【作者】 林建；

【导师】 杨倩；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2013， 博士

【摘要】 目前我国的奶山羊品种与发达国家相比差距明显,缺乏国际竞争力,突出表现为产量奶低,多数羊的年产奶量不足300kg,经济效益不高。为解决这一问题,本研究通过转基因技术培育高产奶量的奶山羊。本试验选取在乳腺发育和泌乳中起着重要作用的类胰岛素生长因子(IGF-1)基因作为转入的目的基因。通过分子生物学的方法,构建了乳腺特异性表达类胰岛素生长因子的载体pIN；然后分别转染人乳腺癌细胞系(Bcap-37)和山羊乳腺上皮细胞(GMEC)以验证乳腺特异性表达载体pIN表达IGF-1的能力,进一步的研究将载体pIN灌注泌乳期山羊乳腺以验证其生物学功能。在确认了载体pIN可以表达IGF-1后,我们使用脂质体法将载体pIN转染山羊胎儿成纤维细胞和小羊耳皮成纤维细胞,通过抗性筛选和单克隆挑选获得转IGF-1基因阳性克隆细胞。将获得的转IGF-1基因阳性克隆细胞通过体细胞核移植技术移植到去核的卵母细胞中,融合激活后待其发育到囊胚期,移植至代孕母羊,最后获得4只克隆奶山羊。提取克隆奶山羊的血液基因组,通过荧光定量PCR、Southern-blot以及TAIL-PCR等方法一方面鉴定本研究获得的克隆羊为转IGF-1基因奶山羊,另一方面检测转IGF-1基因奶山羊中外源IGF-1基因插入的拷贝数以及部分插入位点的侧翼序列。总的来说,转IGF-1基因奶山羊的培育,为通过转基因技术提高山羊奶产量奠定了基础。本研究中主要试验内容分为以下五部分：1.山羊乳腺特异性表达载体的构建及其验证本研究的目的是构建山羊乳腺特异性表达载体pIN,并在体内体外检验载体pIN的生物学活性,为下一步生产高产奶量转基因奶山羊奠定基础。第一步,以萨能奶山羊为材料,采用RT-PCR的方法从奶山羊的肝脏组织中扩增465bp的类胰岛素生长因子1(insulin-like growth factors-1,igf-1)基因；同时以真核表达载体pCDNA3.1为模板,扩增1505bp的真核筛选标记neo基因。第二步,以乳腺特异性表达载体pBC1为骨架载体,先将扩增的igf-1基因插入p-酪蛋白5’端启动子的下游,再将克隆的neo基因克隆到β-酪蛋白3’端终止子的下游,构建乳腺特异性表达载体pIN。第三步,采用脂质体法将构建好的乳腺特异性表达载体pIN转染人乳腺癌细胞系(Bcap-37)和山羊乳腺上皮细胞,同时使用乳腺灌注法将载体pIN导入泌乳期山羊乳腺组织中去检测乳腺特异性表达载体pIN的生物学功能。体外试验结果显示：在Bcap-37细胞中,载体pIN转染组的IGF-1蛋白和mRNA表达量均显著高于对照组(p<0.05)；在山羊乳腺上皮细胞中,转染载体pIN的细胞可以成功诱导表达IGF-1。体内试验结果进一步证实本研究所构建的乳腺特异性表达载体pN可以在山羊乳腺组织中成功表达IGF-1,为增加羊奶产量奠定基础。2.双亲性小分子DMSO和薄荷醇增加转染效率的研究简单、快速和高效地将目的基因转入细胞核将会有利于加速转基因阳性细胞的筛选过程。转染增强剂的使用可以有效的促进外源基因的转运。在众多增强剂中,双亲性小分子化合物二甲基亚枫(DMSO)和薄荷醇可以有效的提高基因的转染效率。本研究首先使用MTT法检测DMSO和薄荷醇的细胞毒性,摸索出对细胞没有伤害或者伤害很小的最适使用浓度；接着在人乳腺癌细胞系(Bcap-37)上,使用荧光定量PCR和流式细胞检测法去评价DMSO和薄荷醇对转染效率的影响。研究结果表明2%(V/V)的DMSO和12.5μM薄荷醇可以显著提高外源基因的转染效率。荧光定量PCR结果显示,在薄荷醇后处理和DMSO前处理的Bcap-37细胞上,生长激素(GH)的mRNA表达量提高了10倍以上；而在DMSO后处理和薄荷醇前处理的Bcap-37细胞上,GH的mRNA表达量则提高了30倍以上。荧光显微镜观察结果和流式细胞检测结果进一步证明DMSO和薄荷醇处理细胞可以增加表达绿色荧光的细胞数量。与单独脂质体转染组相比较,DMSO后处理组和薄荷醇前处理组表达绿色荧光的细胞比例增加了15%。进一步细胞周期分析发现,DMSO和薄荷醇处理均可以显著影响细胞周期,大大的改变了细胞周期停滞的比例。这一结果表明DMSO和薄荷醇可能是通过影响细胞周期来增加转染效率的。总的来说,DMSO和薄荷醇处理细胞均可以显著增加转染效率,其中DMSO后处理细胞的方式可以更有效的增加转染效率3.核定位信号肽增加转染效率的研究转基因阳性细胞筛选效率低下的问题严重限制转基因动物的发展；其中外源DNA片段(尤其是大分子DNA片段)转运进入细胞核是转基因阳性细胞筛选中最重要的限速步骤。研究发现核定位信号(NLS)可以协助大分子亲核蛋白的入核转运；补骨脂素(SPB)可以非共价结合DNA片段以保护其在转运过程中不被核酸酶降解。因此,本研究人工合成经典的核定为信号肽"CGGPKKKRKVP (NLS)"以及核定为信号肽-补骨脂素复合物"SPB-PKKKRKV(SPB-NLS)"去协助大分子DNA片段的转运,希望能够增加转染效率,继而可以应用到转基因阳性细胞的筛选中去。本研究通过荧光定量PCR,激光共聚焦观察以及流式细胞检测等技术对NLS及SPB-NLS的生物学功能进行检测。荧光定量PCR结果显示：在NLS和SPB-NLS介导的转染中,生长激素(GH)的mRNA表达量分别增加了69%和330%。流式细胞检测发现绿色荧光阳性细胞的数量在SPB-NLS组中增加了32.4%；然而在NLS组中,其阳性细胞的数量却减少了75%。进一步试验(western-blot)证实SPB-NLS介导的转染可以显著的增加转染基因的表达效率(在人乳腺癌细胞的研究中,SPB-NLS介导的转染增加了GFP的表达量；在山羊乳腺上皮细胞的研究中,SPB-NLS介导的转染增加了IGF-1的表达量)。最后,本研究通过激光共聚焦显微镜观察发现SPB-NLS介导的转染可以增加外源基因如何的效率。总的来说,本研究证实SPB-NLS是一种优良的转染增强剂,很有希望被广泛用于外源基因的转染以及转基因阳性细胞的筛选。4.转IGF-1基因阳性克隆细胞的筛选及转IGF-1基因奶山羊的制备转基因动物的制备一直以来就是人们关注的焦点和难点,尤其是转基因大型家畜的制备。本研究首先分离培养奶山羊胎儿成纤维细胞和小羊耳皮成纤维细胞,摸索不同浓度的G418对两种细胞的毒性,选取2周内能将细胞全部杀死的临界浓度作为抗性筛选浓度(800ng/mL G418:胎儿成纤维细胞,600ng/mL G418:小羊耳皮成纤维细胞)。然后通过电转染法将乳腺特异性表达载体pIN转染到小羊耳皮成纤维细胞和胎儿成纤维细胞中去。转染48小时后,更换培养基并添加G418进行抗性筛选,筛选10天左右出现明显的单克隆细胞团。