【作者】 田永芝；

【导师】 贾斌； 刘明军；

【作者基本信息】 石河子大学 ， 遗传育种， 2013， 博士

【摘要】 随着社会经济和科学研究的发展，多基因共表达技术在生物医药和农业生产中的作用越来越重要。目前，常用的多基因共表达方法有内核糖体进入位点(IRES)元件载体、双向或双启动子共表达、多个质粒共转染及多病毒载体共感染等。但是这些方法都遇到了多基因共表达不平衡及表达量不足的问题，如IRES下游基因的表达对其在IRES之后的特异定位敏感，有可能造成表达沉默，或下游蛋白的表达水平远低于同一开放阅读框中上游蛋白的表达水平。2A肽则能够改善以上缺陷。2A肽属于cis-水解酶作用元件（CHYSELs），多基因载体在2A肽调控下各基因可独立表达。与IRES相比，2A肽序列短（54-90bp）、能够调节多基因独立表达且表达效率高，具有其它多基因共表达技术无可比拟的优点。目前，大多数转基因动物的制备均以单基因表达为目标，而多基因共表达制备转基因动物的研究较少。利用2A肽生产多基因修饰动物已在小鼠和猪上报道，但在绵羊上未见报道。慢病毒载体转基因技术是目前家畜生产中效率最高的转基因技术，2A肽介导的多基因共表达与慢病毒载体相结合制备转基因动物，具有转基因效率高、多基因同时表达的特点。在一个转基因动物上同时表达多个功能基因，能够通过转基因实现多个基因协同作用或多基因聚合作用，提高转基因动物的利用效率，减少转基因动物生产成本，缩短转基因动物的生产周期。本研究中，我们选用三色荧光蛋白报告基因（tdTomato、zsYellow1和acGFP1）作为2A肽连接的靶基因，以便于2A肽介导的多基因在细胞和动物体内表达的观察和检测。首先，将报告基因tdTomato、zsYellow1和acGFP1与2A肽连接，克隆构建了三基因共表达慢病毒载体（pLEX-2A-TYG）；将pLEX-2A-TYG重组慢病毒感染CHO和293T细胞，在激光共聚焦显微镜下观察三色荧光蛋白的表达；在此基础上利用慢病毒卵周隙注射方法进行绵羊转基因，首次获得了携带三色荧光的转基因绵羊，通过PCR、Westernblotting、Southernblotting、RealTimeRT-PCR等方法对转基因整合、多基因表达及DNA甲基化等进行了分析；针对转基因羊出现的基因表达沉默现象，通过DNA甲基化序列分析（BisulfitesequencingPCR,BSP），检测了转基因羊外源基因启动子区和编码区的甲基化水平，利用甲基转移酶抑制剂（5-azaC）和乙酰化酶抑制剂（TSA）分别处理分离的转基因羊成纤维细胞，对多基因共表达转基因羊的DNA甲基化机制进行了探索。本研究主要研究结果如下：1设计并合成了长为102bp的2A-linker片段。经PCR、酶切、克隆，成功构建了由2A-linker连接的三色荧光蛋白基因（tdT．omato、zsY．ellow1和acG．FP1）共表达慢病毒载体（pLEX-2A-TYG)。2利用该重组载体pLEX-2A-TYG转染293T和CHO细胞，荧光显微镜观察和Westernblotting检测结果表明，tdTomato、zsYellow1和acGFP1能够在细胞中独立、高效表达。3运用脂质体转染法制备重组慢病毒，将pLEX-2A-TYG质粒与包膜质粒pMD2.G和包装质粒pSPAX2共转染293T细胞，获得了高滴度（3×109IU/ml）重组慢病毒颗粒。将生产的重组慢病毒感染293T和CHO细胞，Confocal荧光显微镜检测结果显示，2A肽介导的三荧光蛋白基因可以在感染细胞中独立高效表达。4为了检测2A肽介导的三色荧光蛋白报告基因载体是否可以在动物体内表达，我们利用慢病毒卵周隙注射技术生产转基因绵羊。选取供体羊5只，受体羊37只。利用重组慢病毒卵周隙注射获得可移植转基因胚胎37枚，移植入37只受体母羊中。出生羔羊7只，其中2只（#6和#7）经PCR检测为阳性转基因羊。5在紫外灯下观察转基因羔羊耳部、头部、唇部、蹄部等部位，均没有观测到荧光。以转基因羔羊尾部皮肤组织为样本，对转基因羊中三色荧光蛋白基因的表达进行Westernblotting及RT-PCR检测，均未检测到三种荧光蛋白。6为了分析造成转基因表达沉默的原因，我们利用BSP法对转基因羊尾组织基因组进行了甲基化分析。结果显示，转基因启动子区甲基化水平为76.25%（#6）和64.70%（#7）；编码区甲基化水平在#6转基因羊中tdTomato、zsYellow1和acGFP1分别为97.5%、98.1%、和99.2%；#7羊中tdTomato、zsYellow1和acGFP1分别为97.6%、99.5%和99.6%。结果表明，转基因羊外源基因存在超甲基化现象。我们推测，转基因超甲基化导致了荧光蛋白基因在转基因羊中的表达沉默。7为了证明DNA超甲基化是否为转基因表达沉默的原因，我们利用DNA甲基转移酶抑制剂（5-azaC）和去乙酰化酶抑制剂（TSA）对分离的转基因羊成纤维细胞进行处理。首先采取2只阳性（#6和#7）和1只阴性羔羊的尾部皮肤，分离培养了成纤维细胞，用5-azaC和TSA按不同浓度、不同作用时间，分别或同时对分离的转基因羊成纤维细胞进行处理。经荧光显微镜观察和流式细胞术分析，结果显示，经5-azaC和TSA分别处理后，部分成纤维细胞可见三色荧光的表达，且荧光细胞比例随着药物处理浓度及时间的增加而增加，两者呈一定的正相关。同时，当5-azaC和TSA共同作用时，荧光细胞数显著高于5-azaC或TSA单独作用时的荧光细胞数，表明5-azaC与TSA存在协同作用。以上结果表明，细胞中荧光蛋白报告基因的表达与DNA甲基化和组蛋白去乙酰化存在明显关联；5-azaC与TSA共同作用时存在协同作用。综上所述，我们利用2A肽介导的多基因共表达慢病毒载体成功制备了三色荧光蛋白转基因羊，为未来多基因共表达转基因研究提供了新材料。在研究中，我们发现2A肽介导的多基因共表达慢病毒载体在体外真核细胞中能够独立高效表达，但在转基因羊中表达沉默。转基因羊成纤维细胞经5-azaC和TSA处理后，部分细胞荧光蛋白报告基因的表达被激活，提示我们DNA甲基化在转基因动物中对转基因的表达调控发挥着至关重要的作用。研究转基因表达调控机制，降低转基因DNA超甲基化水平，规避转基因表达沉默，是本实验未来研究的重点方向。更多还原

【Abstract】 With the development of economic and science research, it is often critical to make transgenic animals that express multiple genes under the control of a single promoter in biomedical research and agriculture. Now, different approaches, such as internal ribosomal entry site (IRES) elements, bidirectional or double promoters, or coinfection with multiple viral vectors are commonly used. These strategies, however, mostly suffer from the fundamental problem that coexpression of the heterologous proteins is unreliable and far from