Publication: Desarrollo y aplicación de la tecnología CRISPR para el estudio de procesos reproductivos en mamíferos
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2021-03-16
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Universidad Complutense de Madrid
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Abstract
Las aplicaciones de la mutagénesis dirigida se han visto obstaculizadas por dificultades técnicas. La baja eficiencia de la recombinación homóloga (HR), el único método disponible para llevar a cabo esta tarea antes del descubrimiento de las endonucleasas específicas de sitio, hace necesario el uso de células intermedias. Este requisito ha restringido en gran medida la mutagénesis dirigida al modelo del ratón de laboratorio, la única especie en la que se disponía de células madre pluripotentes como células intermediarias. El desarrollo de endonucleasas específicas sitios en los últimos años ha facilitado en gran medida la tarea de lograr una mutación en un lugar específico del genoma. Estas tecnologías incluyen la incorporación más notoria y reciente: CRISPR-Cas9. Mediante esta tecnología, pueden lograrse directamente modificaciones en un lugar específico del genoma mediante la transmisión de los componentes de CRISPR a un cigoto, evitando así la necesidad de células intermedias. Este avance facilita enormemente el proceso en ratones y, especialmente, en otras especies en las que, en ausencia de células madre, se requería la transferencia nuclear de células somáticas [en inglés “somatic cell nuclear transfer” (SCNT)], para generar mutagénesis dirigida. Además, la generación de mutantes en un solo paso permite el estudio de la función génica durante el desarrollo embrionario sin necesidad de mantener colonias de animales mutantes...
The applications of targeted mutagenesis have been hampered by technical difficulties. The low efficiency of homologous recombination (HR), the only available method before the discovery of site-specific endonucleases, requires the use of intermediary cells. This requirement has largely restricted targeted mutagenesis to the mouse model, the only species where pluripotent stem cells were available as intermediary cells. The development of site-specific endonucleases in recent years has greatly facilitated the task of achieving a mutation at a specific locus of the genome. These technologies include the latest and most notable incorporation: CRISPR-Cas9. By this technology, site-specific genome modifications can be directly achieved by delivering of CRISPR components into a zygote, thereby avoiding the need of intermediary cells. This improvement greatly facilitates the process in mice and, specially, in other species where, in the absence of stem cells, somatic cell nuclear transfer was required to generate site-specific mutagenesis. Besides, the generation of mutants in a single step allows the study of the gene function during embryo development without the need of maintaining colonies of mutant animals...
The applications of targeted mutagenesis have been hampered by technical difficulties. The low efficiency of homologous recombination (HR), the only available method before the discovery of site-specific endonucleases, requires the use of intermediary cells. This requirement has largely restricted targeted mutagenesis to the mouse model, the only species where pluripotent stem cells were available as intermediary cells. The development of site-specific endonucleases in recent years has greatly facilitated the task of achieving a mutation at a specific locus of the genome. These technologies include the latest and most notable incorporation: CRISPR-Cas9. By this technology, site-specific genome modifications can be directly achieved by delivering of CRISPR components into a zygote, thereby avoiding the need of intermediary cells. This improvement greatly facilitates the process in mice and, specially, in other species where, in the absence of stem cells, somatic cell nuclear transfer was required to generate site-specific mutagenesis. Besides, the generation of mutants in a single step allows the study of the gene function during embryo development without the need of maintaining colonies of mutant animals...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Fisiología Animal, leída el 13-01-2021