【作者】 云彦；

【导师】 雷安民；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2010， 硕士

【摘要】 自Dolly诞生以来,多种哺乳动物已被成功克隆,但该技术仍然存在着克隆效率低下的问题。现在普遍认为克隆效率低是由核移植过程中受体卵母细胞对供体细胞核的不正确或不完全重编程所致,故而提高卵母细胞质量、并促进其对供体细胞核的完全重编程是提高核移植效率、完善该技术的重要突破点。卵特异型连接组蛋白(H1foo),作为一种仅存在于卵母细胞及早期胚胎中的连接组蛋白,对染色体结构及基因表达调控有重要功能。在小鼠上,H1foo在卵母细胞中的表达已被证明是成熟分裂所必需的。在爪蟾及小鼠核移植过程中,H1foo对体细胞型连接组蛋白的快速置换现象被认为是供体核发生重编程的必要过程。本研究以牛卵母细胞为材料,分别对H1foo在卵母细胞成熟及核移植过程中重编程的作用进行了探讨。首先,我们通过向成熟过程中的卵母细胞显微注射H1foo siRNA或体外合成的H1foo mRNA来干扰或超表达H1foo,以确定其在卵母细胞成熟中的作用。同时,我们以RFP(red fluorescent protein)-H1e(somatic linker histone H1e)稳定转染或正常的成纤维细胞为核供体,以超表达H1foo-Venus或正常的成熟卵为受体进行核移植试验,电融合后通过荧光观察以确定H1foo对H1e的置换过程。试验结果如下:1.本试验通过自制的卵母细胞裂解液,利用RT-PCR技术成功从微量(5个)牛卵中克隆得到H1foo CDS全长序列。荧光显微镜观察、RT-PCR、Western Blotting检测表明构建的真核表达载体pH1foo-Venus能够在细胞中正确表达及定位。将H1foo-Venus体外转录为mRNA后显微注射牛卵母细胞,其表达产物能准确定位于卵母细胞与极体的染色体上。同时我们利用脂质体共转染的方法来筛选H1foo的有效siRNA序列,结果显示siRNA-3干扰效率最高,可分别在mRNA和蛋白水平上沉默H1foo表达的90%和60%。最后我们通过显微注射的方法将H1foo mRNA或siRNA-3注射至成熟过程中的牛卵中,结果显示:与未注射对照组相比(71.2%),H1foo-Venus注射组和siRNA-3注射组的卵母细胞成熟率分别为88.7%和36.0%,表明H1foo超表达能显著促进卵母细胞成熟,而干扰其表达则明显降低卵母细胞的成熟率。2.由于体细胞型连接组蛋白H1e为单一外显子基因,故我们以牛成纤维细胞基因组DNA为模板成功克隆得到H1e CDS全长序列。荧光显微镜观察、RT-PCR检测表明构建的真核表达载体pdsred1-H1e能够在细胞中正确表达及定位。然后将其转染牛皮肤成纤维细胞,经G418连续筛选得到稳定表达H1e-RFP的细胞株,检测表明该细胞株正确表达H1e-RFP。最后我们以pDsRed1-H1e稳定转染或正常的成纤维细胞为核供体,以超表达H1foo-Venus或正常的成熟卵为受体进行核移植试验,结果显示:(1)以正常的MII期去核卵和稳定表达H1e-RFP的成纤维细胞进行核移植,电融合后5h可观察到红色荧光开始弥散变弱,8h后红色荧光即H1e从供体核上完全消失。(2)以表达H1foo-Venus的MII期去核卵和正常的成纤维细胞进行核移植,电融合后5h可观察到绿色荧光即H1foo已集中于供体细胞核上,8h后绿色荧光变强。(3)以表达H1foo-Venus的MII期去核卵和稳定表达H1e-RFP的成纤维细胞进行核移植,电融合后可观察到红色荧光即H1e的逐渐消失和绿色荧光即H1foo在供体核上的不断积累。8h后,红色荧光完全消失。本试验结果表明:H1foo表达能促进牛卵母细胞成熟,其表达是卵母细胞成熟所必需的;与在小鼠上研究结果一致,在牛体细胞核移植过程中,组蛋白互换现象同样发生。这说明H1foo在卵母细胞成熟及核移植过程中的作用并非仅局限于啮齿类动物,而可能是在哺乳动物上高度保守的,这为进一步探究核移植重编程的机理提供了相关证据。更多还原