quantitative. In the case of IRES-mediated coexpression, for example, expression of the downstream cistron is sensitive to its specific positioning after the IRES and will not be come true; the upstream cistron in these bicistronic mRNA open reading frames (ORFs) is more strongly transcribed than that of downstream protein.2A peptide could improve the above defectiveness.2A peptide, known as cis-acting hydrolase element’(CHYSEL), could mediates expression of multi-gene indepently. Compared to IRES,2A peptide has a shorter sequence (54-90bp), genes in the2A based multi-gene vector could express more independently and efficiently, which owns the advantages that others couldn’t gain over it.At present, most of the production of transgenic animals are mediated by single-cistron vector, and animals mediated by multi-cistron vector are rare. hi2A peptide mediated transgenic animals, except mice and pigs, however, there is no report in sheep. Now, lentivirus vector based transgenic technology owns the highest efficiency in transgenic livestock production. Combining the lentivirus vector with2A-mediated polycistronic co-expression, the transgene efficiency would be high and multiple genes expressed coordinately. In one animal that express multiple exogenous gene at the same time, it is feasible to make many genes synergy or polymerization, which could improve the transgene utilization efficiency, shorten the production cost and animal production cycle. In the study, we make the fluorescent reporter gene (tdTomato, zsYellowl and acGFPl) as target gene, to facilitate testing the gene expression mediated by2A peptide. Firstly, tdTomato, zsYellowl and acGFPl were linked by2A peptide, subcloned and constructed the multi-gene vector pLEX-2A-TYG. Second, the lentiviral vector was transfected in to the293T and CHO cells, and the expression of the vector was investigated under confocal microscope. Upon this, the first tri-gene transgenic sheep were produced by injecting the lentiviral particles into the perivitelline space of sheep zygotes. Several methods such as PCR, Western blotting, Southern blotting, Real Time RT-PCR were used to analyze the transgene integration, copy numbers, transgene expression for trangene lambs. To analyze the transgene silencing, methylation level of the promoter and coding region in the vector was examined by Bisulfite sequencing PCR, BSP. Isolated fibroblasts from transgene lambs were treated by DNMT inhibitor5-azacytidine (5-azaC) and deacetylase inhibitor TSA, the DNA methylation mechanism was explored. Main points in the study were listed in the following:1We first constructed the fragment2A-linker with the size of102bp.2A-linker mediated tri-gene lentiviral pLEX-tdTomato-zsYellow1-acGFP1(pLEX-2A-TYG) was constructed successfully PCR, enzyme digestion and clone reaction.2The Tri-gene vector was transfected with the293T and CHO cells, which were detected by inverted fluorescent microscope and western blotting. Results showed that fluorescent proteins genes tdTomato, zsYellowl and acGFPl contained in the tri-gene vector could express high efficiently and independently.3Lentiviral particles were produced by cotransfection pLEX-2A-TYG vector with pMD2.G