此时将G418的浓度降至维持浓度(300ng/mL),维持3-5天后,挑取单克隆细胞至48孔细胞培养板。待单克隆细胞扩大培养至6孔板后,将一部分单克隆细胞提取基因组DNA进行PCR验证,另一部分单克隆细胞冻存后用于核移植试验。本试验共筛选到46个单克隆细胞株,挑选其中生长状态良好的12个单克隆细胞进行PCR鉴定；PCR鉴定结果显示igf-1和neo基因整合进入细胞基因组的阳性克隆有2个。挑选验证阳性的单克隆细胞进行体细胞核移植制备转IGF-1基因奶山羊。本试验共获得4只转IGF-1基因奶山羊。5.转IGF-1基因奶山羊的鉴定转基因动物基因组中不但含有特定的外源基因序列,还包括特定的启动子、调控元件和标记基因等,这些是进行转基因鉴定的基础。在鉴定转基因动物时,除了检测外源基因的存在、表达与否外,还需要对转入基因的完整性、整合位点以及拷贝数等情况进行分析。本研究首先通过普通PCR和Southern-blot鉴定所获得的克隆羊为转IGF-1基因奶山羊；然后通过绝对荧光定量PCR和热不均一交错PCR(TAIL-PCR)进一步检测体细胞核移植技术生产的转IGF-1基因奶山羊中插入的外源基因拷贝数和整合位点。普通PCR检测结果表明转IGF-1基因奶山羊基因组中整合有igf-1基因和neo基因；Southern-blot结果进一步证实所获得的克隆羊为转IGF-1基因奶山羊。绝对荧光定量可以精确的分析外源基因在基因组中的拷贝数,本研究首先制备标准曲线(log2N(拷贝数)=-1.0244△Ct+5.3576(R2=0.9963)),接着检测转IGF-1基因奶山羊中外源IGF-1基因的拷贝数,结果显示4只转IGF-1基因奶山羊中外源基因拷贝数均为8个拷贝；进一步TAIL-PCR成功鉴定了转IGF-1基因奶山羊中外源基因的部分整合位点。3轮特异性的TAIL-PCR得到的多条特异性条带,经测序和BLAST比对得到4个特异性位点,这四个位点分别位于牛基因组的2,11,16和18四条染色体中。本研究初步建立了绝对荧光定量PCR和TAIL-PCR检测外源基因拷贝数和整合位点侧翼序列分析的体系,为今后研究外源基因在转基因奶山羊的中遗传和表达奠定了基础。更多还原

【Abstract】 Nowadays, the development of dairy goat industry in Chinese is slow and lacks of international competitiveness, especially in goat milk production. The milk yield of one goat is less than300kg each year, which decreased the economic benefits. To solve this problem, our study tries to improve milk yield with transgenic technology. Transgenic technology is a promising strategy to enhance the performance of mammary glands. An essential component of breeding favorable varieties is selecting a right target gene. Insulin-like growth factors-1(IGF-1) can stimulate cell growth, reproduction and regeneration, and further influence the secreting cells of the goat mammary gland, thus it is an ideal choice as a target and chosen to produce IGF-1transgenic goats. Firstly, we constructed a mammary gland specific expression plasmid pIN with the method of molecular biology. Then plasmid pIN was transfected into the Bcap-37cell line and goat mammary epithelial cells to validate its function in expressing goat IGF-1. Furthermore, the plasmid was injected into goat mammary gland to convince its bioactivity of expressing IGF-1. After analyzing the bioactivity of plasmid pIN, we transfected it into goat fetal fibroblasts and goat ear skin fibroblasts by liposome. Using neomycin antibiotic selection, we picked up two IGF-1-positive clone cells. Then we transplanted the GF-1-positive clone cell into enucleated oocytes through the methods of somatic cell nuclear transfer. When the oocytes were activated and came into the blastocyst stage, we transplanted them into surrogate ewe and obtained five cloned dairy goats. Lastly, we extracted the genomic DNA of clone goats to confirm that these clone goats were IGF-1transgenic goats, using PCR and Southern-blot. At the same time, we detected the copy number and the flank sequence of IGF-1transgenic goats with real-time PCR and TAIL-PCR. Overall, our results cultivated4transgenic goats and laid the foundation for increasing goat milk yeild by transgenic technology.The main experiments were divided into the following five parts. 