Modificaciones epigenéticas en clonación porcina

The somatic cells nuclear transfer technique (SCNT) or animal cloning, involves transferring the nucleus of a somatic donor cell into the cytoplasm of an oocyte that has been previously removed its nucleus, in order to obtain the necessary machinery to generate an embryo. However, it is a highly ine...

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Autor principal: Buemo, Carla Paola
Otros Autores: Fernandez y Martín, Rafael
Formato: Tesis doctoral acceptedVersion
Lenguaje:Español
Publicado: Facultad de Farmacia y Bioquímica 2016
Materias:
Clonación en porcinos
Agregación embrionaria
Reprogramación
Modificaciones epigenéticas
Porcine cloning
Embryo aggregation
Reprogramation
Epigenetic modification
Ciencia de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_2729
http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_2729.dir/2729.PDF
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Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_2729
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic Clonación en porcinos
Agregación embrionaria
Reprogramación
Modificaciones epigenéticas
Porcine cloning
Embryo aggregation
Reprogramation
Epigenetic modification
Ciencia de la vida
spellingShingle Clonación en porcinos
Agregación embrionaria
Reprogramación
Modificaciones epigenéticas
Porcine cloning
Embryo aggregation
Reprogramation
Epigenetic modification
Ciencia de la vida
Buemo, Carla Paola
Modificaciones epigenéticas en clonación porcina
topic_facet Clonación en porcinos
Agregación embrionaria
Reprogramación
Modificaciones epigenéticas
Porcine cloning
Embryo aggregation
Reprogramation
Epigenetic modification
Ciencia de la vida
description The somatic cells nuclear transfer technique (SCNT) or animal cloning, involves transferring the nucleus of a somatic donor cell into the cytoplasm of an oocyte that has been previously removed its nucleus, in order to obtain the necessary machinery to generate an embryo. However, it is a highly inefficient method and requires technical improvements.\nCloning has various applications in animal production. However, is still not entirely clear the mechanisms of nuclear remodeling and reprogramming that must suffer to become an embryo. A variety of factors probably contribute to this inefficiency, from the quality of oocytes to the type of donor cell. The failure to adequately reprogram the transplanted nucleus could be due to a deficient imprinting in somatic nuclei reconstituted embryo. To resolve the reprogramming failures we utilized in embryos the embryo aggregation technique and applied drugs that modify the epigenetic.\nAt the beginning of this study, partenogenetics embryos were used with these drugs that induce changes in methylation and histone acetylation, obtaining a combination of drugs (1 uM 5 Azacitidine + 1 uM PD0325901) which showed a tendency to increase in vitro embryo development. Subsequently, we continued to perform embryo aggregation, generating two groups: (1x) control and (3x) aggregates. The aggregated embryos increased in vitro development rates not involving a greater use of oocytes, also increased size measured by embryonic diameter, however, no statistically differences were found in the number of cells, apoptosis cell or DNA fragmentation between both groups.\nWhen we worked in swine clones, embryo aggregation in the one cell stage showed that were not required additional oocytes to produce higher rates of blastocyst resulting in an increase in the diameter and number of embryonic cells, while the level apoptosis was statistically lower in 3x aggregates embryos . This could prove partial compensation possibly caused by the combination of three different epigenetically embryos that had higher pluripotency gene expression . Moreover, by improving in vitro development, number of cells, embryonic diameter and decrease levels of apoptosis was improving embryonic reprogramming, obtaining better quality embryos.\nIn the last chapter of this thesis, we combined the embryo aggregation method and the use of drugs that induce epigenetic modifications in the early embryo development. This was encompass several aspects involving nuclear reprogramming, gene expression, showing marks relative to histones and DNA methylation. As a result, the treated 3x group showed an increase in embryonic size without involving changes in cell number. Also it showed an increase in gene expression related to trophoblast, involved in embryonic placentation, as in antiapoptotic genes and in the MAPK pathway genes involved.\nAnother change was displayed in the redistribution of acetylated histone H3 lysine 27 (H3K27ac). This is preferably located in the nuclear perisferia. Histone H3 with a methyl group at lysine 4 (H3K4me1) did not modify their distribution within the core, showing a homogeneous distribution. Regarding DNA methylation, Oct4 and Dnmt1 presented a marked demethylation, showing that the drug 5 Azacitidine, acting on methylation, generated changes in the embryos.\nIn conclusion, this work generated a contribution to the study of embryonic reprogramming that must suffer embryos during cloning, covering different variables that affect the reprogramming process.
