High-throughput Reporter Assay Research Articles

RAEdb: a database of enhancers identified by high-throughput reporter assays.

High-throughput reporter assays have been recently developed to directly and quantitatively assess enhancer activity for thousands of regulatory elements. However, there is still no database to collect these enhancers. We developed RAEdb, the first database to collect enhancers identified by high-throughput reporter assays. RAEdb includes 538 320 enhancers derived from eight studies, most of which were from six human cell lines. An activity score was assigned to each enhancer based on reporter assays. Based on these enhancers, 7658 epromoters (promoters with enhancer activity) were identified and stored in the database. RAEdb provides two ways of searches: the first is to search studies by species and cell line; the other is to search enhancers or epromoters by position, activity score, sequence and gene. RAEdb also provides a genome browser to query, visualize and compare enhancers. All data in RAEdb is freely available for download.

Human genome-wide measurement of drug-responsive regulatory activity

Environmental stimuli commonly act via changes in gene regulation. Human-genome-scale assays to measure such responses are indirect or require knowledge of the transcription factors (TFs) involved. Here, we present the use of human genome-wide high-throughput reporter assays to measure environmentally-responsive regulatory element activity. We focus on responses to glucocorticoids (GCs), an important class of pharmaceuticals and a paradigmatic genomic response model. We assay GC-responsive regulatory activity across >108 unique DNA fragments, covering the human genome at >50×. Those assays directly detected thousands of GC-responsive regulatory elements genome-wide. We then validate those findings with measurements of transcription factor occupancy, histone modifications, chromatin accessibility, and gene expression. We also detect allele-specific environmental responses. Notably, the assays did not require knowledge of GC response mechanisms. Thus, this technology can be used to agnostically quantify genomic responses for which the underlying mechanism remains unknown.

High-throughput characterization of genetic effects on DNA-protein binding and gene transcription.

Many variants associated with complex traits are in noncoding regions and contribute to phenotypes by disrupting regulatory sequences. To characterize these variants, we developed a streamlined protocol for a high-throughput reporter assay, Biallelic Targeted STARR-seq (BiT-STARR-seq), that identifies allele-specific expression (ASE) while accounting for PCR duplicates through unique molecular identifiers. We tested 75,501 oligos (43,500 SNPs) and identified 2720 SNPs with significant ASE (FDR < 10%). To validate disruption of binding as one of the mechanisms underlying ASE, we developed a new high-throughput allele-specific binding assay for NFKB1. We identified 2684 SNPs with allele-specific binding (ASB) (FDR < 10%); 256 of these SNPs also had ASE (OR = 1.97, P-value = 0.0006). Of variants associated with complex traits, 1531 resulted in ASE, and 1662 showed ASB. For example, we characterized that the Crohn's disease risk variant for rs3810936 increases NFKB1 binding and results in altered gene expression.

QuASAR-MPRA: accurate allele-specific analysis for massively parallel reporter assays.

The majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets. We have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays. http://github.com/piquelab/QuASAR/tree/master/mpra. fluca@wayne.edu or rpique@wayne.edu. Supplementary data are available online at Bioinformatics.

Characterization of noncoding regulatory DNA in the human genome.

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1pro, with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Abstract PR12: Targeting eIF2α phosphorylation for cancer therapy

