Optimization of luciferase activity in a tomato transient assay system

The gene encoding firefly luciferase is a commonly used reporter for transient expression assays in plants. We have found that the concentration of buffers normally used in luciferase assays is too low to adequately buffer acidic plant organs. This results in low apparent luciferase activity as well as high variability among replicates. In a transient assay system based on particle bombardment of ripe tomato fruit, luciferase activity driven by the 35S promoter of cauliflower mosaic virus was increased as much as 130 fold by increasing the concentration of the buffer from 50 mM to 300 mM. Using 300 mM buffer, expression levels of luciferase driven by three different plant promoters were found to reflect expression patterns in intact plants.     

Multichannel perfusion culture bioluminescence reporter system for long-term detection in living cells

We constructed a multichannel perfusion culture system. Using this device, we developed a perfusion device to detect six samples at the same time and demonstrated the response to dexamethasone of the circadian-related promoter activity in Rat1 fibroblast cells. We could detect the sequential phase shifts of the circadian peaks that are dependent on the timing of the drug treatments. We also demonstrate a temporal dual reporter assay using two kinds of secreted luciferase in the perfusion culture. The combination of secreted luciferase and multiple perfusion culture assay system reveals the effects of transient drug treatment for the pharmacological assay.     

一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建

双荧光素酶报告基因系统能够提供灵敏的读数,但该系统需要依赖组成型表达的内参对读数进行归一化。然而,大多数内参并不是在所有条件下都组成型表达。为此,文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先,突变BmV gP78启动子上的激素应答相关元件,获得了在家蚕细胞中稳定表达的组成型启动子BmV gP78M;然后,用BmV gP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列,成功构建了pRL-V gP78M内参质粒;最后,通过细胞转染实验证实pRL-V gP78M内参在家蚕细胞系中稳定表达,并且pRL-V gP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终,获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-V gP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时,该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。     

Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multi-cloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

人p53启动子萤光素酶报告基因的构建及其活性测定

目的：克隆p53基因的启动子,插入萤光素酶报告基因载体,并检测启动子活性。方法：采用PCR技术从人肝癌细胞系HepG2基因组中扩增人p53启动子,插入萤光素酶报告基因载体pGL4．0-empty,将重组质粒转染293T、ZR75-1、HepG2、A549细胞,测定p53启动子的转录活性。结果：构建了p53启动子的萤光素酶报告基因;通过测序及质粒酶切鉴定,所构建的p53启动子正确;活性实验表明,报告基因在多种细胞中显示构建的p53启动子活性,并呈现一定的剂量效应;转录因子USF能以剂量效应方式提高p53报告基因的转录活性。结论：克隆了人p53启动子,为进一步研究调控p53的转录因子奠定了基础。     

Rapid and highly sensitive detection of cadmium chloride induced cytotoxicity using the HSP70B' promoter in live cells

One unique to detect cytotoxicity is to utilize reporter gene assays for promoters that respond to stress-induced effects. In the present study, we discovered that the DNA sequence from nt -287 to +110 of the heat shock protein 70B' (HSP70B') gene could be used as a functional promoter to detect cytotoxicity of cadmium chloride. We thus detected cytotoxicity induced by cadmium chloride with the luciferase assay using this functional HSP70B' promoter, as well as the cell viability test based on the quantification of intracellular ATP. The luciferase assay using the functional HSP70B' promoter resulted in nearly maximal luciferase activity after only 12 h of exposure to cadmium chloride, however, with intracellular ATP quantification, the decrease in cell viability only reached a plateau after 24 h of exposure. Cytotoxicity detection limits for cadmium chloride with the functional HSP70B' promoter assay or cell viability based on ATP quantification were 130 ng/mL and 530 ng/mL, respectively. Our results therefore suggest that the novel reporter gene assay using a functional region of the HSP70B' promoter has significant advantages for the detection of cytotoxicity in terms of both speed and sensitivity, when compared to the cell viability test based on ATP quantification.     

