【作者】 张学明；

【导师】 吴应积；

【作者基本信息】 内蒙古大学 ， 动物学， 2009， 博士

【摘要】 应用动物乳腺生物反应器生产药用蛋白已成为生物高技术领域的研究热点。然而,传统的基因随机整合方法制作乳腺生物反应器,外源基因的表达调控受到整合部位邻近的DNA序列影响,表达水平差异大、且大多停留在较低水平。而用基因打靶方法制作的转基因动物外源基因表达不受位置效应影响,能够显著提高表达水平。胶质细胞源性神经营养因子（GDNF）对多巴胺能神经元具有营养及保护作用,在帕金森病及其它神经损伤疾病的治疗方面显示出了巨大的应用前景。本研究构建了人gdnf在牛胎儿成纤维细胞β-casein基因座的定位整合的基因打靶载体,将打靶载体转入人乳腺肿瘤上皮细胞系Bcap-37细胞中,检测了打靶载体构建的合理性和有效性,验证了所构建的打靶载体具有生物学功能。在此基础上,将打靶载体转入牛胎儿成纤维细胞,经正负筛选获得了非红荧光抗性克隆,应用PCR法鉴定非红荧光抗性克隆获得了中靶克隆。以中靶体细胞为核供体,利用核移植技术制备牛基因打靶克隆囊胚。本研究结果为制备人gdnf在牛β-casein基因座定位整合的牛乳腺生物反应器奠定了坚实的基础。1.人gdnf在牛β-casein基因座定位整合载体pNRTCNbG的构建选用pPGK-neoLoxP作为骨架载体构建人gdnf基因打靶牛β-casein基因座的正负筛选打靶载体pNRTCNbG。该打靶载体的特点是以牛β-casein基因上游包括启动子、外显子1、内含子1和部分外显子2的2.2 kb调控序列和下游5.7kb序列为5′和3′同源臂;neo抗性基因为正筛选因子,位于两同源臂之间;HSV-tk基因和DsRed2基因为双负筛选因子,分别位于5′和3′同源臂外侧;人gdnf基因置于5′同源臂下游,SV40 polyA序列插入到人gdnf基因下游作为人gdnf基因转录终止信号。经PCR检测、限制性内切酶图谱及DNA测序分析结果表明,所构建载体结构正确。2.打靶载体pNRTCNbG的生物学功能分析为了检测打靶载体pNRTCNbG的生物学功能,用脂质体介导法将其导入人乳腺肿瘤上皮细胞系Bcap-37细胞中,经G418抗性筛选8-10天后,获得了表达红色荧光蛋白的稳定转染pNRTCNbG的人乳腺上皮细胞单克隆。将单克隆细胞扩大培养后,经PCR检测结果表明,牛β-casein基因启动子驱动的人gdnf基因已整合到表达红色荧光蛋白的人乳腺上皮细胞中。用催乳素、胰岛素及氢化可的松诱导培养转基因细胞,经RT-PCR和Western Blot检测表明,牛β-casein基因启动子驱动的人gdnf基因能够在人乳腺上皮细胞中转录翻译并分泌到胞外。这些结果表明,我们构建的人gdnf基因在牛β-casein基因座定位整合载体pNRTCNbG具有生物学功能,同时也表明人乳腺肿瘤上皮细胞系Bcap-37能够用于乳腺表达载体的生物学功能检测。3.人gdnf基因在牛胎儿成纤维细胞β-casein基因座的定位整合采用组织块贴壁法分离培养了牛胎儿成纤维细胞。应用PCR方法鉴别了培养细胞的性别。选择雌性牛胎儿成纤维细胞连续培养75天,进行了形态观察和染色体分析。结果表明分离培养的牛胎儿成纤维细胞具有良好的生理特性,能够用于基因打靶。本研究首次使用DsRed2基因作为负筛选因子。为了比较HSV-tk和DsRed2对同源重组的富集作用,利用线性化打靶载体pNRTCNbG电击转染牛胎儿成纤维细胞,将转染细胞分为两等份,分别加入G418和G418+GANC筛选获得抗性克隆后,在荧光显微镜下排除红荧光克隆,富集中靶克隆。结果表明,HSV-tk和DsRed2富集效率分别为2倍和4倍;而且,与GANC相比较,DsRed2蛋白对富集的克隆细胞无毒性作用。之后,利用线性化打靶载体pNRTCNbG电击转染了3.2×10⁷个牛胎儿成纤维细胞,用G418筛选8-10天后,共获得了3057个G418抗性克隆,在荧光显微镜下排除红荧光抗性克隆后,获得了773个非红荧光抗性克隆,经PCR扩增、PCR产物测序等方法鉴定,证实有5个单克隆为打靶克隆,相对打靶效率为0.65%（5/773）,绝对打靶效率为1.6×10^-7（5/3.2×10⁷）。4.人gdnf基因打靶克隆囊胚的制备以中靶牛胎儿成纤维细胞为核供体,去核卵母细胞为胞质受体制作转基因克隆胚胎,获得了基因打靶克隆囊胚。为了确定合适的重构胚激活与发育培养方案,我们首先进行了孤雌胚的激活与体外发育培养。排出第一极体的成熟牛卵母细胞置于含有7%乙醇的发育液中激活7 min,然后放入含2 mM6-DMAP的发育液（SOFaa+BSA）中培养4 hr后,再移入40μL SOFaa+BSA的发育液小滴中培养36hr,卵裂率为74.9%;将卵裂胚胎移入SOFaa+4%FBS+卵丘细胞的共培养体系中,发育培养7-9天,囊胚率为24.9%。结果表明这一培养系统能够很好地支持牛卵母细胞的孤雌激活与体外发育培养。核移植重构胚采用该方案进行激活和体外发育培养。本研究采用抽吸法共回收牛卵母细胞2627枚,成熟培养18 hr成熟率为70.2%;去核操作卵母细胞186枚,用于融合的重构胚为154枚,融合134枚,融合率为87.0%;将134枚融合重构胚进行发育培养,获得5枚囊胚,囊胚率为3.7%。随机挑选2枚克隆囊胚,用于PCR检测克隆囊胚是否为转基因囊胚,结果全部为阳性。更多还原

【Abstract】 The highlight has been given to the recombinant human proteins produced by the mammary glands of genetic,ally modified transgenic livestock mammals in the top field of biological technology.However,for random integration of mammary gland bioreactor,inserted gene expression level varied and stayed at a low level because of affecting by flanking DNA sequences.In contrast with random integration,site-specific integration of mammary gland bioreactor created by gene targeting at high expressing locus can remarkably improve gene expression.Glial cell line-derived neurotrophic factor（GDNF）,a distant member of the transforming growth factor[beta]（TGF-[beta]） super family,is a novel type of neurotrophic factor cloned in 1993.Researches revealed that it may have potential application in the treatment of Parkinson’s disease and other diseases of the central nervous system as a survival factor for central dopaminergic neurons.In the present study,we constructed a gene targeting