利用肿瘤特异性和可热调控的siRNA表达载体条件性敲除基因
发布时间：2018-11-09 13:05
【摘要】：RNAi是在生物进化上高度保守的特异性转录后基因沉默过程,并已经成为强大的基因操作工具。它几乎可以用来研究任何一种基因的分子途径以及表型和基因型之间的关系。但仍存在些许不足之处,严重限制了RNAi在基因治疗领域中的广泛应用。其中RNAi在时间和空间上的不可控性以及非靶向性是需要亟待解决的难题。诱导型RNAi技术可以通过诱导物介导sh RNA的表达进而实现对靶基因的有效调控。与诱导型调控系统结合的RNAi体系作用机制更精细,这可能会开辟出一个更广阔的应用领域。本研究选取合适的热休克元件和肿瘤特异性启动子,将两者有效结合,构建一种温度诱导型的RNAi质粒载体。具体实验内容和结果如下:①选择h TERT肿瘤特异性启动子的核心序列,它几乎只在大多数的癌症细胞中启动表达。热休克蛋白启动子中含有可以响应热的热休克元件,实验中选择三种热休克元件:一种来源于小HSP家族的Hsp27;另一种是三个“NGAAN-NTTC”HSE基本单位的重复排列;最后一种是由“AGAAC”和“GTTCT”交替排列得到的HSE序列。将HSE序列可以插入到h TERT启动子序列-65和-85位点之间或者放在首位。②构建ph TERTp-EGFP、ph TERT/HSE(AB)p-EGFP、ph TERT/HSE(MN)p-EGFP和ph TERT/HSE(XY)p-EGFP四种质粒,并将其转染至MCF-7细胞中,通过检测EGFP的蛋白表达量比较不同启动子的启动能力。数据显示h TERT/HSE(MN)和h TERT/HSE(XY)启动子既可以降低h TERT启动子的本底表达水平又可以很好的响应过高热。此外,这两种启动子在Ha Ca T和HL7702人正常细胞中表达EGFP的量远远低于Hsp70启动子,这表明所构建的ph TERTp-EGFP和ph TERT/HSEp-EGFP质粒具有良好的肿瘤特异性。③构建p Mh TERTpsi SNCG和p Mh TERT/HSEpsi SNCG质粒载体,并选择MCF-7和Hep G2为实验细胞,以SNCG为靶基因,分别从RNA和蛋白水平上检测这种表达系统的敲除效果。实验结果显示无论是在MCF-7还是在Hep G2细胞中,p Mh TERT/HSE(MN)psi SNCG和p Mh TERT/HSE(XY)psi SNCG质粒在37℃条件下几乎不能敲除SNCG,而当经历过热诱导,这两种质粒沉默SNCG基因的能力明显提高,敲除率分别在50%和60%以上。
[Abstract]:RNAi is a highly conserved process of specific post-transcriptional gene silencing in biological evolution and has become a powerful tool for gene manipulation. It can be used to study the molecular pathways of almost any gene and the relationship between phenotypes and genotypes. However, there are still some deficiencies, which seriously limit the wide application of RNAi in gene therapy. The uncontrollability and non-targeting of RNAi in time and space are difficult problems to be solved. Inducible RNAi technique can effectively regulate the target gene by inducing the expression of sh RNA. The mechanism of RNAi system combined with inducible regulation system is more refined, which may open up a wider application field. In this study, the suitable heat shock elements and tumor specific promoters were selected and combined effectively to construct a temperature-induced RNAi plasmid vector. The specific experimental contents and results are as follows: 1 the core sequence of h TERT tumor specific promoter is selected, which only activates expression in most cancer cells. Heat shock protein promoters contain heat shock elements that can respond to heat. Three heat shock elements are selected in the experiment: one is Hsp27; from small HSP family and the other is repetitive arrangement of three basic units of "NGAAN-NTTC" HSE; The last is the HSE sequence, which is arranged alternately by "AGAAC" and "GTTCT". The HSE sequence can be inserted between the -65 and -85 sites of the h TERT promoter sequence or put in the first place. 2 to construct four kinds of plasmids of ph TERTp-EGFP,ph TERT/HSE (AB) p-EGFP, TERT/HSE (MN) p-EGFP and ph TERT/HSE (XY) p-EGFP. After transfection into MCF-7 cells, the promoter ability of different promoters was compared by detecting the protein expression of EGFP. The data showed that h TERT/HSE (MN) and h TERT/HSE (XY) promoters could not only reduce the background expression level of h TERT promoter but also respond to hyperthermia. In addition, the expression of EGFP in Ha Ca T and HL7702 human normal cells was much lower than that in Hsp70 promoter. This indicated that the constructed plasmids of ph TERTp-EGFP and ph TERT/HSEp-EGFP had good tumor specificity. 