1. Construction of mammary gland specific expression plasmid pIN and its expression in vitro and in vivoTransgenic technology provides an opportunity to enhance the performance of IGF-1expression, and then modify mammary gland function. This study aims at constructing a mammary gland-specific expression vector, pGN, and validating its function in expressing goat insulin-like growth factor1(IGF-1) both in vitro and in vivo. The backbone plasmid pBCl contained goat β-casein5’ arm and β-casein3’arm, which can express gene specific in mammary gland. Firstly, the igf-1gene was amplified from liver tissue harvested from a Saanen dairy goat and inserted into the downstream of β-casein5’arm. Then the neo gene was cloned from plasmid pCDNA3.1and placed to the downstream of β-casein3’arm as a positive selection marker. In order to analyze the bioactivity of the pIN plasmid, pIN was transfected into the Bcap-37cell line and goat mammary epithelial cells, coupling with goat mammary gland injection. In vitro experiments not only proved that the expression of IGF-1protein and mRNA in transfected Bcap-37cells was higher than that of the control group, but also confirmed that mammary gland specific expression plasmid pIN could be induce to express IGF-1on goat mammary epithelial cells. In vivo studies showed that the expression of IGF-1in pIN injected group was significantly higher than that of the control group. Together, these results strongly demonstrated that the pIN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing IGF-1both in vitro and in vivo, which laid a foundation for increasing milk production.2. Enhancement of gene transfer efficiency in the Bcap-37cell line by amphiphilic molecules (dimethyl sulphoxide and menthol)Simply and efficiently transfer gene into nucleus will facilitate the progress of positive cells screening in producing transgenic animals. One promising method of fast gene delivery is to apply penetration enhancers. Amphiphilic molecules, such as dimethyl sulfoxide (DMSO) and menthol, could serve as non-toxic vehicles in improving gene transfer efficiency. In this study, the cytotoxic effects of DMSO and menthol were evaluated using MTT assays. Gene delivery efficiency in a human breast cancer cell line (Bcap-37) was investigated by quantitative PCR, fluorescence microscopy and flow cytometry. Results proved that non-toxic concentrations of DMSO (2%, V/V) and menthol (12.5μM) enhanced the efficiency of liposome-mediated gene delivery in Bcap-37cells. Quantitative PCR results showed that the expression of growth hormone (GH) in post-menthol and pre-DMSO treatment groups were10times as that of the liposome group, while in the pre-menthol and post-DMSO treatment groups, a30times increase