Retro-Tet:一种基于逆转录病毒的诱导性基因表达系统

 Retro Tet基因表达系统是一种新型的高效、稳定、无毒、具有严密开 关功能的可诱导性真核细胞基因表达系统 .该系统兼备了逆转录病毒基因表达系统和Tet off Tet on基因表达系统的优点 ,在稳定表达细胞系的筛选、基因的表达与调控及基因功能研究等方面得到了成功的应用 ,同时也为基因治疗提供了一种理想的基因载体系统     

Tet system的调控原理及其在转基因小鼠模型上的应用

基因表达的调控是分子生物学研究的一个重要问题,也是基因治疗和基因功能研究的重要手段。诱导性基因表达系统可以从时间上调控基因的表达,是基因治疗和基因功能研究的重要工具之一。其中,四环素诱导基因表达系统(tetracycline inducible expression system,Tet system)是应用最广泛的一种,它可以在时间和空间上对基因进行严谨和高效地诱导表达。基于该系统获得了不同用途的转基因动物,这些模型动物的建立为研究特定基因的功能及其在疾病发生中的作用打下了实验基础。现就四环素诱导表达系统的原理和在小鼠模型上的研究应用做一综述。     

Tet调控STGC3基因表达CNE2细胞系的建立及其功能初步研究

利用Tet-on调控系统,建立受强力霉素诱导STGC3基因表达的CNE2细胞系,为进一步研究STGC3的功能提供了一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-STGC3转染入CNE2细胞,并用G418和潮霉素分别进行两轮筛选,运用RT-PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度强力霉素诱导CNE2/Tet/pTRE-STGC3细胞,RT-PCR检测STGC3的表达,确定强力霉素的最佳诱导浓度.采用此浓度的强力霉素分别诱导CNE2、CNE2/Tet/pTRE、CNE2/Tet/pTRE-STGC3三组细胞,测定细胞的生长曲线、克隆形成率和细胞周期分布.诱导STGC3基因高表达,CNE2细胞增殖速度显著减慢(P<0.05),克隆形成能力显著降低(P<0.01);流式细胞仪检测结果显示,瘤细胞群体中处于G0/G1期细胞数增加,细胞阻滞于G0/G1期.Tet调控STGC3基因表达CNE2细胞系的成功建立,为进一步研究STGC3基因的功能提供一个理想的细胞模型.     

Tet调控CYP2E1基因表达细胞系的代谢能力分析

应用Tet-On基因表达系统,调控CYP2E1基因在NIH 3T3细胞中的表达水平,探讨CYP2E1在化学致癌物二甲基亚硝胺(N-nitrosodimethylamine, NDMA)代谢中的作用．先后将 Tet-on基因表达系统的调控质粒pRevTet-on和反应质粒pRevTRE-2E1转染NIH 3T3细胞,分别用G418和潮霉素筛选,并通过RT-PCR和Western印迹检测,成功获得了3个具有良好诱导性Tet调控的CYP2E1基因表达细胞系（Tet/3T3-2E1）．应用不同浓度强力霉素(doxycycline, Dox)处理Tet/3T3-2E1细胞诱导CYP2E1表达,HPLC分析细胞对CYP2E1特异性药物探针氯唑沙腙的原位代谢能力及MTT法分析CYP2E1介导的NDMA细胞毒性作用．结果显示,Tet/3T3-2E1细胞CYP2E1的表达及其代谢能力有明显的Dox浓度依赖性,而且NDMA对Dox诱导组细胞有明显浓度依赖性细胞毒性作用,其IC₅₀值为12.06 μmol/L,无Dox诱导组未见明显的NDMA细胞毒性作用．该细胞模型的成功建立对于开展与CYP2E1相关的毒物、致癌物代谢研究,筛选抗毒物、抗癌物等具有重要价值．     

重组DNA技术中可诱导的基因表达调控系统

在重组DNA技术中,可诱导的基因表达调控系统可被用来调节目的基因的表达以达到基因功能研究、转基因动物研究、以及基因治疗研究等目的。该系统主要由诱导剂、可诱导的受体或转录因子、顺式作用元件以及载体系统四部分组成。本文以诱导剂为分类依据,叙述目前主要的6类可诱导的基因表达调控系统:类固醇激素受体诱导的基因表达调控系统、四环素诱导的基因表达调控系统、缺氧诱导的基因表达调控系统、高热诱导的基因表达调控系统、电离辐射诱导的基因表达调控系统和lac基因表达调控系统。     

