GCLC基因上游AHR/ARNT元件负性调控GCLC基因在大鼠支气管上皮细胞的表达

研究谷氨酰半胱氨酸合成酶催化亚单位(GCLC)基因上游调控序列中2个AHR/ARNT元件的功能,从而了解γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因转录调节特征．分别构建缺失2个位点AHR/ARNT元件的GCLC基因上游近端序列的萤光素酶报道基因载体以及含有2个AHR/ARNT元件核心序列的萤光素酶报道基因载体．转染大鼠支气管上皮细胞(RTE),比较检测野生与缺失报道载体的基因转录调控效率;利用电泳迁移率变动实验（EMSA）和超级迁移率变动实验检测AHR/ARNT元件与AHR以及ARNT因子的特异性结合;通过转染AHR因子真核表达质粒进一步确定AHR/ARNT元件与AHR结合在GCLC基因表达中的最终作用．结果显示,相比其野生序列,缺失AHR/ARNT元件（-1 090～-1 085）和双缺失AHR/ARNT元件（-1 090～-1 085,-215～-210）的GCLC上游调控序列报道载体在RTE显著提高萤光素酶表达（均P＜0.05）,而缺失AHR/ARNT元件（-215～-210）则未见显著影响（P＞0.05）; 独立AHR/ARNT元件（-1 090～-1 085）具有转录促进作用（P＜0.05）而独立AHR/ARNT元件（-215～-210）无明显影响（P＞0.05）．转染CMV2-AHR能够抑制野生型和缺失型报道载体的萤光素酶表达（P＜0.05）．EMSA证实GCLC基因上游调控区域的2个AHR/ARNT元件均有核蛋白结合,并且超级迁移率变动实验显示结合的蛋白主要含有转录因子AHR以及ARNT．因此,2个AHR/ARNT元件均可以与异源二聚体AHR/ARNT结合,AHR/ARNT元件（-1 090～-1 085）是GCLC基因中重要的抑制元件．     

New plant promoter and enhancer testing vectors

We describe here a set of binary vectors suitable forAgrobacterium-mediated gene transfer and specially designed for studying plant promoters. These vectors are based on the use of thegus reporter gene, contain multiple unique restriction sites upstream of thegus gene, and minimal promoters for testing the effect of enhancers or activator elements. In addition, an intron-containinggus (uidA) gene was introduced into one of these vectors in order to examine reporter gene activity in tissues whereAgrobacterium contamination may be a problem or in transient expression assays.     

Identification and Characterization of Enhancer-Blocking Insulators to Reduce Retroviral Vector Genotoxicity

The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.     

A33启动子结肠癌特异性探究及SV40增强子对其转录活性影响

为了探究A33核心启动子结肠癌特异性及SV40增强子对其转录水平的影响,该研究通过构建A33核心启动子和带SV40增强子的A33核心启动子(eA33)的荧光素酶报告基因载体pGL3-A33和pGL3-eA33,与内参照pRL-SV40质粒共转染至不同的细胞系中,利用双荧光素酶检测系统检测分析了A33和eA33启动子在不同细胞系中的转录活性。结果显示,A33核心启动子在结肠癌细胞系中具有转录活性低,但结肠癌特异性好的特点,而在其他类型癌细胞中基本没有活性。同时发现,eA33在各类癌细胞中的转录水平与A33相比,均呈大幅度提高,有显著性差异(P<0.01),但SV40增强子能显著增强A33启动子转录活性的同时减低了其结肠癌特异性。这为靶向癌症基因—病毒治疗策略在结肠癌的生物治疗应用中寻找结肠癌特异性的启动子奠定了研究基础。     

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.     

Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene.These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.