud – Paris XI; 2013. Available from: http://www.theses.fr/2013PA112267

9. Vargas, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones.

Degree: 2018, Federal University of Uberlândia

URL: VARGAS, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. 2018. 62 f. Dissertação (Mestrado em Genética e Bioquímica) - Universidade Federal de Uberlândia, Uberlandia, 2018. Disponível em: http://dx.doi.org/10.14393/ufu.di.2018.815 ; https://repositorio.ufu.br/handle/123456789/22391 ; http://dx.doi.org/10.14393/ufu.di.2018.815

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

A clonagem por meio da transferência nuclear de células somáticas (TNCS) é uma biotecnologia da… (more)

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

A clonagem por meio da transferência nuclear de células somáticas (TNCS) é uma biotecnologia da reprodução com várias aplicações e já em uso comercial. No entanto, ainda é uma técnica com baixa eficiência devido a alta frequência de anormalidades fetais e placentárias. Para que o embrião se desenvolva normalmente, o perfil epigenético do genoma somático da célula doadora de núcleo deve ser reprogramado corretamente após a transferência nuclear. No entanto, nos embriões clones essa reprogramação não ocorre de forma eficiente, o que leva a alterações no padrão de metilação do DNA, e consequentemente, alterações no perfil de expressão gênica de genes importantes durante o desenvolvimento embrionário e fetal. Nesse estudo, nós determinamos os níveis de mRNA para 6 genes alvos (DNMT1, DNMT3A, DNMT3B, TET1, TET2 e TET3) relacionados à reprogramação da metilação do DNA em cotilédone fetal de 13 bezerros provenientes de TNCS. O RNA total foi extraído das amostras de cotilédone fetal e submetido à transcrição reversa para análises em PCR em tempo real. Os resultados mostraram padrões de expressão gênica diferentes entre os clones e os animais controles produzidos por inseminação artificial para todos os genes avaliados, exceto para a TET3. Os níveis de mRNA de DNMT1 (p=0,0044), DNMT3A (p=0,0044) e TET2 (p=0,0044) foram maiores nos controles, enquanto que, para DNMT3B (p=0,0308) e TET1 (p=0,0308) os níveis foram maiores nos clones. Além disso, foi identificado que os clones que morreram na primeira semana de vida apresentaram diferenças de expressão gênica em relação aos controles para os genes DNMT1 (p=0,0244), DNMT3A (p=0,0279), DNMT3B (p=0,0364), TET1 (p=0,0103) e TET2 (p=0,0184), enquanto que os que sobreviveram foram diferentes dos controles somente para TET1 (p=0,0344). Esses resultados mostraram que os animais clones que sobrevivem não são apenas fisicamente mais saudáveis, mas também relativamente mais próximos dos controles no nível molecular. Isto sugere que uma expressão adequada das enzimas relacionadas com a reprogramação epigenética do DNA na placenta é essencial para a sobrevivência do animal proveniente de TNCS.

Cloning by somatic cell nuclear transfer (SCNT) is an assisted reproductive technique with several applications in livestock. However, it is still a technique that shows low efficiency due to the high frequency of fetal and placental abnormalities. The somatic genome must be correctly reprogrammed after nuclear transfer to achieve normal embryo development. However, in cloned embryos this reprogramming does not occur efficiently, leading to changes in DNA methylation patterns and consequently alterations in gene expression profile of important genes for embryo and foetal development. In this study, we determined the mRNA levels for six target genes (DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3) related to DNA methylation reprogramming in the placental cotyledon of 13 calves produced by SCNT. Total RNA was extracted from the…

Advisors/Committee Members: Franco, Maurício Machaim, Fonseca, Márcio José Poças, Goulart, Vivian Alonso.

Subjects/Keywords: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA ANIMAL; Transferência nuclear de células somáticas; Clonagem; Expressão gênica; Epigenética; Metilação do DNA; Placenta; Somatic Cell Nuclear Transfer; Cloning; DNA methylation; Epigenetics; DNMT; TET; Gene expression; Genética; Ácido desoxirribonucleico

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Vargas, L. N. (2018). Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. (Masters Thesis). Federal University of Uberlândia. Retrieved from VARGAS, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. 2018. 62 f. Dissertação (Mestrado em Genética e Bioquímica) - Universidade Federal de Uberlândia, Uberlandia, 2018. Disponível em: http://dx.doi.org/10.14393/ufu.di.2018.815 ; https://repositorio.ufu.br/handle/123456789/22391 ; http://dx.doi.org/10.14393/ufu.di.2018.815

Chicago Manual of Style (16^th Edition):

Vargas, Luna Nascimento. “Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones.” 2018. Masters Thesis, Federal University of Uberlândia. Accessed April 10, 2021. VARGAS, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. 2018. 62 f. Dissertação (Mestrado em Genética e Bioquímica) - Universidade Federal de Uberlândia, Uberlandia, 2018. Disponível em: http://dx.doi.org/10.14393/ufu.di.2018.815 ; https://repositorio.ufu.br/handle/123456789/22391 ; http://dx.doi.org/10.14393/ufu.di.2018.815.

MLA Handbook (7^th Edition):

Vargas, Luna Nascimento. “Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones.” 2018. Web. 10 Apr 2021.

Vancouver:

Vargas LN. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. [Internet] [Masters thesis]. Federal University of Uberlândia; 2018. [cited 2021 Apr 10]. Available from: VARGAS, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. 2018. 62 f. Dissertação (Mestrado em Genética e Bioquímica) - Universidade Federal de Uberlândia, Uberlandia, 2018. Disponível em: http://dx.doi.org/10.14393/ufu.di.2018.815 ; https://repositorio.ufu.br/handle/123456789/22391 ; http://dx.doi.org/10.14393/ufu.di.2018.815.

