Sox2基因对绵羊体细胞克隆不同供体细胞表观遗传修饰的影响

发布时间：2018-04-26 06:06

  本文选题：绵羊 + Sox2基因　； 参考：《内蒙古农业大学》2016年硕士论文

【摘要】：哺乳动物体细胞克隆技术自诞生以来,发展迅速且日益趋于成熟,但体细胞克隆效率低始终是制约其在畜牧业生产中应用与发展的瓶颈。核不完全重编程及供体细胞的类型与克隆效率密切相关。表观遗传修饰中DNA甲基化、组蛋白乙酰化是影响核重编程的关键因素。研究证实,sox2基因对维持细胞多能性及胚胎着床后的发育起关键的作用,以高表达sox2基因的神经干细胞为核供体进行核移植时具有较高的克隆效率。为此,本研究以内蒙古地区优势畜种绵羊为实验对象,选择绵羊骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells, BMSCs)、脂肪间充质干细胞(Adipose-derived Stromal Cells,ADSCs)和皮肤成纤维细胞(Sheep Epidermal Fibroblast Cells, SEFCs)为核供体,经电转染将sox2基因导入BMSCs和ADSCs和SEFCs,检测sox2基因对三类核供体细胞DNA甲基转移酶1(DNA Methyl transferase 1，，DNMT1)和组蛋白去乙酰化酶2(Histone Deacetylase 2, HDAC2)基因的表达水平,分析了sox2基因对三类核供体细胞表观遗传修饰的影响。同时以导入sox2基因的BMSCs为核供体构建重构胚,以SEFCs核供体构建的重构胚为对照,分析了sox2基因对重构胚表观遗传修饰及发育能力的影响。主要研究结果如下：(1)分离得到了绵羊BMSCs, ADSCs和SEFCs细胞系,三类细胞生长曲线均呈“S”型;经RT-PCR检测,BMSCs和ADSCs均表达了干细胞特异因子Sox2、Oct4和Nanog基因。(2)构建了pEGFPS-N1-5bx2真核表达载体,对BMSCs、ADSCs和SEFCs三类细胞进行了电转染,经筛选得到过表达sox2基因细胞株,经实时定量PCR检测,三种过表达sox2基因细胞株中的DNMT1和HDAC2基因表达量表现出不同程度的下降,DNNT1分别下降多少68%、25%、43%；HDAC2下降了15%左右。(3)以过表达sox2基因BMSCs为核供体,构建重构胚159个,以SEFCs为核供体构建重构胚182个。检测两类重构胚中DNMT1和HDAC2的表达量,过表达sox2基因BMSCs为供体的克隆胚比以SEFCs构建的克隆胚中DNMT1和HDAC2的表达量低50%左右,由此推测sox2基因的过表达可能有利于克隆胚核的重编程,进而促进重构胚的发育。以上结果对阐明sox2基因影响不同核供体细胞表观遗传修饰的机制奠定了必要的基础,为通过选择及对不同核供体细胞进行遗传修饰从而提高体细胞克隆效率提供了新的方法,进而为日后利用转基因体细胞克隆技术进行优质绵羊种畜的改良及高效快繁提供了科学依据。
[Abstract]:Since the birth of mammalian somatic cell cloning technology, it has developed rapidly and become more and more mature, but the low efficiency of somatic cell cloning has always been the bottleneck of its application and development in animal husbandry. Incomplete nuclear reprogramming and donor cell types are closely related to cloning efficiency. DNA methylation and histone acetylation are key factors affecting nuclear reprogramming in epigenetic modification. It has been proved that sox2 gene plays a key role in maintaining the pluripotency of the cells and the development of embryos after implantation. The neural stem cells with high expression of sox2 gene are used as nuclear donors for nuclear transplantation with high cloning efficiency. Therefore, in this study, the dominant cattle sheep in Inner Mongolia were selected as nuclear donors, including bone Marrow-derived Mesenchymal Stem Cells, BMSCs, adipose mesenchymal stem cells (Adipose-derived Stromal CellsCellsCellsASCs) and skin fibroblasts (Sheep Epidermal Fibroblast Cells, SEFCs) from sheep bone marrow mesenchymal stem cells (BMSCs), adipose mesenchymal stem cells (Adipose-derived Stromal cells) and skin fibroblasts (Sheep Epidermal Fibroblast Cells, SEFCs). Sox2 gene was transfected into BMSCs, ADSCs and SEFCs.The expression of sox2 gene on DNA methyltransferase 1(DNA Methyl transferase 1 (DNMT1) and histone deacetylase 2(Histone Deacetylase 2 (HDAC2) in three kinds of nuclear donor cells was detected. The effect of sox2 gene on epigenetic modification of three nuclear donor cells was analyzed. At the same time, the effect of sox2 gene on the epigenetic modification and developmental ability of reconstructed embryos was analyzed by using BMSCs as nuclear donor and SEFCs nuclear donor as control. The main results were as follows: (1) Sheep BMSCs, ADSCs and SEFCs cell lines were isolated, and the three cell growth curves were all "S" type, and the pEGFPS-N1-5bx2 eukaryotic expression vector was constructed by RT-PCR detection of the stem cell specific factor Sox2Oct4 and Nanog gene. After electrotransfection of BMSCs cells and SEFCs cells, overexpression of sox2 gene was obtained and detected by real time quantitative PCR. The expression of DNMT1 and HDAC2 genes in three kinds of over-expressed sox2 gene cell lines showed a decrease of 68%, and that of DNNT1 decreased by about 15%, respectively. The over expressed sox2 gene BMSCs was used as nuclear donor to construct 159 reconstructed embryos. 182 reconstructed embryos were constructed with SEFCs as core donor. The expression of DNMT1 and HDAC2 in two kinds of reconstructed embryos was detected. The expression of DNMT1 and HDAC2 in cloned embryos with overexpression of sox2 gene BMSCs was about 50% lower than that of cloned embryos constructed with SEFCs, which suggested that the overexpression of sox2 gene might be beneficial to the nuclear reprogramming of cloned embryos. Furthermore, it promotes the development of reconstructed embryos. These results provide a necessary basis for elucidating the mechanism of sox2 gene affecting epigenetic modification of different nuclear donor cells, and provide a new method for improving the cloning efficiency of somatic cells through selection and genetic modification of different nuclear donor cells. It provides a scientific basis for the improvement and efficient rapid propagation of high quality sheep breeds by using transgenic somatic cell cloning technology in the future.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q813

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