【作者】 刘凤军；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2007， 博士

【摘要】 体细胞核移植(SCNT)已经成功应用于转基因动物生产、制作乳腺生物反应器及治疗性克隆等前沿领域。尽管这项技术已经取得了很大成果,但它的总体效率仍旧很低,所以SCNT技术体系仍需优化改进。为了提高体细胞核移植法生产转基因动物的生产效率,本研究对山羊SCNT方案进行了优化,并对利用优化后的SCNT方案生产转人乳铁蛋白(hLF)基因山羊的几个关键技术环节进行了系统地研究。一、山羊体细胞核移植方案的优化1、本实验分别在山羊卵母细胞成熟培养液(OM)及胚胎培养液(mSOFaa)中添加1 % ITS(v/v)。结果添加ITS到成熟液中,对卵母细胞成熟率没有明显提高,但显著提高了激活后孤雌胚胎的囊胚率(58.06 % vs. 48.19 %,P<0.05);添加ITS到胚胎培养液中,显著提高了山羊孤雌胚胎的囊胚率(68.30 % vs. 56.82 %,P<0.05)。结果表明,ITS对山羊卵母细胞体外成熟及胚胎早期发育均有促进作用;另外,ITS可以代替卵母细胞成熟培养液和胚胎培养液中的血清,作为无血清培养体系,用于相关研究。2、为优化并提高山羊SCNT的电融合效率,本实验对微电极融合法进行了改进,顶端直径分别为15μm(A)、100μm(B)、200μm(C)的3种微电极分别以A+A、A+B、B+B和C+C四种不同的组合方案与显微操作仪相连,分别对颗粒细胞(GC)、胎儿成纤维细胞(FF)和乳腺上皮细胞(MGE)构建的重组体进行电融合实验,传统槽式融合法设为对照。同时,在每种组合方案的实验中对比了增加供受体膜接触面紧密性(挤压组)和不挤压组(对照组)对融合率的影响。结果,微电极非挤压组的融合率与槽式融合法无显著差异。在挤压组的4种组合方案中,A+A(94.92 %)和A+B(92.23 %)的融合率显著高于B+B(83.01 %)和C+C(83.90 %)。与槽式融合法相比,A+A挤压融合方案显著提高了重组体的融合效率(GC:72.15 % vs. 89.04 %;FF:77.11 % vs. 94.64 %;MGE:51.23 % vs. 78.02 %)。结果表明,应用直径较细微电极并在融合时轻轻地挤压以增加供受体膜间的紧密性能够显著提高山羊SCNT的电融合效率。3、为了提高乳腺上皮细胞(MGE)核移植的电融合效率,本实验以PHA处理融合前的核质重组体,并用微电极挤压融合法(pMEF)来融合MGE重组体,PHA未处理组及槽式融合法作为对照。实验通过测定PHA对孤雌激活胚发育力的影响来确定PHA的最佳使用剂量及作用时间,结果,100μg/mL和20 min是合适的作用浓度和时间,用于后面的核移植实验;PHA处理重组体显著提高了槽式融合法的融合率(52.82 % vs. 74.02 %,P<0.05),对pMEF的融合率也有提高(72.68 % vs. 78.14 %,P>0.05);与未处理组相比,PHA处理对克隆胚胎卵裂率及囊胚发育率没有显著影响;PHA处理与pMEF联合把总克隆效率从6.67 %提高到12.59 %。结果表明,在融合前以适宜浓度的PHA处理MGE构建的重组体,并以pMEF进行电融合能显著提高MGE核移植的融合率,且不影响克隆胚的体外发育力,从而提高了MGE核移植的整体效率。二、单个转染阳性细胞快速扩大培养技术体系的建立在96孔板中,将转染hLF基因乳腺特异性表达载体pBLM-C1的单个FF和MGE细胞进行克隆。由于哺乳动物的单个细胞在新鲜培养基中不容易生长,本实验使用了适应性条件培养基(v/v:0 %、50 %和100 %)对单个转染细胞进行细胞单克隆的制备;进而把转染细胞单克隆与非转染细胞共培养进行扩大培养,neo基因被用于筛选基因,以PCR鉴定转染细胞基因组DNA。结果:(1)100 %适应性条件培养基能够显著提高细胞单克隆存活率(FF:21.430% vs. 3.70 %;MGE:13.64 % vs. 0.00 %);(2)转染细胞单克隆与非转染细胞共培养,显著提高了转染细胞单克隆扩大培养后的比率(FF:53.33 % vs. 10.00 %;MGE:33.33 % vs. 6.670 %),且明显缩短了扩大培养汇合时间(约20 d);(3)PCR鉴定结果表明,上述方法获得的克隆细胞整合有hLF目的基因。本实验为分离转基因细胞、理想载体的插入及诊断提供了一种可靠的方法,并能降低转基因动物生产的费用及时间。三、转染阳性乳腺上皮细胞的诱导表达及产物蛋白检测为了评价载体效率、外源蛋白表达情况及表达蛋白大小,本实验用含5 mg/L胰岛素、5 mg/L催乳素、1 mg/L氢化可的松的DMEM/F12培养液对转染阳性乳腺上皮细胞进行诱导培养,每6 h收集1次上清液。上清液浓缩后用Western blot检测,结果显示有目的蛋白表达,分子量为42 ku。四、用优化的核移植方案生产转hLF基因山羊本实验利用优化的核移植方案将转染阳性细胞(FF和MGE)移植到山羊MII期去核卵母细胞内,经电融合、激活、体外培养后,所获克隆胚胎一部分用于PCR检测外源基因,一部分(2-8细胞期和槡囊胚期)移植到同期发情受体山羊输卵管和子宫角内。结果,PCR检测表明,外源基因已整合到转基因克隆胚中;共移植401枚处于不同发育时期的转基因克隆胚胎到43只同期发情的受体山羊体内,结果共有11只妊娠,但都在妊娠前3个月流产;对移植记录进行统计分析发现,每只受体移植胚胎平均数在10枚以下的实验组,其妊娠率在20.00 %～26.67 %之间,而胚胎平均数高于20枚的实验组妊娠率高达40.00 %,由MGE细胞构建的转基因克隆的妊娠率与FF细胞无显著差异(20.00 % vs. 21.43 %),输卵管移植(2-8细胞期胚胎)与子宫角(槡囊期胚胎)的妊娠率无明显差异(26.47 % vs. 25.00 %)。结果表明,本研究优化的技术体系可以用于生产转基因克隆动物。更多还原

