| The relationship between cytoplasm and nucleus is the basic problem of biology, and nuclear transfer (NT) and pronuclear transplantation are the important methods to research their relations. So far, the mammalian pronuclear transplantation is mainly studied on mice. According to the different donor types, mammalian nuclear transfer can be divided into two kinds: embryonic nuclear transfer and somatic nuclear transfer. Mouse is an important developmental model animal in biological research. In the reported references of mouse nuclear transfer, the cloning strategies are different when embryonic blastomeres and somatic cells are employed as donors, respectively. In embryonic nuclear transfer, fusion methods are generally adopted, while direct introcytoplasmic injection is often used in the somatic nuclear transfer. Enucleating the nuclei of recipient oocytes accurately is one of the key techniques in nuclear transfer research. Enucleation of mouse MⅡoocytes can be performed under an inverted microscope equipped with Normarski or Hoffmann optics. It has been demonstrated that the mouse oocytes can be enucleated accurately with the treatment of appropriate sucrose. The main objective of the dissertation was to improve and modify the experimental procedures of mouse nuclear transfer and pronuclear transplantation, respectively, and try to establish new possible methods for mouse cloning and pronuclear transplantation. 1. Studies on mouse nuclear transfer with compacted morula blastomeres as donors In this study, C57BL/6 mouse morulae blastomeres and Kunming white mouse MⅡoocytes were used as donors and recipients, respectively, to investigate the effects of different sucrose concentration on the manipulation time of nuclear transfer, and the in vitro development of reconstructed embryos. The experimental results demonstrated that: 1) when the oocytes were enucleated with 1%, 2% and 3% sucrose treatment, respectively, the enucleation rates could be up to 100%, but the mean nuclear transfer manipulation time of every oocyte was different, they were 181.4±5.0s, 125.9±3.9s and 114.2±2.3s, respectively, and had highly significant difference (P<0.01); 2) when the oocytes were treated with different sucrose concentration, the fusion rates of the reconstructed embryos were 61.7%, 61.4% and 57.2%; and the developmental rates of morula/blastocyst were 24.8%, 28.7% and 26.2%, respectively; both of them had no significant difference (P>0.05). When 457 nuclear transfer embryos of pseudopronucleus and 2-cell stages were transplanted into the oviducts of 27 foster mothers. Among them, one foster mother became pregnant, and 3 dead, full-term fetuses were obtained. This result suggested that enucleated oocytes with sucrose treatment could support the embryonic cell NT embryos development to term in vivo. 2. Studies on mouse nuclear transfer with adult male mouse ear fibroblast cells as donors In this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming (white) mouse MⅡoocytes were used as donors and recipients, respectively, to investigate the effects of different passages of donor cells and electrofusion times on the in vitro development of NT reconstructed embryos, when the oocytes were treated with 3% sucrose. The experimental results demonstrated that: 1) when the mouse ear fibroblast cells of 2-4, 5-7 and 8-10 passages were used as donors, respectively, to produce NT embryos, the different passages of donor cells had no significant effects on the in vitro development of reconstructed embryos, and their developmental rates of morula/blastocyst were 15.2%, 13.3%, and 14.0%, respectively, which had no significant difference (P>0.05); 2) when the reconstructed embryos were electrofused, the fusion rates of the first electrofusion and the second electrofusion were 53.5% and 59.8%, respectively, which had no significant difference (P>0.05). But the developmental competence of reconstructed embryos with only once electrofusion was significantly higher than those with twice electrofusions, and their developmental rates of 2-cell were 65.7% and 18.4%, and those of 4-cell were 36.4% and 6.1%, respectively, which were highly significant (P<0.01), and the reconstructed embryos with twice electrofusions could not develop beyond 4-cell. When 361 nuclear transfer embryos of pseudopronucleus and 2-cell stages were transplanted into the oviducts of 34 foster mother mice, each receiving about 8-12 embryos, in the end, one cloned C57BL/6 pup was obtained from one recipient. 3. Studies on mouse pronuclear transplantation In this study, the inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain mouse (black). First, the MⅡoocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by the capacitated sperms of C57BL/6 mouse in vitro, thus, at least one male pronuceus derived from C57BL/6 mouse formed in the enucleated oocytes of Kunming mouse. At the same time, MⅡoocytes of Kunming mouse were parthenogenetically activated using SrCl2 solution, which did not contain cytochalasin B, and the oocyte extruded the second polar body and formed a female pronucleus. Finally, we removed the male pronucleus derived from C57BL/6 sperm, and transplanted it into the perivitelline space of parthenogenetically activated one female...
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