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Study on Related Problems about Oocytes TII Enucleation in the Procedure of Pig Cumulu Cells Nuclear Transfer

Author: HanShuBiao
Tutor: LiYueMin
School: Southwestern University
Course: Cell Biology
Keywords: Pig Oocytes Late to go nuclear Nuclear transfer Nucleoplasm
CLC: Q813
Type: Master's thesis
Year: 2007
Downloads: 71
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Abstract


In the process of nuclear transfer, donor cells with the receptor cytoplasmic two important components of the reconstructed embryos, the receptor cytoplasmic crucial to the reconstructed embryos nuclear gene reprogramming and embryonic development. Currently, most researchers are using as the receptor cytoplasmic M Ⅱ oocytes. However, the method is to extract a large number of cytoplasmic go nuclear, but also irradiated with ultraviolet light to go nuclear to check whether completely reconstructed embryos thus the impact of cell damage. In recent years been used to extract the second polar body and around a small amount of nuclear cytoplasmic activation of M Ⅱ oocytes wait until after the discharge of the second polar body to go nuclear, also known as the T Ⅱ period to go nuclear. Since such a method decimated little cytoplasm, and does not require the UV irradiation, and therefore less damage to cells. This method in many laboratories to check the pig somatic cell nuclear transfer reconstructed embryo development but oocytes T Ⅱ period, the domestic no reports. In this thesis, and pig oocytes T Ⅱ period to nuclear methods and effects were compared on this basis also pig oocyte cell T-Ⅱ period to go nuclear method to produce nuclear transfer embryonic program is optimized to promote the application of the technology to produce pig somatic cell nuclear transfer embryos to provide some data and experience. The experiments on the basis of the original pig somatic cell nuclear transfer technology, the development of several factors on the impact of pig oocytes T Ⅱ period to go nuclear reconstructed embryos (oocytes pre-activate the program, the role of nucleocytoplasmic time after nuclear transfer and nuclear donor cell pretreatment methods) the following experiment was conducted. 1, of porcine oocytes T II period pre-activated material selection. In this experiment, the second polar body discharge ratio as an indicator to select different concentrations of three substances, acting on pigs mature oocytes observed statistical pre-activation effect. Rate of 5%, 7% and 10% ethanol concentration pre-activated porcine oocytes discharge of the second polar body was 20.83% (25/120), 25.00% (30/120) and 22.50% (27/120); using concentration 4μg/mL, 6μg/mL, 8μg/mL and 10μg/mL of Actidione the pre-activation of porcine oocytes after the second polar body were: 16.67% (20/120), 19.17% (23/120), 25.83% (31/120) and 20.83% (25/120); concentration 5μmol/mL, 10μmol/mL, 15μmol/mL and 20μmol/mL calcium ionophore A23187 (CaA) pre-activated porcine zona mother cell after the second polar body is 54.17% (65/120), 58.33% (70/120), 64.17% (77/120) and 57.50% (69/120), the best 15μmol/mL group between the various concentration difference was not significant (p> 0.05). Comparing the above three substances pre-activated porcine oocytes effect found 15μmol/mL the CaA very significantly better than the other two substances (p <0.01). 2 different receptor oocyte cytoplasm of porcine somatic cell nuclear transfer. In this study, in order to reconstruct the embryo cleavage rate and 4 - cells over embryonic development rate of detection of statistical indicators, enucleated oocytes were enucleated M II medium-term and T Ⅱ period as the receptor cytoplasmic cumulus cells by serum starvation building reconstructed embryos as nuclear donors of two receptors cytoplasm pig somatic cell nuclear transfer effect. The results show that: the M Ⅱ medium-term enucleated oocyte the nuclear receptor cytoplasmic cleavage rate and 4 - cells over embryonic development were higher than the T Ⅱ of enucleated oocytes (31.67% vs24.17% 14.17 % vs10.83%), a significant difference (p <0.05). M II medium-term enucleated oocytes more suitable as nuclear transplantation receptor cytoplasmic. 3 the nucleoplasm role porcine somatic cell nuclear transfer. Electric fusion shifts cumulus cells into enucleated oocytes, the receptor cytoplasmic nuclear donor cells in T Ⅱ period were incubated 0h, 1h, 2h, 3h, 4h, 5h, 1.3kV/cm , 80μs, single pulse electrical activation of reconstructed embryos, in order to reconstruct the embryo cleavage rate and embryonic development - cells above the rate of detection of statistical indicators, of comparing different nucleoplasm incubation time on the effects of nuclear transfer. The results show that: nucleocytoplasmic incubated 4h can get a better effect of nuclear transfer, the cleavage rate (35.83% (45/120)) was significantly higher than the other groups (22.50%, 25.83%, 26.67%, 28.83%, 30.83% , p <0.05). - Cells embryonic development rate and incubation 3h group differences not significant (12.50% vs12.50%, p> 0.05). But compared to the other groups differences significant (12.50% vs. 4.17%, 9.17%, 10.00%, 7.50%, p <0.05). T Ⅱ period enucleated oocytes as a nuclear receptor cytoplasmic, nuclear and cytoplasmic incubated for 4h after the the supplementary electrical activation can improve the rate of porcine somatic cell nuclear transfer reconstructed embryos. 4 different donor cells pretreatment methods on the effects of nuclear transplantation. In this study, the cumulus cells after serum starvation as a control group, respectively, with 7% ethanol and calcium ionophore A23187 (5μmol / L) pretreatment donor cells fused nuclear transfer reconstructed embryos, nucleoplasm incubated for 4h reactivation comparing the two pretreatment methods of nuclear transfer reconstructed embryos. The results show that: with the CaA pretreatment group of nuclear transfer reconstructed embryos cleavage rate (37.50% vs30.83%) and 4 - the cells embryo development rate (15.83% vs10.83%) were higher, the difference is significant (p <0.05). The cleavage rate slightly higher than the the ethanol pretreatment group nuclear reconstructed embryos cleavage rate (37.50% vs34.16% of p> 0.05), but 4 - cells over embryonic development rate significantly higher than ethanol pretreatment group (15.83 % vs9.17%, p <0.05). Thus, T II period to the oocyte nuclear as a nuclear receptor cytoplasmic and nuclear donor cells pretreated with calcium ionophore nucleoplasm incubated for 4h and then to activate reconstructed embryos, which is conducive to the development of nuclear transfer reconstructed embryos. In summary, T Ⅱ of enucleated porcine oocytes as a nuclear receptor cytoplasmic, the most suitable procedure is: a concentration of 15μmol/mL calcium ionophore handling oocytes 5min, and then extracting the second pole body and around a small amount of cytoplasm to go nuclear. T Ⅱ period 5μmol/mL calcium ionophore pretreatment into enucleated porcine oocyte perivitelline parameters 1.0kV/cm, 80μs, single pulse electric fusion method shifted donor nucleus donor cells build reconstructed embryos, after which, the nucleoplasm incubated for 4h, last used parameters for 1.3kV/cm 80μs, single pulse electrical activation of reconstructed embryos can significantly improve the ratios of nuclear transfer embryos. Simplify the operation of nuclear transplantation program.

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