in GH mRNA expression was observed. Both DMSO and menthol treatments increased the numbers of cells expressing green fluorescent protein, which was shown by fluorescence microscopy experiments. Compared to the liposome group, the number of positive cells in the pre-menthol and post-DMSO treatment groups was significantly increased by15%. Furthermore, cell cycle analysis demonstrated that there were significant differences among the DMSO-treated group, the menthol-treated group and the normal group, which implied different effects of DMSO and menthol treatments. In conclusion, both non-toxic and harmless DMSO (2%) and menthol (12.5μM) treatments improved gene transfer efficiency, and post-DMSO treatment may be the most effective protocol in increasing gene transferring efficiency.3. Improvement gene transfection efficiency in the Bcap-37cell line with NLS and SPB-NLSLow transfection efficiency severely blocks the development of transgenic animals. The process of exogenous DNA fragments entering nucleus is a major rate-limiting step, especially for the large DNA fragments. Nuclear localization sequence (NLS) peptide mediates the trafficking of nuclear protein, from cytoplasm into nucleus. Succinimidy-4-(psoralen-8-yloxy)-butyrate (SPB) can non-covalent couple with DNA molecules to prevent DNA from degradation when delivered into cell. Peptide "CGGPKKKRKVP (classic NLS)" and peptide derivative "SPB-PKKKRKV" were synthesized to mediate transfection in vitro, aiming at improving large DNA fragments transfection efficiency, especially for producing transgenic animals. To explore their biology function, we compared GH mRNA and GFP protein expression by qRT-PCR and flow cytometry. Results identified NLS group (increased by69%) and SPB-NLS group (330%) significantly improved the expression of GH mRNA. Likewise, SPB-NLS group increased the number of GFP positive cells (32.4%), but NLS group decreased the number of GFP positive cells (75%). Further analysis (western blot) demonstrated the function of SPB-NLS in hard-to-transfect Bcap-37cell (increase the expression of GFP) and target GMEC cells (improve the expression of IGF-1). In conclusion, SPB-NLS served as a transfection enhancing agent, can be widely used in both nuclear delivery and producing genetically modified animals. 4. The screening of IGF-1-positive clone cells and the production of IGF-1transgenic goatsThe production of transgenic animals has always been the focus and difficulty, especially for large-scale livestock (such as goats and castles). In this study, we firstly isolated and cultured dairy goat fetal fibroblasts and ear skin fibroblasts. Then we explored the optimal concentration of G418for positive-cells screening. Results displayed that when the G418concentration came to800ng/mL, fetal fibroblasts would be killed within two weeks. In terms of the ear skin fibroblasts, the