Abstract Solid tumor tumors contain hypoxic regions. Tumor hypoxia is co-present with reduced nutrients and usually intracellular acidification. Lack of O2 and nutrients, and intracellular acidification causes endoplasmic reticulum stress (ER). In addition to canonical hypoxia response, tumor cells activate integrated ER-stress response (IERSR) in a spatially and temporally regulated manner to enter into dormancy under hypoxia and de-activate IERSR to return to a proliferative state once blood supply is restored. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) branch of IERSR is a critical determinant of tumor survival under hypoxia. Temporally and spatially regulated eIF2α phosphorylation attenuates overall translation while favoring translation of a subset of mRNAs coding for transcription factors and accessory proteins. This results in re-programming of gene expression that favors tumor-cell survival. Once the hypoxic conditions are ameliorated eIF2α phosphorylation is reversed and tumor cells exit dormancy. Sustained eIF2α phosphorylation on the other hand not only reduces overall protein synthesis to inhibit proliferation but also re-programs gene expression to induce expression of pro-apoptotic proteins such as CHOP to induce cell death. We therefore hypothesized that pharmacological activation of eIF2α kinases to induce sustained eIF2a phosphorylation will cause selective demise of tumors. To test this hypothesis and develop mechanism specific anti-cancer agents we generated high throughput reporter assays and screened large chemical libraries for inducers of eIF2α phosphorylation. Our Screening effort combined with functional genetic studies, and synthesis and evaluation of focused libraries resulted in identification of highly potent and specific activators of three eIF2α kinases, heme-regulated inhibitor (HRI), Protein kinase R, and PKR-like ER-kinase (PERK). These agents downregulate expression of pro-proliferative proteins such as cyclin D1 and cyclin E while upregulating the expression of pro-apoptotic proteins such as CHOP and tumor suppressor proteins such as BRCA1; all dependent on eIF2α phosphorylation. We will present unpublished data on the structure activity relationship studies of the second generation activators of eIF2α kinases. We will also present in vivo data demonstrating that inducers of eIF2α phosphorylation fully abrogate proliferation of human breast and melanoma xenografts growth with no apparent toxicity. At the molecular level, they induce eIF2α phosphorylation, expression of pro-apoptotic protein CHOP and cell cycle inhibitor p27Kip1 while inhibiting Cyclin D1 expression in xenograft tumors. Tumor cells acquire resistance to targeted therapy by bypassing the signaling node inhibited by treatment; activating alternate oncogenic pathway or downstream signaling node. Because translation initiation is in the apex of many oncogenic and proliferative pathways we hypothesized that translation initiation inhibitors will abrogate proliferation of targeted-therapy resistant cancer cell lines independent of the primary therapy or the mechanism of resistance. Our studies in melanoma cancer cells resistant to B-Raf or B-Raf and MEK inhibitors demonstrate that activators of eIF2α kinases or inhibitors of eIF4F complex inhibit the proliferation of parental and drug resistant cancer-cells with the same efficiency. We will also present these data and on our progress in resolving the crystal structure of full-length HRI which should allow us to accelerate drug development by improving the specificity and potency of our current lead compounds. We conclude that our second generation activators of eIF2α kinases are potent and mechanism specific agents that can be developed for treatment of naïve and drug resistant tumors. This abstract is also being presented as Poster B20. Citation Format: Revital Yefidoff-Freedman, Ronghui Du, Fan Jing, Quingwen Zhang, Bertal H. Aktas. Targeting eIF2α phosphorylation for cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR12.

Efficient production of glycosylated Cypridina luciferase using plant cells

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20–30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 μM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.

MPRAnator: a web-based tool for the design of massively parallel reporter assay experiments.

MotivationWith the rapid advances in DNA synthesis and sequencing technologies and the continuing decline in the associated costs, high-throughput experiments can be performed to investigate the regulatory role of thousands of oligonucleotide sequences simultaneously. Nevertheless, designing high-throughput reporter assay experiments such as massively parallel reporter assays (MPRAs) and similar methods remains challenging.ResultsWe introduce MPRAnator, a set of tools that facilitate rapid design of MPRA experiments. With MPRA Motif design, a set of variables provides fine control of how motifs are placed into sequences, thereby allowing the investigation of the rules that govern transcription factor (TF) occupancy. MPRA single-nucleotide polymorphism design can be used to systematically examine the functional effects of single or combinations of single-nucleotide polymorphisms at regulatory sequences. Finally, the Transmutation tool allows for the design of negative controls by permitting scrambling, reversing, complementing or introducing multiple random mutations in the input sequences or motifs.Availability and implementationMPRAnator tool set is implemented in Python, Perl and Javascript and is freely available at www.genomegeek.com and www.sanger.ac.uk/science/tools/mpranator. The source code is available on www.github.com/hemberg-lab/MPRAnator/ under the MIT license. The REST API allows programmatic access to MPRAnator using simple URLs.Supplementary information Supplementary data are available at Bioinformatics online.

Abstract PR02: Identification of a potent and selective chemical probe for exploring the role of CDK8/19 in cancer biology