vector for the human gdnf gene knock-in at the bovine beta-casein gene locus so that human GDNF protein can be produced at high level in the mammary gland of the gene-targeted bovine.Human mammary tumor epithelial cell line Bcap-37 cells were used to analyze the rationality and validity as well as the biological function of the vector.Then bovine fetal fibroblast cells were transfected with the plasmid DNA.The gene-targeted fibroblast cells were obtained after positive-negative selection and PCR identification.The gene-targeted cells were used as nuclear donor to produce gene-targeted blastocysts by nuclear transfer.The results lay a massive foundation for production of human GDNF protein by gene-targeted bovine mammary gland bioreactor.1.Construction of targeting vector pNRTCNbG for the human gdnf gene knock-in at the bovine beta-casein locusThe pPGK-neoLoxP vector containing neo gene between two LoxP sites was used as plasmid backbone to construct the targeting vector pNRTCNbG.The 5’ and 3’ homologous arms,the beta-casein gene fragments,were amplified from purified bovine genomic DNA by PCR.The 5’ homologous arm was 2.2 kb fragment including promoter,exon 1,intron 1 and part of exon 2 of the bovine beta-casein gene sequence,and the 3’ homologous arm was 5.7 kb fragment including the bovine beta-casein gene 3’ flanking sequence.The human gdnf cDNA amplified by RT-PCR was located at the downstream of the 5’ arm.Moreover,SV40 polyA signal sequence was located at the downstream of the human gdnf gene as its transcriptional ending signal.The neo gene,positive selection marker gene,was located between the 5’ and 3’ homologous arms.The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes,respectively The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing.The results show that the structure of the final constructed vector accords with the designed plasmid map.2.Analysis of the biological function of the gene targeting vector pNRTCNbGIn order to analysis the bioactivity of the vector,linearized pNRTCNbG was transfected into human mammary tumor epithelial cell line Bcap-37 by using lipofectamine.After selection with G418 for 8-10 days,cell clones expressing red fluorescence protein were obtained.PCR analysis demonstrated that gdnf cassette had integrated into the genomic DNA of the transfected Bcap-37 cells.After proliferation culture,the transgenic cells were cultured in induction medium containing serum-free RPMI-1640 medium with prolactin,insulin and hydrocortisone,which can induce recombinant human GDNF expression.RT-PCR and Western-blotting analysis showed that the induced cells produced recombinant human GDNF mRNA and protein.The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells and secrete the protein outsite of the cells.At the same time,it is first time to confirm that human mammary tumor epithelial cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.3.Human gdnf gene knock-in at beta-casein locus in bovine fetal fibroblast cells The fetal fibroblast cells were successfully isolated from bovine fetus tissue.The gender of fetuses was identified using PCR.The fibroblast cells from the female fetus were cultured consecutively for 75 days.Morphological observation and analysis of chromosome