基因组编辑新技术对猪CCR5基因的靶向作用

发布时间：2018-05-05 15:26

  本文选题：基因组编辑 + 耳成纤维细胞　； 参考：《上海海洋大学》2015年硕士论文

【摘要】：动物基因组编辑技术是指研究人员能够依照特定的目的去修饰动物的遗传组成,这是一项综合性的并且富有挑战性的技术。该技术由于能够比较容易并且精确的对基因组进行DNA的添加、删除和置换的操作,使得其在改良畜产品质量,提高生产性能,研究疾病模型,研发生物医药产品等方面都显示出广阔的应用前景。基因定点敲入时,需要为一些外源基因,如报告基因、自杀基因、筛选基因、治疗性基因和外源性优良性状基因提供一个安全可靠的停泊“港口”,在这些“港口”外源基因既可稳定高效表达,又对细胞和组织无已知的副作用。近年来,转基因动物的生物安全性一直是人们讨论的热点问题。从目前已经已发表的文献看来,针对猪基因组安全港位点的研究凤毛麟角。因此,寻找合适的安全港位点对于转基因猪的制备显得尤为重要。在克隆动物的制备中,耳成纤维细胞作为供核体细胞具有取材简便,成本低,对动物生长影响小等优点,并且从耳部组织的采样能够实现活体取样,从而为优良猪种的保种育种奠定基础。本研究第一部分采用组织块培养法分离、培养并建立猪耳成纤维细胞系,然后用电转染的方法将质粒p EGFP-C1导入该细胞系,探索并优化其最适条件和参数。最后成功建立上海白猪耳成纤维细胞系。电转后经过对各组转染效果的观察,发现在DNA量为20 ng,电压为150 V,电阻为500Ω,电容为500μF的条件下进行电转,并且电转后经37℃孵育,其效果最好。第二部分为了研究CCR5基因作为安全港基因的可能性,首先分析了CCR5基因序列,发现该基因位于猪基因组第13号染色体上,基因总长4 565 bp,CDS区位于第二外显子上。然后针对CDS区atg下游约110 bp处分别设计并成功构建了TALEN和CRISPR/Cas9系统的打靶载体,并将其分别电转染猪耳成纤维细胞。然后用Cruise TM酶对细胞池进行酶切验证后,发现TALEN打靶CCR5基因CDS区的结果为阴性,CRISPR的pool验证显示敲除效率很低。初步怀疑这两种技术在该位点上进行打靶可能存在的脱靶效应。第三部分为检测分析上海梅山猪封闭群携带猪内源性逆转录病毒(porcine endogenous retrovirus,PERV)存在与表达的情况,为该猪种作为器官移植供体的生物安全性提供现实依据,笔者从上海嘉定区梅山猪育种中心的梅山猪封闭群内随机采集45头猪的耳组织,并构建耳成纤维细胞系,利用PCR检测PERV前病毒核心蛋白基因(gag)、多聚酶基因(pol)以及囊膜基因(env)的基因组DNA,利用RT-PCR的方法检测m RNA表达状况。结果发现被检测的45份样品均携带完整的3种亚型的PERV前病毒DNA;RT-PCR结果显示,45份样品中有40份同时有PERV-A和PERV-B两种亚型的表达,还有5份样品仅检测到PERV-B亚型的表达,未检测到PERV-C亚型的表达。鉴于生物安全性方面的考虑,该猪种能否作为人异种移植的供体仍有待进一步商榷。
[Abstract]:Animal genome editing is a comprehensive and challenging technique that allows researchers to modify the genetic composition of animals for specific purposes. This technology can easily and accurately add, delete and replace DNA to the genome, so that it can improve the quality of animal products, improve the production performance, and study the disease model. Research and development of biomedical products and other aspects have shown a broad application prospects. When gene knockout, we need to provide a safe and reliable berthing "port" for some foreign genes, such as reporter gene, suicide gene, screening gene, therapeutic gene and exogenous good trait gene. In these ports, exogenous genes can be expressed stably and efficiently without known side effects on cells and tissues. In recent years, the biological safety of transgenic animals has been a hot issue. According to the published literature, few studies have been done on the safe harbour site of pig genome. Therefore, it is very important to find a suitable safe port for the preparation of transgenic pigs. In the preparation of cloned animals, ear fibroblasts as nuclear donor cells have the advantages of simple selection, low cost and little influence on animal growth. So as to lay a foundation for the conservation breeding of superior pig breeds. In the first part of the study, the fibroblast cell line of porcine ear was isolated and established by tissue mass culture method. Then the plasmid p EGFP-C1 was transfected into the cell line by electrotransfection, and the optimum conditions and parameters were explored and optimized. Finally, Shanghai White Pig ear fibroblast cell line was successfully established. After electrotransposing, we observed the transfection effect of each group, and found that under the condition of DNA 20 ng, voltage 150 V, resistance 500 惟 and capacitance 500 渭 F, the best effect was obtained after incubation at 37 鈩，

本文编号：1848218