Abstract The discovery of chemical probes by testing libraries of small molecules against cellular pathway screens has re-emerged as a hit discovery strategy. We previously reported a series of 3,4,5-trisubstituted pyridines identified from a high-throughput cell-based reporter assay of WNT pathway signalling. We were able to optimise this series and identified CCT251545 as a chemical tool that potently inhibits readouts of WNT signalling pathway activity with evidence for in vivo activity. A series of cell-based assays activating WNT signalling at distinct loci identified the TCF locale as the likely target. CCT251545 was not a general inhibitor of the transcription machinery and did not affect expression of TCFs. Regulation of beta-catenin/TCF transcription involves recruitment or loss of DNA binding proteins, histone modification and also interaction with additional protein networks. Given the potential complexity of these multiple networked interactions, we employed an unbiased chemical proteomics strategy to identify molecular targets of CCT251545. Knowledge of the structure-activity-relationships of the series allowed us to identify derivatives that retained cellular potency and were suitable for linker coupling to generate an affinity matrix. SILAC-based quantitative mass spectrometry identified target proteins captured by incubation of the immobilised compound with lysates from cells grown in media with different forms of isotopically-labeled amino acids. Competition experiments with unconjugated analogues allowed us to identify affinities of proteins bound to the immobilised probe. These experiments identified Mediator complex-associated protein kinases CDK8 and CDK19 as targets of the 3,4,5-trisubstituted pyridine series. We show that CCT251545, is a selective and potent ATP competitive chemical probe for these two kinases, with &amp;gt;100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a Type 1 binding mode involving insertion of the CDK8 C-terminus into the ligand-binding site. In contrast to Type II-like CDK8/19 ligands, CCT251545 displays potent cell-based activity. We demonstrate that CCT251545 not only alters WNT-pathway regulated gene expression, but also other CDK8/19 targets including genes regulated by STAT1. Consistent with this we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we show in vivo activity of CCT251545 in WNT-dependent tumours. The potential role for CDK8, and by implication CDK19, as an oncoprotein further highlights the need for an active and specific probe compound that can be used as a complementary reagent to RNAi tools. Here we demonstrate that the small molecule CCT251545 fulfils this requirement as a potent, selective, cell-active chemical probe that can be used with confidence to explore the consequences of CDK8/19 kinase function in cellular and in vivo animal models. Citation Format: Paul A. Clarke, Trevor Dale, Christina Esdar, Dennis Waalboer, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Aurelie Mallinger, Ken Ewan, Robert te Poele, Sharon Gowan, Paul Workman, Kai Schiemann, Suzanne A. Eccles, Dirk Wienke, Julian Blagg. Identification of a potent and selective chemical probe for exploring the role of CDK8/19 in cancer biology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR02.

IMPS-44S100B INHIBITION WITH DULOXETINE, A SEROTONIN-NOREPINEPHRINE REUPTAKE INHIBITOR, ALTERS MACROPHAGE POLARIZATION AND ABROGATES GLIOMA GROWTH IN MICE

S100B, a member of the multigene family of Ca2 + -binding proteins, is overexpressed by most malignant gliomas but its biological role in gliomagenesis is unclear. Recently, we demonstrated that low concentrations of S100B attenuated microglia activation through the induction of STAT3. Furthermore, S100B downregulation in a murine glioma model inhibited macrophage trafficking and tumor growth. Based on these observations, we hypothesized that S100B inhibitors may have antiglioma properties through modulation of tumor microenvironment. To discover novel S100B inhibitors, we developed a high-throughput screening cell-base S100B promoter-driven luciferase reporter assay. Initial screening of 25,904 compounds in NIH and ChemBridge libraries identified 1297 hits with >85% S100B inhibitory activity. One compound, duloxetine (Dul, a serotonin-norepinephrine reuptake inhibitor) was selected for the initial proof-of-concept studies. At low concentrations (1-5 □M) Dul inhibited S100B and CCL2 production by mouse GL261 glioma cell lines, but had minimal cytotoxic activity. In vivo, Dul (30 mg/kg/14 days) inhibited S100B production, altered polarization and trafficking of macrophages and abrogated the growth of intracranial GL261 gliomas. Dul therapeutic efficacy, however, was not observed in the K-Luc glioma model that expresses low levels of S100B. These findings affirm the role of S100B in gliomagenesis and justify the development of S100B inhibitors for glioma therapy.

Probing the microRNA pathway with small molecules

MicroRNA (miRNA)/RNA interference (RNAi) is recognized as one of the most important mechanisms regulating gene expression at the posttranscriptional level in eukaryotic cells. The main components within the miRNA/RNAi pathway are now known and well characterized, but studies on the molecular mechanisms that regulate the activity of the miRNA/RNAi pathway are just beginning to emerge. High-throughput reporter assays have been developed to monitor the activity of the miRNA/RNAi pathway and applied in a proof-of-concept pilot screening, which has led to the identification of some inhibitors and activators that either generally or specifically regulate the activity of the miRNA/RNAi pathway. In addition, combined with multidisciplinary approaches like proteomics, biochemistry, and genetics, some protein co-factors were found to play important roles in the regulation of the miRNA/RNAi pathway. Herein we highlight the high-throughput reporter assays developed in recent years and the resulting discovery of miRNA/RNAi enhancers and inhibitors.

Chemical Biology Approach for Dissection of RNAi/miRNA Pathway

RNA interference (RNAi)/miRNA has been recognized as one of the most important regulation mechanisms of gene expression at posttranscriptional levels in eukaryotic cells. Although the main components within the RNAi/ miRNA pathway have been identified and characterized, studies on the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have begun to emerge in recent years. High-throughput reporter assays have been developed to monitor the activity of the RNAi/miRNA pathway and applied for proof-of-concept pilot screening, leading to identification of some inhibitors and activators that are generally or specifically regulate activity of the RNAi/ miRNA pathway. Additionally, in combination with multidisciplinary approaches such as proteomics, biochemistry and genetics, to identify the targeting components, some protein co-factors that to play significant roles in the regulation of the activity of the RNAi/miRNA pathway have been identified. Herein we highlight the high throughput reporter assays developed in recent years and the resulting discovery of the RNAi/miRNA enhancers and inhibitors, with attention on developing novel RNAi- or miRNA-based therapeutic interventions.