Synthesis and biological evaluation of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.     

小鼠HMGB1启动子荧光素酶报告基因的构建及功能鉴定

利用PCR技术扩增小鼠高迁移率族蛋白1(HMGB1)基因启动子序列,构建小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1.经PCR、酶切及测序鉴定后,用脂质体法将pGL3-basic-HMGB1转入巨噬细胞264.7中,并应用萤光素酶测定系统检测其活性.检测结果显示pGL3-basic-HMGB1具有启动子活性.小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1的成功构建,为进一步研究HMGB1提供基本材料.     

Gaussia分泌型萤光素酶的研究进展及其应用

Gaussia分泌型萤光素酶是近年发现的一种来源于海洋桡脚类动物Gaussia princeps的新型分泌型萤光素酶,是目前已知的可自然分泌的最小萤光素酶,因其分子小、灵敏度高、半衰期短和可高效分泌,而成为一种理想的报告基因,广泛应用于体内外研究。我们就Gaussia分泌型萤光素酶的发光原理、荧光特性及其应用等进行简要综述。     

The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.     

低密度脂蛋白受体/报告基因系统建立及中药降脂药物的筛选

为了从中药中筛选降脂药物,本文构建了低密度脂蛋白受体／荧光素酶(LDLR／Luc)报告基因系统。将LDLR／Luc报告质粒转染至HepG2细胞,并优化细胞接种密度、细胞培养时间以及检测底物浓度等条件,获得较高荧光素酶活性;进一步选择普伐他汀作为阳性对照药物,可明显提高LDIR／Luc转录活性,表明成功地建立了靶向ldlr转录活性的报告基因筛选体系。应用该体系对500余种中药提取物进行了筛选,决明子、泽泻、穿龙薯蓣等提取物显示阳性,与过去报道采用动物模型研究的结果一致。     

Chimeric receptors as a tool for luminescent measurement of biological activities of steroid hormones

Some applications of chimeric cellular models are presented to study the biological activities of steroid hormones. We have used several chimeric constructs encoding the DNA binding domain of Gal4 yeast protein fused to the hormone binding domain of various steroid receptors (MR, PR, GR and ER). Interactions of these chimeric receptors with a 17-mer DNA sequence, specific for Gal-4, control expression of the firefly luciferase as a reporter gene. Stable transfected cell lines expressing the firefly luciferase under the control of different steroids were established and an efficient and easy sub-cloning was allowed with the help of an imaging system using a single-photon-counting camera. In the cell lines obtained, the bioluminescent response can be easily measured and thus used to measure specific biological activities of steroid agonists or antagonists. We observed that the responses are effector-concentration-dependent and their biological activities will be compared to those of native receptors.     

Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.     

启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用

垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide,PACAP）特异受体PAC1-R (PACAP receptor 1)是神经系统疾病药物开发的重要靶点。为了研究其表达的生物学机制,本研究克隆了PAC1-R基因转录起始位点上游从-2 500到+26的2 526 bp启动子片段,构建PAC1-R启动子驱动的荧光素酶基因报告载体pGL3-PAC1-Rp,并确证PAC1-R启动子荧光素酶报告系统在小鼠脑神经瘤细胞Neuro-2a和人神经母细胞瘤细胞SH-SY5Y中工作正常。运用此PAC1-R启动子荧光素酶报告系统,首次发现低浓度过氧化氢（hydrogen peroxide,H2O2）有效激活PAC1-R启动子,此作用可被转录因子特化蛋白1（specificity protein 1,SP1）抑制剂光神霉素A（mithramycin A）所抑制,提示SP1参与介导H2O2对PAC1-R启动子的激活作用。生物信息学分析显示,PAC1-R启动子含有多个SP1结合位点。PAC1-R启动子的荧光素酶报告系统的构建为深入探索PAC1-R高表达的作用与机制奠定了基础,低浓度H2O2对PAC1-R启动子激活作用的发现有助于深入诠释低浓度活性氧的生理学作用。