Tesis doctoral de Mireia Perez Camps
At present, the study of the nuclear reprogramming mechanisms is of interest due to the knowledge of these processes are the clue to act efficiently in questions as the cancer or the regenerative medicine. Moreover, this type of studies are very important in the multiple and oriented transgenic animal obtaining. although several animal models can be used to carry out these studies, zebrafish has been selected due the low economic cost, as well as some of the developmental characteristics (short embryonic development, embryo transparency..) And the sequenced genome, among others. It is known that the zebrafish model has also some inconvenient as the availability of the nuclear transplant (nt) technique and, at other level, the chimaerism. The development of these two techniques are expected in this thesis, what justify the objectives. to this end several experimental studies entitled: ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish, evaluation of presumptive caudal fin blastema cells as candidate donors in intraspecies zebrafish (danio rerio) chimaeras, definition of three somatic adult cell nuclear transplant methods in zebrafish (danio rerio): before, during and after egg activation by sperm fertilization, transplant of adult fibroblast into the central region of metaphase ii eggs resulted in mid blastula transition (mbt) embryos, electroactivation of zebrafish (danio rerio) eggs, comparison of different activating stimuli efficiency in zebrafish nuclear transplant, reconstruction of heteroparental gynogenetic diploid condition by nuclear transplant in zebrafish. in the first two studies related to the chimaerism, the final efficiency of the technique was optimized penalizing the recipient embryo with uv radiation and performing the manipulation into a medium with the same osmolarity than inside of the zebrafish embryos. Germ-line chimaerism rates obtained reached as high as 50%. the chimaerism technique was employed in this thesis to test the pluripotency of the blastema cells derived from the fin amputation. These cells seem to not reach the pluripotent stage in their reprogramming process. in the four studies devoted to the development of the nt technique, it was showed that the transplant can performed into activated eggs, during it activation and, even, before activation, avoiding the difficulty of detecting the micropyle. In addition, it was observed that the efficiency of the of the parthenogenote development extraordinary depends on the activating stimulus used, being maximum with the sperm and minimum with water. In this way, an activation reinforce treatment based on the application of the electric pulses into ionic medium which, unfortunately, involved the egg lysis when it was applied to manipulated eggs, but not in intact egg. during the development of the present thesis emerged, in the research lab, a nt variant which was considered of maximum interest, and that involved the take up again a research line developed in mammals in our lab years ago: the hemmicloning. In the actual case, using zebrafish, the technique involves the haploid gynogenetic larvae obtaining to be used for the establishment of primary cultures. These haploidsomatic cells were used as donors to reconstruct the diploid condition of non enucleated oocytes by nt. It has to be noted that the combination of somatic and resident nucleus is more than the assayed in this thesis. This last technique will allow realizing studies about the reprogramming and gametic imprinting which are not possible in mammals.
Datos académicos de la tesis doctoral «Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish«
- Título de la tesis: Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish
- Autor: Mireia Perez Camps
- Universidad: Politécnica de Valencia
- Fecha de lectura de la tesis: 22/12/2009
Dirección y tribunal
- Director de la tesis
- Fernando García Ximénez
- Tribunal
- Presidente del tribunal: Miguel angel Silvestre camps
- ignacio Salvador vidal (vocal)
- rita pilar Cervera Juan es (vocal)
- María José Escribá pérez (vocal)