Emergence of Chemical Biology Approaches to the RNAi/miRNA Pathway

RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression posttranscriptionally. Gene regulation by RNAi is now recognized as one of the major regulatory pathways in eukaryotic cells. Although the main components of the RNAi/miRNA pathway have been identified, the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have only begun to emerge within the last couple of years. Recently, high-throughput reporter assays to monitor the activity of the RNAi/miRNA pathway have been developed and used for proof-of-concept pilot screens. Both inhibitors and activators of the RNAi/miRNA pathway have been found. Although still in its infancy, a chemical biology approach using high-throughput chemical screens should open up a new avenue for dissecting the RNAi/miRNA pathway, as well as developing novel RNAi- or miRNA-based therapeutic interventions.

Sensitive and Convenient Yeast Reporter Assay for High-Throughput Analysis by Using a Secretory Luciferase from Cypridina noctiluca

The yeast reporter assay has been widely used in various applications such as detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system. The molecular characteristics of the reporter enzyme are critical determinants for this assay. We herein report the establishment of a novel yeast reporter assay using a secretory luciferase, Cypridina noctiluca luciferase (CLuc), as an alternative to the conventional beta-galactosidase. The CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay. A yeast high-throughput reporter assay was established with a laboratory automation system, and the transcriptional activity of hundreds of yeast promoter fragments was comprehensively determined. Our results indicate that the yeast CLuc reporter assay is a promising tool for large-scale and sensitive analysis in the development of new drugs and in various fields of biotechnology research.

A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

BackgroundRegulation of gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific DNA-protein interactions. In this study we finely map the genome-wide targets of the CREB protein across all known and predicted human promoters, and characterize the functional consequences of a subset of these binding events using high-throughput reporter assays. To measure CREB binding, we used HaloCHIP, an antibody-free alternative to the ChIP method that utilizes the HaloTag fusion protein, and also high-throughput promoter-luciferase reporter assays, which provide rapid and quantitative screening of promoters for transcriptional activation or repression in living cells.ResultsIn analysis of CREB genome-wide binding events using a comprehensive DNA microarray of human promoters, we observe for the first time that CREB has a strong preference for binding at bidirectional promoters and unlike unidirectional promoters, these binding events often occur downstream of transcription start sites. Comparison between HaloCHIP-chip and ChIP-chip data reveal this to be true for both methodologies, indicating it is not a bias of the technology chosen. Transcriptional data obtained from promoter-luciferase reporter arrays also show an unprecedented, high level of activation of CREB-bound promoters in the presence of the co-activator protein TORC1.ConclusionThese data suggest for the first time that TORC1 provides directional information when CREB is bound at bidirectional promoters and possible pausing of the CREB protein after initial transcriptional activation. Also, this combined approach demonstrates the ability to more broadly characterize CREB protein-DNA interactions wherein not only DNA binding sites are discovered, but also the potential of the promoter sequence to respond to CREB is evaluated.

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.

Progress in methodology. Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.

Seven-transmembrane G-protein-coupled receptors play a central role in physiology by facilitating cell communication through recognition of a wide range of ligands. Even more important, they represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and hence their functions, remain to be identified. These receptors are referred to as "orphan" receptors. A pre-requisite for the identification of ligands activating orphan receptors is powerful assay systems. Until now, reporter gene assays have not been in common use in this process. Here, we summarize our development of improved reporter gene assays. We optimized reporter gene assays in respect of (i) the promoter region of the construct, (ii) the reporter enzyme used, (iii) and the assay procedure. Furthermore, an unique fluorescence-based clone selection step was introduced, allowing rapid selection of the most sensitive reporter cell clones when establishing stable reporter cell lines. Mathematical formulae are provided to enable a simple and reliable comparison between different cell lines, when tested with a compound of interest. The resulting reporter cell lines responded in a very sensitive way to the stimulation of various test receptors. The reporter system was termed HighTRACE (high-throughput reporter assay with clone election). Its high assay quality makes it suitable as a primary screening tool. Ligands for two recently unknown 7TM receptors were identified using the HighTRACE system i.e., two cell surface free fatty acid receptors, GPR40 (FFA1R) and GPR43 (FFA2R). The identification was accomplished using a reverse pharmacology approach.