【作者】 徐冬兰；

【导师】 成勇；

【作者基本信息】 扬州大学 ， 临床兽医学， 2008， 硕士

【摘要】 转基因动物乳腺生物反应器是当前生物工程研究的热点。当人们将外源基因转染入哺乳动物细胞以后,总是希望外源基因能高效表达,并通过转基因动物克隆技术制作转基因动物,使其要求导入的外源基因在动物个体高水平表达所需要的新性状。然而事与愿违的是,许多转基因实验都导致了外源基因在哺乳动物细胞水平或动物个体水平不表达。研究表明在许多情况下外源基因整合进基因组后,其表达和转基因沉默有关。世界各国对转基因沉默机理及其防制展开了广泛研究,克服转基因沉默来提高外源基因的表达己经成为基因工程的一个重要课题。因此,有效预防转基因沉默,对提高外源基因的表达,提高制作转基因动物的成功率具有重要的理论和实际意义。本研究构建了BLG/hLF乳腺特异性表达载体BLC-14,建立了体外培养奶山羊乳腺上皮细胞(GMEC)系,并将构建的乳腺特异性表达载体BLC-14转染奶山羊乳腺上皮细胞,获得了阳性单克隆细胞株并用TSA处理,通过细胞水平的整合和表达检测,探讨了组蛋白去乙酰化酶抑制剂TSA能否克服转基因沉默,解除转染入山羊乳腺上皮细胞基因组中的BLG/hLF基因表达受抑制的现象,从而提高BLG/hLF基因在乳腺上皮细胞中的表达水平,为研制乳腺特异性高效表达载体提供依据,为后期转基因体细胞核移植制备高表达转基因克隆动物乳腺生物反应器提供具有表达潜能的体(供核)细胞。1乳腺特异性表达载体的构建本实验利用实验室已有的人乳铁蛋白cDNA、奶山羊β-乳球蛋白(β-BLG)5`端(4.2Kb)、3`端(1.8Kb)表达调控序列、Neor、增强子序列构建出了BLG/hLF乳腺特异性表达载体BLC-14。2奶山羊乳腺上皮细胞系的建立用胶原酶消化法从经过催乳的刚成年奶山羊或者处于泌乳中后期的青年奶山羊乳腺实质组织中分离得到乳腺上皮细胞。采用分步消化法进行纯化,经过一到二次选择性传代培养,获得纯化的乳腺上皮细胞。用DMEM/F12+10%FCS培养液体外培养20代仍保持正常细胞繁殖扩增能力,生长良好。3 BLG /hLF基因转染奶山羊乳腺上皮细胞及TSA的处理BLG/hLF乳腺特异性表达载体BLC-14通过电转染法导入纯化的P1或者P2奶山羊乳腺上皮细胞中,共转染5个细胞系。经400μg﹒mL-1G418筛选培养液筛选三周后,得到有抗性的细胞株。挑取单克隆细胞株并进行扩大培养,共获得89株。随机取7株细胞DNA进行PCR检测,结果均为阳性。将单克隆细胞株分成实验组与对照组,实验组加入5μM催乳素、1μMTSA的诱导培养液,对照组只加入5μM催乳素的诱导培养液。诱导培养24小时后,利用间接ELISA检测,实验组与对照组均获得11株细胞能稳定表达人乳铁蛋白。实验组的平均表达水平是0.781μg﹒mL-1(106个细胞),未用TSA处理的对照组表达水平是0.558μg﹒mL-1(106个细胞)。经统计学分析,TSA处理后的实验组要明显高于对照组。实验结果表明组蛋白去乙酰化酶抑制剂TSA能提高BLG/hLF基因在奶山羊乳腺上皮细胞中的表达水平。所得到的高表达BLG/hLF的单克隆细胞将用作细胞核移植的供核细胞进行转基因克隆羊的制作。更多还原

【Abstract】 Mammary gland bioreactor is a hot issue in biological engineering areas currently. We always hope that the foreign genes could be highly expressed in mammalian cells and individuals when foreign genes are transfected into mammalian cells and the transgenic animals are made. But things go contrary to one’s wishes, the foreign genes do not express in many transgenic experiments. Researches show that in many cases the expression of the foreign genes which have been integrated into the genome in mammalian cells are relevant to the transgene silencing. Countries in the world are carrying out extensive research on the mechanism and control of the transgene silencing, in order to overcome it to improve the expression of foreign genes. It has become one of the most important problems to the application of transgenic animals in genetic engineering areas at home and abroad. Therefore, from above, the prevention of transgene silencing has great theoretical and practical significance to enhancing the expression of foreign gene and to improve the success rate of transgenic animals.We constructed a mammary gland-specific expression vector BLC-14, established the goat mammary epithelial cell lines in vitro, transfected the mammary gland-specific expression vector BLC-14 into the goat mammary epithelial cells, obtained transfected cell clonies and treated with TSA. By testing the experssing level of the hLF at cell level, whether the histone deacetylases inhibitor TSA could overcome the transgene silencing and enchance the expression of BLG/hLF gene in the goat mammary epithelial cells was investigated. It provides the basis for the further study of constructing high experssed mammary gland-specific vector and offers the potential foreign genes expression cells to the latter part study of producing high expressed transgenetic animals by somatic cell nuclear transfer.1 Construction of mammary gland-specific expressional vectorcDNA sequence of human lactoferrin gene, 5` and 3` flank regulatory sequence ofβ-lactoglobulin gene, neor gene and the sequence of enhancer which had been harboured in our laboratory were used to constructing mammary gland-specific expressional vector BLC-14.2 Establishment of goat mammary epithelial cell lines in vitroPrimary goat epithelial mammary cells were obtained using collegenase I -digesting method.Goat mammary gland tissue from the middle-lactogening adult goat was digested and cultured in medium DMEM/F12 containing 10% fetal calf serum. By tripsin digesting, the pured mammary epithelial cell line could be obtained after 1～2 passages. After a long time observation, it was found that the cell proliferate vigorously in vitro even after 20th passages.3 Transferring BLC-14 into goat mammary epithelial cell lines and the treatment of TSABLC-14 vectors containing neor gene as the selective marker were transfected into 1th or 2th passage goat mammary epithelial cells by electroporation. After three weeks,neomycin resistant single colonies were formed in 400μg﹒mL-1 G418. 89 colonies were obtained, which were picked out and proliferate in 200μg﹒mL-1 medium. DNA samples from 7 clonies were screened by PCR and all of them had been identified to be the transgenic cells. Then colonies divided into two groups as experimental group and comparative group. After inducing comparative group by 5μM luteotropic and experimental group by 5μM luteotropic and 1μM TSA, BLG/hLF expression was tested by ELISA. The results showed that the ELISA positive of the two group were both 11. The experssing level of the experimental group was 0.781μg﹒mL-1 per 106 cells, and the comparative group was 0.558μg﹒mL-1 per 106 cells.Through statistical analysis, the experssing level of the experimental group which was treated with TSA was significantly higher. From above, it was indicated that histone deacetylases inhibitor TSA could enchance the experssing level of the transgene at cell level. The clonies of the highest expression would be used as donor cells in nuclear transfer in the later study.更多还原