3 the plasmid vectors of p Mh TERTpsi SNCG and p Mh TERT/HSEpsi SNCG were constructed, and MCF-7 and Hep G2 were selected as experimental cells and SNCG as target gene. The knockout effects of the expression system were detected at RNA and protein levels, respectively. The results showed that both, p Mh TERT/HSE (MN) psi SNCG and p Mh TERT/HSE (XY) psi SNCG plasmids could hardly knock out SNCG, at 37 鈩，
本文编号：2320497
[Abstract]:RNAi is a highly conserved process of specific post-transcriptional gene silencing in biological evolution and has become a powerful tool for gene manipulation. It can be used to study the molecular pathways of almost any gene and the relationship between phenotypes and genotypes. However, there are still some deficiencies, which seriously limit the wide application of RNAi in gene therapy. The uncontrollability and non-targeting of RNAi in time and space are difficult problems to be solved. Inducible RNAi technique can effectively regulate the target gene by inducing the expression of sh RNA. The mechanism of RNAi system combined with inducible regulation system is more refined, which may open up a wider application field. In this study, the suitable heat shock elements and tumor specific promoters were selected and combined effectively to construct a temperature-induced RNAi plasmid vector. The specific experimental contents and results are as follows: 1 the core sequence of h TERT tumor specific promoter is selected, which only activates expression in most cancer cells. Heat shock protein promoters contain heat shock elements that can respond to heat. Three heat shock elements are selected in the experiment: one is Hsp27; from small HSP family and the other is repetitive arrangement of three basic units of "NGAAN-NTTC" HSE; The last is the HSE sequence, which is arranged alternately by "AGAAC" and "GTTCT". The HSE sequence can be inserted between the -65 and -85 sites of the h TERT promoter sequence or put in the first place. 2 to construct four kinds of plasmids of ph TERTp-EGFP,ph TERT/HSE (AB) p-EGFP, TERT/HSE (MN) p-EGFP and ph TERT/HSE (XY) p-EGFP. After transfection into MCF-7 cells, the promoter ability of different promoters was compared by detecting the protein expression of EGFP. The data showed that h TERT/HSE (MN) and h TERT/HSE (XY) promoters could not only reduce the background expression level of h TERT promoter but also respond to hyperthermia. In addition, the expression of EGFP in Ha Ca T and HL7702 human normal cells was much lower than that in Hsp70 promoter. This indicated that the constructed plasmids of ph TERTp-EGFP and ph TERT/HSEp-EGFP had good tumor specificity. 3 the plasmid vectors of p Mh TERTpsi SNCG and p Mh TERT/HSEpsi SNCG were constructed, and MCF-7 and Hep G2 were selected as experimental cells and SNCG as target gene. The knockout effects of the expression system were detected at RNA and protein levels, respectively. The results showed that both, p Mh TERT/HSE (MN) psi SNCG and p Mh TERT/HSE (XY) psi SNCG plasmids could hardly knock out SNCG, at 37 鈩，
本文编号：2320497
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