optimal G418concentration was600ng/mL. After confirmed the optimal concentration, we transfected mammary gland specific expression vector pIN into goat fetal fibroblasts and ear skin fibroblasts. In the following of10-14days screening, we picked up monoclonal cell mass, then cultured them in48-well cell culture plates and dropped the concentration of G418to300ng/mL. The picked monoclonal cells were cultured until full of a6-well cell culture plates. Then the monoclonal cells were divided into two parts. One part was used to extract genomic DNA for identify, the other part was frozen for nuclear transplantation. Our study finally screened46monoclonal cell lines. From these monoclonal cell lines, we selected12monoclonal cell lines with good growth state by PCR identification. The PCR results showed that there were2positive clones in the cell’s genome which IGF-1gene has integrated into. After identification, we transplanted the IGF-1-positive clone cell into enucleated oocytes through the methods of somatic cell nuclear transfer. At last, we obtain4IGF-1transgenic goats.5. The identification of IGF-1transgenic goatsTransgenic technology has recently been employed to create animal lines with new genes inserted into their chromosomes. The genome of transgenic animals comprised of exogenous gene, specific promoter, regulatory elements and marker genes, which were the basis for the identification of transgenic animals. The identification of transgenic animals contained two parts. The first part was detecting the presence and the expression of the exogenous gene, the second part was analyzing the integrity site and the copy number of transferred gene. In this study, we firstly tried to prove the clone goats to be IGF-1transgenic goats by PCR and Southern-blot. Then we used the methods of real-time PCR and TAIL-PCR to analyze the copy number and the integrity site of transferred IGF-1gene. PCR results demonstrated that the igf-1gene and neo gene were integrated into the genome of IGF-1transgenic goats. Further experiments (Southern blot) proved that these clone goats were IGF-1transgenic goats. Absolute quantitative PCR could be used to precisely analysis the copy number of exogenous gene. To begin with, we established the standard curve (log2N (copy number)=-1.0244△Ct+5.3576(R2=0.9963)), then detected the copy number of four IGF-1transgenic goats. Results showed that the four IGF-1transgenic goats contained8copy IGF-1gene in their genome. At last, we attempted to identify the integrated site of exogenous IGF-1gene. TAIL-PCR results identified4specific integrating sites which were listed in bovine chromosome2,11,16and18. Overall, our study initially established a system about detecting the copy number and the integrating site’s flanking sequences of exogenous gene, which laid a solid foundation for the genetic research of transgenic dairy goats.更多还原