Council of Science Editors:

Vargas LN. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. [Masters Thesis]. Federal University of Uberlândia; 2018. Available from: VARGAS, Luna Nascimento. Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones. 2018. 62 f. Dissertação (Mestrado em Genética e Bioquímica) - Universidade Federal de Uberlândia, Uberlandia, 2018. Disponível em: http://dx.doi.org/10.14393/ufu.di.2018.815 ; https://repositorio.ufu.br/handle/123456789/22391 ; http://dx.doi.org/10.14393/ufu.di.2018.815

10. Marco Roberto Bourg de Mello. "Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo".

Degree: 2003, University of São Paulo

URL: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29062005-161626/

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O objetivo deste estudo foi avaliar a viabilidade in vitro e in vivo de embriões bovinos reconstruídos com oócitos enucleados em Metáfase II e núcleos… (more)

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O objetivo deste estudo foi avaliar a viabilidade in vitro e in vivo de embriões bovinos reconstruídos com oócitos enucleados em Metáfase II e núcleos de células somáticas (fibroblastos) fetais e adultas. Para tanto, oócitos de ovários colhidos em matadouro foram maturados in vitro por 17 horas e enucleados pela remoção do primeiro corpúsculo polar (CP) e da região do oolema contendo a placa metafásica. Como núcleo doador, foram utilizados fibroblastos de orelha de vaca da raça Nelore e de feto colhido em abatedouro. Para a reconstrução dos embriões, cada célula doadora de núcleo, após indução à G0, foi inserida sob a zona pelúcida de cada oócito enucleado e o complexo citoplasma receptor - núcleo doador (CCN) fundido e ativado por eletrofusão (2 pulsos de 4 KV/cm durante 20µs). Após ativação elétrica, cada CCN foi incubado em solução de ciclohexemide (10µg/ml) e citocalasina D (2,5µg/ml) por 1 hora e, em seguida, em solução de ciclohexemide (10µg/ml) por mais 4 horas. Os embriões reconstruídos e ativados, assim como os fecundados in vitro (controle), foram co-cultivados em monocamada de células da granulosa e TCM 199 acrescido de 10% de SFB por 7-9 dias. Após o co-cultivo por 7-9 dias, parte dos embriões (controle e reconstruídos) foi fixada e corada para determinação do número de células e parte transferida para receptoras. Um total de 668 embriões foram reconstruídos com célula fetal e 569 com fibroblasto adulto. Após eletrofusão, 212 embriões reconstruídos com célula fetal e 181 com célula adulta fundiram e 32 (15,1%) e 30 (16,6%) atingiram o estádio de blastocisto, respectivamente. O número médio de células dos blastocistos foi 129,3, 101,3 e 114,3, respectivamente, para célula fetal, adulta e embriões FIV (controle), não havendo diferença estatística significante entre os grupos (P<0,05). Após a transferência de 18 blastocistos de célula fetal e 21 de célula adulta, as taxas de prenhez aos 90 dias foram 16,7% (3) e 19% (4), respectivamente, não havendo diferença estatística significante entre os grupos (P<0,05). A primeira prenhez com célula fetal deu origem a um bezerro saudável, aos 290 dias, pesando 34kg. Uma das receptoras morreu aos 229 dias de gestação em conseqüência de hidroalantóide e outra abortou aos 252 dias. As prenhezes de embriões reconstruídos com célula adulta ainda estão em andamento. Estes resultados indicam que fibroblastos fetal e adulto podem ser usados como doadores de núcleo com semelhantes taxas de desenvolvimento in vitro e in vivo.

The aim of this study was to evaluate the in vitro and in vivo viability of bovine nuclear transferred embryos from metaphase II oocytes and fetal and adult fibroblasts. Oocytes from ovaries collected at slaughterhouse were matured in vitro for 17 hours and enucleated after aspiration of first polar body (PB) and small volume of cytoplasm containing metaphase plate. Fibroblasts from Nelore cow and foetus collected at slaughterhouse were used as nuclei donor. In Nuclear Transfer, each nuclei donor cell, after serum starvation, was inserted under…

Advisors/Committee Members: José Antonio Visintin, Mayra Elena Ortiz D'Avila Assumpção, Lygia da Veiga Pereira Carramaschi, Joaquim Mansano Garcia, Flavio Vieira Meirelles.

Subjects/Keywords: Bovinos; Clonagem animal; Embrião animal; Fibroblastos; Núcleo Celular (Transferência); Bovine; Cloning; Embryos; Fibroblasts; Nuclear Transfer

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mello, M. R. B. d. (2003). "Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo". (Doctoral Dissertation). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29062005-161626/

Chicago Manual of Style (16^th Edition):

Mello, Marco Roberto Bourg de. “"Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo".” 2003. Doctoral Dissertation, University of São Paulo. Accessed April 10, 2021. http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29062005-161626/.

MLA Handbook (7^th Edition):

Mello, Marco Roberto Bourg de. “"Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo".” 2003. Web. 10 Apr 2021.

Vancouver:

Mello MRBd. "Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo". [Internet] [Doctoral dissertation]. University of São Paulo; 2003. [cited 2021 Apr 10]. Available from: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29062005-161626/.

Council of Science Editors:

Mello MRBd. "Clonagem em bovinos: uso de fibroblastos fetal e adulto como fonte doadora de núcleo". [Doctoral Dissertation]. University of São Paulo; 2003. Available from: http://www.teses.usp.br/teses/disponiveis/10/10131/tde-29062005-161626/

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