number were carried out.The results showed that the cells possessed normal morphology,proliferation characteristics and chromosome number.So the bovine fetal fibroblast cells gave a good fit to gene targeting manipulation.DsRed2 was used as a marker gene for negative selection in the present study for the first time.In order to compare enrichment efficiency of targeting events between selections with DsRed2 gene and HSV-tk gene,linearized pNRTCNbG was introduced into the bovine fetal fibroblast cells by electroporation.The transfected cells were divided into two aliquots.The cells in each aliquot were selected by addition with G418 or G418 plus GANC in cell culture medium,respectively.After selection with G418 or G418 plus GANC for 8-10 days,the resistant clones in which targeting events had occurred were enriched by eliminating cells clones expressing red fluorescence protein via a fluorescent microscope.The results indicated that enrichment factors for DsRed2 and HSV-tk used as negative selection marker genes were 4-fold and 2-fold, respectively.Moreover,compared with GANC,DsRed2 protein had not cytotoxic effect on those gene-targeted clones.Subsequently,3.2×10⁷ bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation.After selection with G418 for 8-10 days,total 3057 cell clones were obtained.After eliminating the cell clones expressed red fluorescent protein under a fluorescent microscope,773 cell clones were harvested,in which 5 cell clones involved in gene targeting events were conformed by PCR analysis and PCR product sequencing.Therefore,the relative targeting frequency was 0.65%（5/773）,and the absolute targeting frequency was 1.6×10^-7（5/3.2×10⁷）4.Preparation of human gdnf gene-targeted blastocysts by somatic cell nuclear transferThe cloned blastocysts were prepared by somatic cell nuclear transfer,using the gene-targeted cells as the nuclear donor cells and enucleated oocytes matured in vitro as the recipient cytoplasm.In order to make sure the proper method of activation and development culture of reconstructed embryos,activation and development culture of parthenogenetic oocytes were carried out.The oocytes matured in vitro were activated with 7%ethanol for 7 minutes followed by incubation in SOFaa-BSA medium containing 2 mM 6-DMAP for 4 hours.After the activation the embryos were cultured in SOFaa-BSA medium for 36 hours,the cleaved embryos（cleaved rate was 74.9%） were co-cultured with cumulus cells in SOFaa medium containing 4%FBS for 7 to 9 days.The blastocyst formation rate was 24.9%.The results indicated the procedure could be used for activation and in vitro development of reconstructed embryos.In the present study,total of 2627 bovine oocytes were aspirated from follicles（2-8 mm in diameter）.70.2%of them were matured after 18 hours culture.186 in vitro matured oocytes were enucleated, and 154 of them were electrofused with donor cells.Among them,134（87.0%） couplets were fused.134 reconstructed embryos were activated and cultured in vitro,and 5（3.7%） of them developed to blastocyst stage.2 of the blastocysts were chosen at random to detect whether the cloned blastocysts were transgenic blastocysts by PCR.The results showed that all of them were cloned transgenic blastocysts.更多还原