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Yang, Feikun (2005): Nuclear Transfer in Rabbits with Different Types of Donor Cells. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Production of cloned transgenic rabbits from somatic nuclei was hampered since the nuclear transfer technique had extremely low rate of success. The objectives of the present study were to evaluate the developmental potential of cloned embryos from different types of donor cells and to produce cloned rabbits from cultured somatic cells. In vivo matured MII oocytes were treated with demecolcine to induce small cytoplasmic extrusion containing all maternal chromatin. Enucleation was carried out by the removal of the cytoplasmic extrusion. Various types of cells were used as nuclear donors. After electric pulses, embryonic blastomeres were introduced into enucleated oocyte cytoplasm having escaped from meiotic arrest by electric pulses followed by treatment with 6-DMAP/CHX. Nuclei from in vitro cultured cumulus cells (RCCs) and fetal fibroblast cells (RFFs) either grown to confluency or deprived of serum from the culture medium (starved-RFFs) or treated with sodium butyrate (NaBu-RFFs) were introduced into enucleated oocytes by electric pulses, and the reconstructed oocytes were activated by electric pulses followed by 6-DMAP/CB. The developmental potential of nuclear transfer embryos was assessed by the rates of in vitro blastocyst formation and in vivo development to term. The results showed that the chemically assisted enucleation protocol was successfully used with MII oocytes since high extrusion and enucleation rates were obtained. High cleavage rates were obtained in any type of nuclear donors (87% for embryonic blastomere, 78% for RCC, 93% for confluent RFF, 87% for starved-RFF, 95% for NaBu-RFF). However, higher blastocyst rates were obtained from embryonic blastomere and NaBu-RFF derived embryos (59% and 49%, respectively) than that from other type of somatic donors (RCC: 32.3%, confluent RFF: 33.1%, starved-RFF: 32.5%). When cloned embryos were transferred into pseudo-pregnant mothers, live offspring derived from embryonic blastomeres and day 27-fetuses derived from cultured RCCs were produced. No full-term development was achieved when RFFs were used as nuclear donors. Additionally, When 4-10-cell stage cloned embryos were aggregated with one or two blastomeres of 6-12-cell stage in vivo fertilized embryos, although a greater proportion of aggregated embryos developed to blastocysts as compared with cloned, non-aggregated embryos (78% vs. 61%, P<0.05), out of four conceptuses in resorption and one normal fetus, none had the same genetic background as donor cells. However, the aggregation of 4-10-cell stage cloned embryos with a single blastomere of 6-12-cell stage parthenogenetic embryos initiated more pregnancies than that in cloned-in vivo fertilized embryos (67% vs. 0% and 27%; P>0.05) and two of them developed to term, although the development to blastocyst was similar to that of cloned embryos (58% vs. 61%, P>0.05), and even lower than that of cloned-in vivo fertilized embryos (58% vs. 78%, P<0.05). One of the two newborns, an overgrowth pup, died of respiratory failure within one hour while another one lived about two weeks until an accidental death. Genotypic analyses confirmed that both pups are clones derived from fetal fibroblasts. Overall, results from our study show that embryos cloned from embryonic cells had higher developmental potential than those cloned from somatic cells. Embryos cloned from somatic cells did not differ in their developmental capacity. After successive optimisation of all steps of nuclear transfer, we produced the first cloned rabbits from cultured somatic cells including cumulus cell and fetal fibroblast cells. These achievements will open a new way to more wide use of rabbit model for basic and applied research.