Feasibility of Nuclear Transfer Techniques Fit to the Generation of Transgenic-Cloned Livestock Using Donor Nucleus from Adult Somatic Cells

• Main Authors: Shen Perng-Chih, 沈朋志
• Other Authors: Cheng Teng-Kuei
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/13181238365162878364

博士 === 國立臺灣大學 === 畜產學研究所 === 91 === The work described in this thesis was undertaken to evaluate the feasibility of nuclear-transfer-based techniques, using adult somatic cells as donor nuclei, for generation of cloned-transgenic calves and kids. Efforts were made to verify several potential factors for ensuring the NT-embryos to be generated with a better survival, both in vitro and in vivo, during their subsequent development. There were the strength of electrical field for nuclear fusion and activation, methods for chemical activation of the cloned embryos, breeds that donor cells and recipient eggs derived from, and the opportune moment for executing the enucleation. The changes of DNA methylation and expression pattern of endogeneous genes in the cloned embryos were also investigated. Results from experiments conducted to evaluate the factors affecting the efficiency in production of NT-embryos and their subsequent surviving capacity appeared that: 1. Increase of the field strength from 1.67 to 2.33 kV/cm for electrical activation not only resulted in much higher cleavage rates of the cloned caprine-embryos (19.6 vs 65.6%, p<0.01) but also ensuring a better pregnancy rate (0 vs 28.6%) after transfer of them into recipient does. Three cloned kids were born under this electrical activation condition. 2. Higher proportion of the cloned bovine embryos developed to blastocyst stage when activation had been performed 4 h post the electrofusion than that of activation and electrofusion been performed simultaneously (26.2% vs. 5.1%, p<0.01). 3. There was no difference in the proportion of the NT-embryos developing to blastocyst stage (averaged around 30.5 ~ 42.2%) among methods used for activation, including the electrical- activation and the chemical-activation using ionomycin, calcium ionorphore (A23187) combined with 6-dimethylaminopurine (6-DMAP), respectively. 4. The NT-embryos derived from recipient oocytes of Taiwan native yellow cattle had a better blastocyst rate then those recipient oocytes derived from Holstein cattle (p＜0.05). Transfer of the NT-embryos into estrus-synchronized fosters gave birth to three cloned calves. All of them were derived from oocytes of Taiwan native yellow cattle with two donor nuclei from Holstein and one from Taiwan native yellow cattle. 5. The effects of enucleation timing on development of NT-embryos showed that blostocyst rate were not different among the NT-embryos, which were produced by either enucleation before activation (EBA) or enucleation after activation (EAA) (46.3% vs. 44.7 %, p＞0.05). 6. The DNA methylation rates of NT-embryos produced by EBA (60.2%) or EAA (55.7%) methods were significantly higher than those embryos produced from bovine oocytes matured and fertilized (24.1%)(p＜0.05). 7. The blastocyst rates of NT-embryos produced from donor cells treated with serum starvation and trichostatin A (TSA) (37.5% and 29.7%, respectively) were significantly higher than those treated with 5-Aza-2’-deoxycytidine (5-Azad) (8.2%) and 5-Azad＋TSA (16.7%)(p＜0.05). The methylation rate of satellite I gene of NT-embryos produced from TSA treated donor cells (46.1%) was lower than those from serum starvation and 5-Azad＋TSA treatments (65.8% and 72.2%, respectively). In addition, two foreign genes, the green fluorescence protein (GFP) and the human coagulation factor Ⅷ (hFⅧ), had been transfected into bovine ear fibroblast cells before they were subjected to the nuclear transfer for generation of cloned-transgenic calves. The result showed that the GFP expression rate of the ear fibroblast cells, which had been passaged after transfection, was higher than those without passage (22.3% vs.14.0%)(p<0.01). Transfection of GFP and hFⅧ genes into the donor nuclei did not affect the developmental capability of NT-embryos ( p＞0.05). One hundred and seventy cloned embryos carrying hFⅧ transgene were transferred into 70 recipient cows. Eight of them became pregnant, with only one gave birth to a healthy transgenic-cloned calf although only a partial fragment of the hFⅧ transgene gene was integrated within the genome of this cloned calf according to results based on the PCR analysis. In conclusion, this study has successfully produced three cloned goats and four cloned calves by nuclear transfer. Unfortunately, one of three cloned kids and three of four cloned calves merely survived for 1-6 days post the neonatal. The two kids and one calf survived are 12- and 4- months of age at writing, respectively. Although the gene transfection technique and screening model of donor cells have been established in this study, however, we are failure to obtain a complete sequence of hFⅧ transgene in the genome of the transgenic cloned calf. Further studies are required to gain those particular clones of donor cells harboring the intact transgene before being subjected to the nuclear transfer and this will ensure the desired transgenic offspring to be successfully cloned.