鼻咽癌相关新基因NPCEDRG表达对CNE2细胞生长的影响

为研究鼻咽癌相关新基因NPCEDRG的功能,探讨其对鼻咽癌细胞生长特性的影响,利用Tet-on调控系统,建立受强力霉素(deoxycycline,Dox)诱导NPCEDRG基因表达的CNE2细胞系.运用RT-PCR选择背景表达低、诱导活性高的细胞克隆,以不同浓度Dox诱导CNE2/Tet/TRE-NPCEDRG细胞,确定Dox的最佳诱导浓度.借助形态学观察、细胞生长曲线、软琼脂克隆形成试验和流式细胞仪分析等方法,对Dox诱导NPCEDRG高表达后CNE2细胞的生物学行为进行了检测.结果显示,NPCEDRG高表达后CNE2细胞增殖速度显著减慢(P<0.05),克隆形成能力显著降低(P<0.01),瘤细胞群体中处于G0/G1期细胞数增加,S期细胞数减少,细胞阻滞于G0/G1期.Tet调控NPCEDRG基因表达CNE2细胞系成功建立,恢复NPCEDRG表达能部分逆转CNE2的恶性表型,证明NPCEDRG是一个鼻咽癌相关的抑瘤基因.     

过表达Runx2促进C2C12细胞成骨分化

利用Tet-on(Tetracycline-on)基因表达系统,通过强力霉素(doxycycline,DOX)诱导Runx2基因在C2C12细胞中的表达,探究Runx2促成骨分化功能,为其分子机制的研究提供一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-Flag-Runx2转染入C2C12细胞,并用G418和潮霉素分别进行2轮筛选,运用实时荧光定量PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度DOX诱导C2C12/Tet/pTRE-Flag-Runx2细胞,蛋白免疫印迹检测Runx2的表达,确定DOX的最佳诱导浓度与时间,并检测C2C12细胞的成骨分化能力.结果表明,诱导细胞最佳DOX浓度为10μg/ml;最佳诱导时间为12h;诱导后Runx2基因高表达,C2C12细胞向成骨方向分化(P0.05).成功建立Tet调控Runx2基因表达C2C12细胞系,为进一步研究Runx2基因功能分子机制提供理想的细胞模型.     

真核细胞内可诱导基因表达系统研究进展

建立可诱导基因表达系统是研究基因功能的重要手段。迄今为止的许多真核细胞内可诱导基因表达系统,包括以真核调控元件及原核基因调控元件为基础的可诱导表达系统,双系统、组织特异性及可诱导基因打靶系统等。     

基于四环素调控系统的病毒载体在基因治疗中的应用

基因治疗已经发展成为处理各种疾病的理想治疗模式.病毒载体是将基因递送到体内或体外的分裂和非分裂细胞的有效工具.病毒载体基因治疗在临床环境成功应用的一个重要问题是严谨和持续地调控基因表达.目前,已经报道的无毒且严谨的调控转基因表达系统中,四环素转录调控系统是在体内和体外研究最好而且高效的调控系统.Tet调控系统已经被编码在慢病毒属、腺病毒属、腺相关病毒属和逆转录病毒属,而且可以有效地对基因进行调控.将主要讨论已经在Tet调控系统调控下递送基因的主要载体系统及其在基因治疗上的应用.     

米非司酮基因诱导调控系统

在基因表达和基因治疗研究中,需要目的基因在特定的时间以适当的水平实现其表达,目的基因过度表达或不适当的表达将影响实验结果,在疾病治疗中甚至会产生致命的副作用,实现对目的基因的表达时间和表达水平的精确调控是一个非常关键的问题。目前生物学家们已构建了多种新型的基因表达调控系统,其中米非司酮诱导调控系统具有诱导效率高,背景表达低,安全性高等诸多优点,是基因调控研究中的重要进展,也是目前最有应用前景的调控系统之一。本文就其结构设计和应用研究方面的进展作一简要综述。     

Tet system in the brain: Transgenic rats and lentiviral vectors approach

Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues. genesis 47:274–280, 2009. © 2009 Wiley‐Liss, Inc.     

Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells.

Retroviral vectors that contain the tetracycline-inducible (Tet) system were developed. The two components of the Tet system were organized within the vectors in a manner that stringently maintains tetracycline-dependent regulation. Regulated expression of an indicator gene inserted into the retroviral vectors was examined in several different cell types. In infected NIH 3T3 cells, levels of induction in the absence of tetracycline were observed to be as much as 336-fold higher than levels in the presence of tetracycline, which were extremely low. Tetracycline-dependent regulation was observed in all other transduced cell types and ranged from 24- to 127-fold. The generation of retroviral vectors containing regulatory elements that allow for the regulated expression of heterologous genes and that have the ability to infect virtually all dividing target cells should greatly facilitate the biochemical and genetic examination of a broad range of genes. Moreover, these inducible retroviral vectors should prove useful in gene therapy applications.     

血小板生成素基因在NIH/3T3细胞中的表达与调控

应用 Tet- On基因表达系统 ,调控血小板生成素 (TPO)基因在 NIH/3T3细胞中的表达时间与水平 .籍脂质体介导的基因转移方法 ,p Tet- On质粒转染 NIH/3T3细胞株 ,得到稳定细胞株NIH/3T3- Tet- On.p TRE/TPO与 p TK- Hyg质粒共转染 NIH/3T3- Tet- On细胞株 ,得到双稳定细胞株 NIH/3T3- Tet- On- TPO.在培养基中加入或不加强力霉素 ,RT- PCR、Western印迹及 ELISA法检测培养上清 TPO表达 .结果表明 ,当培养基中不加强力霉素时 ,TPO无明显表达 (0 .1 μg/L) ;当培养基中加入 2 mg/L强力霉素时 ,TPO表达明显增高 (1 0 .8μg/L) .TPO表达水平与强力霉素浓度有关 ,随强力霉素浓度增高 ,TPO表达增加 .TPO表达水平还与强力霉素作用时间有关 ,加入强力霉素 6 h后 ,TPO表达明显增加 (1 .2μg/L) ,随培养时间延长 ,TPO表达增加 ,2 4 h达到峰值(1 0 .8μg/L) ,而且这种诱导作用是可逆的 .为进一步进行 TPO基因表达调控的体内研究奠定基础 ,有望为 TPO基因治疗提供一条可控的安全途径     

Silencing and un-silencing of tetracycline-controlled genes in neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P(tets)). We have discovered that stably integrated P(tet) becomes functionally silenced in the majority of neurons when it is inactive during development. P(tet) silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced P(tet) gene silencing, possibly by inducing promoter accessibility.     

一株受四环素及其衍生物诱导表达的Tet-on鼻咽癌细胞系

Ｔｅｔ－ｏｎ基因表达系统是新近发展的一种真核生物体外表达系统．该系统利用四环素及其衍生物对所感兴趣基因进行诱导表达．这种诱导表达具有严密、高效、可控性强、表达泄露小等优点．对于研究一些致死基因及毒性强的基因在细胞或体内的表达提供了极好的工具．利用Ｔｅｔ－ｏｎ基因表达系统,构建了一株鼻咽癌细胞系,该细胞系的建立为进一步研究一些鼻咽癌发病相关基因,如ＥＢ病毒潜伏膜蛋白基因、瘤基因、抑瘤基因等与鼻咽癌的相关性,提供了理想的细胞模型．     

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.     

Simple and fast method to test the receptor for advanced glycosylated endproducts (RAGE) for its tumor suppressive potential using the Tet-On system

The Tet gene expression system, that allows tightly controlled gene expression in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 293 cells in semi-solid medium. Establishing a Tet-On gene expression system involves two consecutive stable transfections. Here, we describe an alternative procedure to obtain a Tet-On gene expression system in a single transfection step for the use in tumor biology. The plasmids necessary for the regulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 clones were analyzed using PCR revealing 8 colonies to be double stably transformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression system could be completed in half of the time required for the original two step method. The generated 293 double stable cells were used in the clonogenic assay for the testing of the tumor suppressive potential of RAGE.