【Abstract】 Since the cloned sheep Dolly was generated using somatic cell nuclear transfer (SCNT), a number of mammalian species have been successfully cloned. However, the success rate is still very low and the relevant mechanisms involved in reprogramming are largely unknown. The main cause of these problems may be attributed to the uncompleted reprogramming of the donor genome. Therefore, trying to improve the quality of oocyte and promote the efficient reprogramming of donor nuclear will response well to these problems. As one of the linker histones, the expression of oocyte specific linker histone (H1foo) is restricted in oocytes and early embryos, which makes them have a specific chromosome structure and gene expression pattern. In mouse oocyte, H1foo is indispensable for meiotic maturation. During the process of SCNT either amphibians Xenopus or mammalians mouse, the rapid linker histone H1 transitions can be observed, which is considered to be essential for donor nuclear reprogramming.In our study, the bovine oocyte is used to determine the function of H1foo during the process of oocyte maturation and SCNT. To examine the effect of H1foo expression on oocyte maturation, we microinjected effective siRNA against H1foo or H1foo mRNA transcripted in vitro into the maturing bovine oocytes. Meanwhile, to determine whether the replacement occurs during bovine nuclear transfer, we performed the nuclear transfer, using RFP-taged somatic linker histone H1e stable transfected donor cells and MII oocytes expressing exogenous fusion protein H1foo-Venus. After fusion, the replacement of linker histones can be real-time monitored under fluorescence microscope. The results of the project are as follows:1. We successfully established an improved RT-PCR, which is efficient for gene cloning from trace oocytes. The complete coding sequence of H1foo was cloned from 5 bovine oocytes, which was totally identical with previous reports. After the recombinant plasmid pVenus-H1foo was transfected into Hela cells, H1foo was efficiently expressed identified by Fluorescence microscopy observation、RT-PCR and Western Blotting. H1foo was localized accurately on chromosomes of the oocyte and polar body after H1foo mRNA transcripted in vitro was microinjected into bovine oocytes. Then we co-transfected the pVenus-H1foo and the 4 siRNAs into Hela cells respectively to screen a valid candidate sequence. Fluorescence microscopy observation、RT-PCR and Western Blotting indicated that siRNA-3 had a higher efficiency, which decreased the H1foo expression 90% and 60% in mRNA and protein level, respectively. Finally, we microinjected H1foo mRNA or siRNA-3 into the maturing bovine oocytes, and the results showed that compared to un-injected control group (71.2%), the maturation rate of H1foo-Venus injected group and siRNA-3 injected group is 88.7% and 36.0%, respectively, which indicated that knowdown of H1foo significantly impaired oocyte maturation, and overexpression of H1foo can apparently promote oocyte maturation.2. Since there are no introns existing in linker histone H1e, we successfully amplified the complete coding sequences of H1e from genome DNA of the bovine fibroblast cells. Fluorescence microscopy observation and RT-PCR detection showed that the recombinant plasmid pdsred1-H1e was efficiently expressed in Hela cells. The plasmid pdsred1-H1e was transfected into bovine skin fibroblast cells, and the stable transfected cell lines were successfully established after 3-4 weeks of selection with neomycine (G418). RT-PCR and Western Blotting showed that H1e-RFP mRNA and fusion protein were expressed in these cell lines, and the fluorescence was localized accurately on nuclear. Finally, to determine whether the replacement occurs during bovine nuclear transfer, the different treated fibroblasts were injected into the perivitelline space of different treated enucleated MII-arrested oocytes. Under fluorescence microscope, release of H1e in the donor nucleus, acquisition of H1foo by donor chromosome and H1foo-to-H1e transition were observed. However, unlike that in mouse, the replacement in bovine was slower. Subdued but diffused red fluorescence (H1e) in donor nucleus was still detectable, while green fluorescence (H1foo) has incorporated into the donor chromosome at 5 h, and the complete replacement occurred until 8 h after fusion.Our results indicate that overexpression of H1foo can apparently promote bovine oocyte maturation, while knowdown of H1foo significantly impaired oocyte maturation, which is agreement with that in mouse. During the bovine SCNT, the linker histones transition was observed, which demonstrates that the phenomenon perhaps is sufficiently conserved among different species rather than specific just in rodent oocytes. Our findings provided further evidence for its important role in nuclear reprogramming.更多还原