and packaging plasmid pSPAX2into293T cells using lipofectamine2000, with a high titre (3×109IU/ml). The lentivirals were used to infect293T and CHO cells,2A peptide mediated tdTomato, zsYellowl and acGFPl expressed efficiently and independently, which were detected by confocal microscope.4To test whether the multi-gene vector work well in vivo, transgenic sheep was produced by injecting the lentiviral particles into the perivitelline space of sheep zygotes. There are5donors and37recipient Merino sheep for use.37transgenic embryos were injected lentiviral particles, and transferred to37recipient rams. Of seven lambs born, two (#6and#7) of which were identified with exogenic gene by PCR.5Lambs were all examined by portable UV light, no fluorescent was found in the skin of the ears, heads, lips, feet and other bodies that can be seen. The skin of the transgenic lambs were gained and used as sample, we analyzed transgene expression by western blotting and RT-PCR. Unfortunately, no signal was detected.6To test the cause of transgene silencing, the methylation level of genome DNA in the tails of transgenic lambs were analyzed by BSP. Results showed that methylation levels in the CMV were76.25%(#6) and64.70%(#7); in the coding region of tri-gene construct, methylation levels were for lamb#6, tdTomato97.5%, zsYellow198.1%and acGFP199.2%; for lamb#7, tdTomato, zsYellowl and acGFPl were97.6%,99.5%and97.6%, respectively. It denoted that the hypermethylation existed in the tails of transgenic sheep. We implied that the silencing of transgene expression would caused by DNA methylation in the transgenic sheep.7To test whether DNA methylation played a critical role in the transgene expression, skins of two transgenic and one non-transgenic lamb were cut, the fibroblasts of which were isolated and purified. The cells were treated with5-azaC and TSA with different concentration and time, respectively. The results detected by inverted fluorescent microscope and FACS showed that number of the fluorescent (GFP+) cells increased with the drug concentration and incubation time increasing. When5-azaC and TSA co-incubated in the cells, GFP+cells were more than that treated with5-azaC or TSA alone, which suggested that there is a co-operation between5-azaC and TSA. Results above showed that expression of the fluorescent protein gene has an obvious negative relationship with DNA methylation and histone deacetylation;5-azaC and TSA had a coordinated effect when they work together.In conclusion, we successfully produced multi-gene sheep by2A peptide mediated tri-gene vector, which would provide new materials for transgenic research. Unfortunately, reporter genes of the tri-gene mediated by2A peptide expressed successfully in the cells, but silenced in the tails of the transgenic sheep. Expression of the reporter gene in part of fibroblasts isolated from transgenic sheep was reactivated when they were treated by DNMT inhibitor and deacetylase inhibitor, which denotes that DNA methylation plays an important role in transgene expression. The mechanism of transgene regulation, down-regulation of DNA methylation and methods of avoiding silencing of transgene would be studied in the future.更多还原