COS-1细胞SV40T抗原的RNAi研究及转基因动物模型的建立

发布时间：2019-04-26 21:37

【摘要】：自从发现双链21碱基小干涉RNA(small interfering RNA,siRNA)可以引发特异性的基因沉寂,双链RNA(double—strand RNA,dsRNA)介导的RNA干涉(RNA interference,RNAi)已经逐渐在抑制基因表达方面得到了广泛的应用。目前广泛应用的体外合成RNA转染细胞的方法有合成成本高、转染效率低、不能持续发挥作用等缺点。构建siRNA表达载体,转染到细胞内转录后可形成大量的发卡结构的小RNA(short hairpin RNA,shRNA),而这些shRNA可特异地抑制基因的表达。猿猴病毒SV40可引起生物形成多种肿瘤,其致瘤作用主要通过其早期区域基因编码产物大T抗原而实现。SV40T支持病毒的复制,可与众多肿瘤抑制蛋白如Rb和p53结合,使其丧失功能,致使细胞分裂加速,形成肿瘤。本研究中,我们设计了针对病毒SV40T抗原编码区的三个不同位置处的21nt序列,并将已合成的三个序列插入到干涉表达载体,这种载体能产生针对SV40T抗原编码区的小干涉RNA。然后将能产生小干涉RNA的质粒瞬时转染COS-1细胞,通过半定量RT-PCR、间接免疫荧光、流式细胞仪、Western—blot等方法观测SV40T基因的抑制效果。实验结果表明,SV40T特异性小干涉RNA表达质粒转染的细胞无论从定性还是从定量上所测得的表达水平都明显低于阴性对照,这揭示了小干涉RNA确实能引起SV40T基因的沉默。因而这一结果为研究利用RNA干涉进行基因治疗提供了基础。我们利用经过细胞转染验证有效的小干涉表达质粒经线性化后采用受精卵显微注射技术成功获得了转基因小鼠,此转基因小鼠模型可通过生殖器将目的基因稳定遗传给后代。通过对转基因小鼠后代的体内表达检测,证实目的基因在动物体内的大部分组织都有表达。此转基因小鼠模型的RNA干涉表达系统研究平台的成功建立,为进一步探讨SV40T的致瘤作用创造丁条件,为研究SV40T与其它相关基因的相互作用提供了前提和基础。
[Abstract]:Since it was discovered that double-stranded 21-base small interfering RNA (small interfering RNA,siRNA can induce specific gene silence, double-stranded RNA (double-strand RNA,dsRNA)-mediated RNA interference (RNA interference, RNAi has been widely used in inhibiting gene expression. At present, the widely used method to synthesize RNA transfected cells in vitro has the disadvantages of high synthesis cost, low transfection efficiency, and can not continue to play a role, and so on. The expression vector of siRNA was constructed and transfected into intracellular transcribed cells to form a large number of small hairpin RNA (short hairpin RNA,shRNA, and these shRNA specifically inhibited the expression of the gene. Simian virus SV40 can induce many kinds of tumors, and its tumorigenicity is mainly achieved by large T antigen, which is encoded by its early region gene. SV40T supports the replication of SV40T, which can bind to many tumor suppressor proteins such as Rb and p53. Causes it to lose its function, causes the cell division to accelerate, forms the tumor. In this study, we designed the 21nt sequences at three different positions in the coding region of viral SV40T antigen, and inserted the synthesized three sequences into the interference expression vector. This vector can produce small interference RNA. targeting the coding region of SV40T antigen. Then the plasmid which could produce small interference RNA was transiently transfected into COS-1 cells. The inhibitory effect of SV40T gene was observed by semi-quantitative RT-PCR, indirect immunofluorescence, flow cytometry and Western-blot. The results showed that the expression level of SV40T-specific small interference RNA expression plasmid transfected cells was significantly lower than that of negative control both qualitatively and quantitatively, which indicated that small interference RNA could indeed induce the silencing of SV40T gene. This result provides a basis for the study of gene therapy using RNA interference. The transgenic mice were successfully obtained by linearization of the small interference expression plasmid, which was proved to be effective by cell transfection, and then the transgenic mice were successfully obtained by microinjection of fertilized eggs. This transgenic mouse model can stably transmit the target gene to the offspring through the genitalia. It was confirmed that the target gene was expressed in most tissues of transgenic mice by detecting the expression of the target gene in vivo. The successful establishment of the research platform of RNA interference expression system in transgenic mice provides a prerequisite and basis for further study of the tumorigenicity of SV40T and the study of the interaction between SV40T and other related genes.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R-332

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