author2 Fernandez y Martín, Rafael
author_facet Fernandez y Martín, Rafael
Buemo, Carla Paola
format Tesis doctoral
Tesis doctoral
acceptedVersion
author Buemo, Carla Paola
author_sort Buemo, Carla Paola
title Modificaciones epigenéticas en clonación porcina
title_short Modificaciones epigenéticas en clonación porcina
title_full Modificaciones epigenéticas en clonación porcina
title_fullStr Modificaciones epigenéticas en clonación porcina
title_full_unstemmed Modificaciones epigenéticas en clonación porcina
title_sort modificaciones epigenéticas en clonación porcina
publisher Facultad de Farmacia y Bioquímica
publishDate 2016
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_2729
http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_2729.dir/2729.PDF
work_keys_str_mv AT buemocarlapaola modificacionesepigeneticasenclonacionporcina
_version_ 1766017509710888960
spelling I28-R145-HWA_27292019-09-25 The somatic cells nuclear transfer technique (SCNT) or animal cloning, involves transferring the nucleus of a somatic donor cell into the cytoplasm of an oocyte that has been previously removed its nucleus, in order to obtain the necessary machinery to generate an embryo. However, it is a highly inefficient method and requires technical improvements.\nCloning has various applications in animal production. However, is still not entirely clear the mechanisms of nuclear remodeling and reprogramming that must suffer to become an embryo. A variety of factors probably contribute to this inefficiency, from the quality of oocytes to the type of donor cell. The failure to adequately reprogram the transplanted nucleus could be due to a deficient imprinting in somatic nuclei reconstituted embryo. To resolve the reprogramming failures we utilized in embryos the embryo aggregation technique and applied drugs that modify the epigenetic.\nAt the beginning of this study, partenogenetics embryos were used with these drugs that induce changes in methylation and histone acetylation, obtaining a combination of drugs (1 uM 5 Azacitidine + 1 uM PD0325901) which showed a tendency to increase in vitro embryo development. Subsequently, we continued to perform embryo aggregation, generating two groups: (1x) control and (3x) aggregates. The aggregated embryos increased in vitro development rates not involving a greater use of oocytes, also increased size measured by embryonic diameter, however, no statistically differences were found in the number of cells, apoptosis cell or DNA fragmentation between both groups.\nWhen we worked in swine clones, embryo aggregation in the one cell stage showed that were not required additional oocytes to produce higher rates of blastocyst resulting in an increase in the diameter and number of embryonic cells, while the level apoptosis was statistically lower in 3x aggregates embryos . This could prove partial compensation possibly caused by the combination of three different epigenetically embryos that had higher pluripotency gene expression . Moreover, by improving in vitro development, number of cells, embryonic diameter and decrease levels of apoptosis was improving embryonic reprogramming, obtaining better quality embryos.\nIn the last chapter of this thesis, we combined the embryo aggregation method and the use of drugs that induce epigenetic modifications in the early embryo development. This was encompass several aspects involving nuclear reprogramming, gene expression, showing marks relative to histones and DNA methylation. As a result, the treated 3x group showed an increase in embryonic size without involving changes in cell number. Also it showed an increase in gene expression related to trophoblast, involved in embryonic placentation, as in antiapoptotic genes and in the MAPK pathway genes involved.\nAnother change was displayed in the redistribution of acetylated histone H3 lysine 27 (H3K27ac). This is preferably located in the nuclear perisferia. Histone H3 with a methyl group at lysine 4 (H3K4me1) did not modify their distribution within the core, showing a homogeneous distribution. Regarding DNA methylation, Oct4 and Dnmt1 presented a marked demethylation, showing that the drug 5 Azacitidine, acting on methylation, generated changes in the embryos.\nIn conclusion, this work generated a contribution to the study of embryonic reprogramming that must suffer embryos during cloning, covering different variables that affect the reprogramming process. Fil: Buemo, Carla Paola. