Miniaturization of a Functional Transcription Assay in Yeast (Human Progesterone Receptor) in the 384- and 1536-Well Plate Format

Berg, Michael G.; Undisz, Katrin; Thiericke, Ralf; Moore, Thomas C.; Posten, Clemens

doi:10.1177/108705710000500203

Cited by 22 publications

(7 citation statements)

References 6 publications

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“…Additionally, the microarray format uses 50-fold less assay volume in comparison to the miniaturized 1536-well plate format, leading to potential savings in screening costs. [36, 37]…”

Section: Resultsmentioning

confidence: 99%

A sandwiched microarray platform for benchtop cell-based high throughput screening

Wu¹,

Wheeldon²,

Guo³

et al. 2011

Biomaterials

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The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies. Here, we present a simple microarray sandwich system suitable for screening chemical libraries in cell-based assays at the benchtop. The microarray platform delivers chemical compounds to isolated cell cultures by ‘sandwiching’ chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays was generated without cross-contamination between neighboring assays. After chemical exposure, cell viability was analyzed by fluorescence detection of cell viability indicator assays on a per microwell basis in a standard microarray scanner. We demonstrate the efficacy of the system by generating four hits from toxicology screens towards MCF-7 human breast cancer cells. Three of the hits were identified in a combinatorial screen of a library of natural compounds in combination with verapamil, a P-glycoprotein inhibitor. A fourth hit, 9-methoxy-camptothecin, was identified by screening the natural compound library in the absence of verapamil. The method developed here miniaturizes existing HTS systems and enables the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. We anticipate broad application of such a system as it is amenable to combinatorial drug screening in a simple, robust and portable platform.

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“…Additionally, the microarray format uses 50-fold less assay volume in comparison to the miniaturized 1536-well plate format, leading to potential savings in screening costs. [36, 37]…”

Section: Resultsmentioning

confidence: 99%

A sandwiched microarray platform for benchtop cell-based high throughput screening

Wu¹,

Wheeldon²,

Guo³

et al. 2011

Biomaterials

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“…Luminescent or fluorescent β‐galactosidase substrates are now available and offer a route to increase the sensitivity of detection and range of applications for monitoring gene expression with a range of strains containing lacZ fusions (Oender et al , 2006; Serebriiskii and Golemis, 2000). At the same time, advances in microplate technology have allowed both assay miniaturization and the possibility to save time, reagents and increase dramatically the number of samples analysed (Berg et al , 2000; Brouchon‐Macari et al , 2003). We were interested in using a reporter assay that would be compatible with the robotically controlled liquid‐handling systems used in high‐throughput compound library screens, and which therefore required minimal sample handling and no centrifugation steps.…”

Section: Introductionmentioning

confidence: 99%

A Saccharomyces cerevisiae cell‐based quantitative β‐galactosidase assay compatible with robotic handling and high‐throughput screening

Almeida

Burgess

Shema

et al. 2007

Yeast

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Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-β-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 µl yeast cell culture and with <100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.

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“…For instance, the FUS1-LacZ reporter construct provides a colorimetric readout of agonist-induced β-galactosidase activity by using chlorophenolred-β-D-galactoside (CPRG) [55] or onitrophenyl-β-D-galactopyranoside (ONPG) [76] as substrates. The sensitivity of this bioassay can be improved by using more expensive fluorogenic β-galactosidase substrates, such as fluorescein-di-(β-D-galactopyranoside) (FDG) [77]. Another example is the FUS1-luc reporter that enables agonist-induced luciferase synthesis.…”

Section: Expression Of Gpcrs In Protease-deficient Yeast Strainsmentioning

confidence: 99%

Yeast System as a Screening Tool for Pharmacological Assessment of G Protein Coupled Receptors

Minic

Sautel²,

Salesse³

et al. 2005

CMC

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G-protein-coupled receptors (GPCRs) constitute the largest but the most divergent class of cell surface proteins. Although they are thought to share a common 3D-structure composed of seven transmembrane helical domains, they can be activated by extracellular signals as diverse as light, peptides, proteins, lipids, organic odorants, taste molecules, nucleotides or nucleosides. They are involved in an extraordinarily large number of physiological functions and are therefore potential drug targets for many human diseases. During the last decade various GPCRs have been successfully expressed in S. cerevisiae. Yeast is an attractive expression system because it offers the genetic engineering tools typical of a microorganism while possessing an eukaryotic type of secretory pathway and post-translational machinery. This host is particularly attractive for in-vivo manipulation of these receptors due to the high homology between the yeast pheromone signaling pathway and that of mammalian GPCRs. When expressed in yeast, mammalian GPCRs have been shown to couple functionally to either the endogenous yeast Galpha (Gpa1), or co-expressed mammalian Galpha subunits (wild-type or chimeric), and are characterized by a similar pharmacology in response to agonists or antagonists as in native cells. Heterologous expression of wild type or mutant GPCRs in S. cerevisiae allows a rapid assessment of their ability to detect and transduce extracellular stimulations, through the use of a reporter system. Furthermore, this approach is amenable to high-throughput screening of new drugs, which would provide a determinant advantage in the field of therapeutic research, and also for investigation of the still unknown ligands of orphan receptors. This review will focus on the latest developments of yeast-based technology to screen for potential GPCR agonists/antagonists.

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Miniaturization of a Functional Transcription Assay in Yeast (Human Progesterone Receptor) in the 384- and 1536-Well Plate Format

Cited by 22 publications

References 6 publications

A sandwiched microarray platform for benchtop cell-based high throughput screening

A sandwiched microarray platform for benchtop cell-based high throughput screening

A Saccharomyces cerevisiae cell‐based quantitative β‐galactosidase assay compatible with robotic handling and high‐throughput screening

Yeast System as a Screening Tool for Pharmacological Assessment of G Protein Coupled Receptors

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