Publication: Aplicación del sistema de recombinación específica de secuencia β-six al estudio de la regulación de la expresión génica en células eucariotas y modelos animales
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2016-08-08
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Universidad Complutense de Madrid
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Abstract
Las recombinasas específicas de secuencia son herramientas muy valiosas en la generación de modificaciones génicas condicionales. Estos sistemas permiten controlar la recombinación de forma específica de tejido, temporalmente, o ambas, y sortean diversas limitaciones de los sistemas de knockout (KO) convencionales, como la letalidad embrionaria o la generación de mecanismos compensatorios. Actualmente los sistemas Cre/loxP y Flp/FRT son los más empleados tanto en modelos animales como vegetales. La necesidad de realizar modificaciones más complejas en un mismo organismo hace que sea primordial caracterizar otras recombinasas que complementen a las existentes. La b recombinasa (b-rec) es originaria del plásmido pSM19035 de Streptococcus pyogenes. A diferencia de Cre y Flp, que en ausencia de factores adicionales catalizan la integración en un nuevo sustrato, la b-rec necesita un sustrato superenrollado y un cofactor de la reacción, una proteína asociada a la cromatina (como la procariota Hbsu o la eucariota HMG1). Se ha demostrado que la b-rec cataliza de forma específicamente intramolecular (resolución o inversión) la recombinación en células eucariotas, tanto de sustratos episomales como integrados en la cromatina, lo que indica que el entorno eucariota es capaz de proveer del cofactor y del superenrollamiento necesarios para que la b-rec realice su función. En este trabajo hemos determinado que la tasa de recombinación mediada por la b-rec no se ve afectada en absoluto por la deficiencia en el cofactor HMG1, alcanzando el mismo valor de recombinación en MEF KO en HMG1 que en wt. Este y otros datos confirman que en el entorno eucariota hay otras proteínas accesorias que pueden actuar de cofactores y sugiere que estas reacciones pueden ocurrir en la mayor parte de tejidos y tipos celulares. Para estudiar detalladamente el potencial de la b-rec en eucariotas desarrollamos un sistema de RAGE (activación génica mediada por recombinación) dependiente de la actividad b-rec; este sistema ha resultado funcional tanto en sustratos episomales como en sustratos integrados en la cromatina. También hemos generado un vector retroviral que porta la proteína de fusión b-Egfp, permitiendo de forma rápida y eficiente la integración y expresión funcional de nuestra proteína...
There are two families of conservative site-specific recombination (SSR) systems (tyrosine and serine recombinases). The tyrosine recombinases (e.g., Cre and Flp) use an invariant active-site tyrosine residue to attack the DNA phosphodiester in the DNA-cleavage step of the recombination reaction, whereas the serine recombinases (e.g., Res, β-rec, Hin) use an invariant serine residue. Tyrosine recombinases catalyse site-specific integration and excision, whereas serine recombinases catalyse excision or inversion. SSR is a powerful tool for conditional genomic modifications. It allows programmed recombination in a tissue/lineage-specific and/or inducible manner that overcome some limitations of classical knockout systems, such as embryonic lethality or generation of compensation mechanisms. Cre/loxP and Flp/FRT are currently the most established systems, in both animal models and in plants. Due to the need of more complex modifications, it is necessary to characterize other site-specific recombination systems that could be used as an alternative to or in combination with existing models. b recombinase (b-rec) is a site specific recombinase from the Gram-positive plasmid pSM19035, that requires a supercoiled substrate and a chromatin-associated protein (e.g., bacterial Hbsu or eukaryotic HMG1). The b-rec induces specific intramolecular recombination in mammalian cells, in both episomal plasmids and chromatin-associated substrate structures, implying that the mammalian cell environment can provide such a host factor, and that global nuclear genomic DNA supercoiling is sufficient for b-rec function. In this work we demonstrated that b-rec-mediated recombination is efficiently performed in an HMG1 knockout cell line. This and previous data clearly suggests that in eukaryotic environments there are other chromatin-associated proteins that could act as helpers in b-mediated reactions, and therefore it is expected that those reactions could occur in most cell types and tissues...
There are two families of conservative site-specific recombination (SSR) systems (tyrosine and serine recombinases). The tyrosine recombinases (e.g., Cre and Flp) use an invariant active-site tyrosine residue to attack the DNA phosphodiester in the DNA-cleavage step of the recombination reaction, whereas the serine recombinases (e.g., Res, β-rec, Hin) use an invariant serine residue. Tyrosine recombinases catalyse site-specific integration and excision, whereas serine recombinases catalyse excision or inversion. SSR is a powerful tool for conditional genomic modifications. It allows programmed recombination in a tissue/lineage-specific and/or inducible manner that overcome some limitations of classical knockout systems, such as embryonic lethality or generation of compensation mechanisms. Cre/loxP and Flp/FRT are currently the most established systems, in both animal models and in plants. Due to the need of more complex modifications, it is necessary to characterize other site-specific recombination systems that could be used as an alternative to or in combination with existing models. b recombinase (b-rec) is a site specific recombinase from the Gram-positive plasmid pSM19035, that requires a supercoiled substrate and a chromatin-associated protein (e.g., bacterial Hbsu or eukaryotic HMG1). The b-rec induces specific intramolecular recombination in mammalian cells, in both episomal plasmids and chromatin-associated substrate structures, implying that the mammalian cell environment can provide such a host factor, and that global nuclear genomic DNA supercoiling is sufficient for b-rec function. In this work we demonstrated that b-rec-mediated recombination is efficiently performed in an HMG1 knockout cell line. This and previous data clearly suggests that in eukaryotic environments there are other chromatin-associated proteins that could act as helpers in b-mediated reactions, and therefore it is expected that those reactions could occur in most cell types and tissues...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Microbiología II, leída el 26-01-2016