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Fernandez y Martín, Rafael Rivolta, Carina Facultad de Farmacia y Bioquímica Salamone, Daniel Felipe Buemo, Carla Paola 2016-12-20 La técnica de transferencia de células somáticas (SCNT) o clonación animal, consiste en transferir el núcleo de una célula somática donante dentro del citoplasma de un ovocito al que se le ha eliminado previamente su núcleo, con el objetivo de obtener la maquinaria necesaria para generar un embrión. Sin embargo, es una técnica altamente ineficiente y requiere de mejoras.\nLa clonación tiene diversas aplicaciones en producción animal. No obstante, siguen sin estar del todo claro los mecanismos de remodelación y reprogramación nuclear que debe sufrir el embrión para que llegue a ser viable. Una variedad de factores probablemente contribuyen a esta ineficiencia, desde la calidad de los oocitos hasta el tipo de célula donante. El fracaso para reprogramar el núcleo trasplantado adecuadamente podría deberse a la deficiente impronta somática en los núcleos del embrión reconstituído. Para resolver las fallas en la reprogramación se recurrió a la técnica de agregación embrionaria y al uso de drogas modificadoras de la epigenética embrionaria.\nAl principio del trabajo se utilizó embriones partenogénicos, en ellos se probaron fármacos que inducen modificaciones en la metilación y la acetilación de histonas, obteniendose una combinación de drogas (5 Azacitidina 1?M + PD0325901 1 ?M ) que demostró una tendencia a aumentar el desarrollo embrionario in vitro. Posteriormente se prosiguió a realizar la agregación embrionaria, generándose dos grupos: el control (1x) y los agregados (3x), en los que tres embriones reconstituídos formaban parte del mismo embrión. En los embriones agregados aumentaron las tasas de desarrollo in vitro no implicando un mayor uso de oocitos, se incrementó el tamaño medido en diámetro embrionario, sin embargo, no se encontraron diferencias estadísticas significativas en el número de células ni en la apoptosis celular o fragmentación del ADN entre ambos grupos.\nCuando se pasó a trabajar en los clones porcinos, la agregación embrionaria en la fase de una célula demostró que no fueron necesarios oocitos adicionales para producir mayores tasas de blastocistos obteniendose un aumento en el diámetro y en el número de células embrionarias, mientras que el nivel de apoptosis fue estadísticamente más bajo en los embriones agregados 3x. Ésto podría demostrar una compensación parcial causada posiblemente por la combinación de tres embriones epigenéticamente diferentes que presentaban una mayor expresión de genes de pluripotencia. Por otra parte, al mejorar desarrollo in vitro, número de células, el diámetro embrionario y disminuir los niveles de apoptosis se estaba mejorando la reprogramación embrionaria, obteniendose embriones de mejor calidad.\nEn el último capítulo de la tesis se combinó el método de agregación embrionaria y los fármacos inductores de modificaciones epigenéticas en el embrión temprano. Se trató de abarcar varios aspectos que involucran la reprogramación nuclear mostrando expresión génica en relación a marcas de histonas y a metilación del ADN. Como resultado, el grupo 3x tratado con las drogas presentó un aumento en el tamaño embrionario, sin implicar cambios en el número celular. También presentó un incremento en la expresión génica en genes claves para el desarrollo de trofoblasto, implicados en la placentación embrionaria, al igual que en genes antiapoptóticos y genes implicados en la vía MAPK. Otro de los cambios visualizados fue la redistribución de la histona H3 acetilada en la lisina 27 (H3K27ac). Ésta se ubicó preferentemente en la perisferia nuclear. La histona H3 con un grupo metilo en la lisina 4 (H3K4me1) no modificó su distribución dentro del núcleo, mostrando una distribución homogénea. Respecto a la metilación del ADN tanto el gen que codifica para Oct4 como el que codifica para la enzima Dnmt1 presentaron una desmetilación marcada, mostrando que la droga 5 Azacitidina, que actúa sobre la metilación, estaba generando cambios en los embriones estudiados.\nComo conclusión, éste trabajo generó un aporte al estudio de la reprogramación embrionaria que deben sufrir los embriones provenientes de clonación, abarcando distintas variables que afectan el proceso de reprogramación. application/pdf Targovnik, Héctor Cética, Pablo Vitullo, Alfredo Clonación en porcinos Agregación embrionaria Reprogramación Modificaciones epigenéticas Porcine cloning Embryo aggregation Reprogramation Epigenetic modification spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencia de la vida Modificaciones epigenéticas en clonación porcina info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_2729 http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_2729.dir/2729.PDF

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