【Abstract】 Somatic cell nuclear transfer (SCNT) so far has been successfully used in producing transgenic animal, making mammary gland bioreactors, and therapeutic cloning etc. To date, although some achievements were reported, the overall cloning efficiency has still remained very low. Optimal SCNT procedures still need to be improved for production of transgenic animals and other purposes. To improve the efficiency of production of transgenic animals using SCNT, this study optimized goat SCNT protocol, and systemically studied the key technical links of production of transgenic animals using SCNT technology.1. Optimization of goat SCNT protocol(1) 1 % ITS was added to oocyte maturation medium (OM) and embryo culture medium (mSOFaa). In results, addition of ITS to OM did not improve the maturation rate, but significantly increased the blastocyst rate of parthenogenesis embryos (P<0.05); addition of ITS to mSOFaa also significantly increased the cleavage rate and blastocyst rate of parthenogenesis embryos (P<0.05). In conclusion, addition of ITS to the medium of oocyte maturation and embryo culture can improve the development of oocyte maturation in vitro and embryo culture; ITS may be applied to free-serum culture of oocytes maturation and embryo in vitro.(2) To improve the electrofusion efficiency of goat SCNT, microelectrode fusion protocol was improved in this study, the microelectrodes whose diameter was 15μm (A), 100μm (B), 200μm (C) were respectively combined and formed A+A, A+B, B+B and C+C. Fusion efficiency from different combinations was evaluated, and pressurization and non-pressurization were compared in each combination. Chamber fusion was control. In results, no significant difference was recorded in the fusion rate of non-pressurized microelectrode fusion groups and chamber fusion group (73.1 % in AA, 75.3 % in AB, 73.30% in BB, 74.2 % in CC, and 74.8 % in CF). The fusion rates in the AA (94.9 %) and AB (92.2 %) groups were significantly higher than that in the AA (83.0 %) and CC (83.9 %) groups. The highest fusion rate and the lowest degeneration rate were obtained in the AA group. These results showed that the pressurized fusion protocol carried out by a pair of tip-end micro-electrodes is optimal to improve fusion efficiency in SCNT. (3) To increase the fusion efficiency of couplets reconstructed with mammary gland epithelial (MGE) cells as donor cells, the effects of phytohemagglutinin (PHA) treatment to couplets reconstructed with MGE cells before electrofusion on the fusion and subsequent development of goat NT embryos was explored in this study. The toxicity of PHA was evaluated by testing its effects on the development of parthenogenetic goat oocytes treated with different doses and durations. The effective dose and duration of PHA treatment (1000μg/mL, 20 min incubation) were selected and used to compare fusion efficiency and embryo development following SCNT. Two different electrofusion protocols, chamber fusion and pressurized microelectrode fusion, were also compared, both without and with PHA treatment (100μg/mL, 20 min). Fusion rate of nuclear donor MGE cells to oocyte recipients was increased from 52.8 % to 74.0 % for chamber fusion protocol (P<0.05) but was not significantly different for microelectrode fusion protocol (72.7 % vs. 78.1 %) after PHA treatment. Fusion rate