Inferring gene expression dynamics from reporter protein levels

We present a mathematical method for inferring the dynamics of gene expression from time series of reporter protein assays and cell populations. We show that estimating temporal expression dynamics from direct visual inspection of reporter protein data is unreliable when the half-life of the protein is comparable to the time scale of the expression dynamics. Our method is simple and general because it is designed only to reconstruct the pattern of protein synthesis, without assuming any specific regulatory mechanisms. It can be applied to a wide range of cell types, patterns of expression, and reporter systems, and is implemented in publicly available spreadsheets. We show that our method is robust to a several possible types of error, and argue that uncertainty about the decay kinetics of reporter proteins is the limiting factor in reconstructing the temporal pattern of gene expression dynamics from reporter protein assays. With improved estimates of reporter protein decay rates, our approach could allow for detailed reconstruction of gene expression dynamics from commonly used reporter protein systems.     

A synthetic xylanase as a novel reporter in plants

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene (sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUSPlus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.Abbreviations Act1 Rice actin promoter - AZCL-xylan Azurine cross-linked xylan - AU absorbance units - Blt4.9 Barley lipid transfer protein promoter - GEB GUS extraction buffer - GFP Green fluorescent protein - GluB-1 Rice glutelin B-1 promoter - GUS -Glucuronidase - LUC Luciferase - sXynA Synthetic xylanase A gene - Ubi-1 Maize ubiquitin promoter - XAB Xylanase assay buffer - XYN Xylanase Communicated by P. Lakshmanan     

A reporter gene assay for screening of PDE4 subtype selective inhibitors

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.     

A single-vector EYFP reporter gene assay for G protein-coupled receptors

We here present an improved and simplified assay to study signal transduction of the G_s class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any G_s protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2).     

Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.     

Enhancer-mediated reporter gene expression in Arabidopsis thaliana: a forward genetic screen

Gene expression is controlled and regulated by interactions between cis-regulatory DNA elements (CREs) and regulatory proteins. Enhancers are one of the most important classes of CREs in eukaryotes. Eukaryotic genes, especially those related to development or responses to environmental cues, are often regulated by multiple enhancers in different tissues and/or at different developmental stages. Remarkably, little is known about the molecular mechanisms by which enhancers regulate gene expression in plants. We identified a distal enhancer, CREβ, which regulates the expression of AtDGK7, which encodes a diacylglycerol kinase in Arabidopsis. We developed a transgenic line containing the luciferase reporter gene (LUC) driven by CREβ fused with a minimal cauliflower mosaic virus (CaMV) 35S promoter. The CREβ enhancer was shown to play a role in the response to osmotic pressure of the LUC reporter gene. A forward genetic screen pipeline based on the transgenic line was established to generate mutations associated with altered expression of the LUC reporter gene. We identified a suite of mutants with variable LUC expression levels as well as different segregation patterns of the mutations in populations. We demonstrate that this pipeline will allow us to identify trans-regulatory factors associated with CREβ function as well as those acting in the regulation of the endogenous AtDGK7 gene.     

A new electrochemical assay method for gene expression using HeLa cells with a secreted alkaline phosphatase (SEAP) reporter system

A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4 × 4 array of microwells having a size of 100 μm × 100 μm (diameter × depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay.     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

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.     

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。     

A split luciferase-based reporter for detection of a cellular macromolecular complex

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.     

Alkaline phosphatase vs luciferase as secreted reporter molecules in vivo

Secreted alkaline phosphatase (SEAP) and Metridia luciferase (MLuc) are useful reporter molecules in vitro, but little is understood about their usefulness in vivo. In this study, we investigated in vivo activity of recombinant SEAP and MLuc in blood and urine. When SEAP-transfected cells or recombinant SEAP were injected into rats, substantial increase in the level of serum SEAP was observed. In contrast, activity of SEAP was not detected in urine of rats injected with either the SEAP-transfected cells or recombinant SEAP. SEAP activity was also undetectable in urine of SEAP-injected Nagase analbuminemic rats in which glomerular permeability to macromolecules is enhanced. When MLuc-transfected cells were implanted into rats, activity of MLuc was undetectable not only in urine but also in serum. Even immediately after intravenous injection of recombinant MLuc, activity of MLuc was not detected in serum. Subsequent experiments revealed that, in contrast to SEAP, MLuc was rapidly inactivated either by rat serum, fetal bovine serum, or human serum. Albumin was identified as the molecule responsible for the inhibition of MLuc activity. These data elucidated advantages and limitations of secreted reporter molecules SEAP and MLuc under in vivo situations.     

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice

BACKGROUND: The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins. METHODS: We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)-deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo, and immunological responses after gene delivery to mice. RESULTS: In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T-lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP. CONCLUSIONS: Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter gene for preclinical mouse studies.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase,superoxide dismutase and catalase gene promoters in V79 cells

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.