between both fusion protocols has no significant difference after PHA treatment (74.0 % vs. 78.10 %). There were not significant differences in subsequent cleavage rates and blastocysts development among fused embryos both with and without PHA treatment. The cloning efficiency was significantly improved from 6.7 % to 12.6 % when the recombination of PHA and microelectrode fusion protocol was used. We concluded that the treatment of MGE cells with PHA within a determined range of dose and duration before electrofusion and pressurization has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.2. Establishment of technical system on the multiplication culture of single transfected mammalian cell(4) 96-well cell culture plates were used to isolate cell lineages obtained from a single fetal fibroblast and MGE cell transfected with the pBLM-C1 plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. PCR assays were performed to detect the transfected fragment in positive cells. In results, when a single transfected cell was cultured in 0 %, 50 % and 100 % conditioned medium respectively, the transfected colony rate in 100 % group was significantly increased; the co-culture of single transgenic cell and non-transgenic cells markedly improved the colony rate after multiplication; PCR detection proved that the foreign aim gene has been integrated in transfected colonies. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production. 3. Induced expression and detection of foreign protein from transfected MGE cells(5) The transfected cells were induced by hormone (prolactin + insulin + hydrocortisone) to express human lactoferrin gene. The supernatant was collected 1 time every 6 hours,then was centrifuged for 10～15min (1000 r/min),which was stored in -20℃. The result of western blot analysis on cultured cells supernatant showed that transfected cells can express human lactoferrin protein which the molecular weight is 42 ku.4. Production of transgenic hLF gene goat using optimized SCNT protocol(6) To detect transgenic embryos and obtain viable transgenic goat, this experiment transferred the single transfected fetal fibroblast or mammary gland epithelial cell into the enucleated oocyte using the optimized SCNT technology in above experiments. Reconstructed karyoplast-cytoplast couplets were fused, activated, and cultured in vitro. Partial cloned embryos were used for the dectection of foreign gene, and remaining coned embryos were transferred into synchronized recipients. In results, PCR detection proved that the foreign aim gene has been integrated in cloned embryos. 401 embryos were transferred into 43 recipients, and 11 of them were confirmed pregnancy on 30-45 day by ultrasonography. Regretfully, all of gravid goats aborted within the first 3 months of gestation. In conclusion, though no transgenic goat birth, the technological system using in this study can be used for the production of transgenic goat.更多还原