CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.National Institute of Environmental Health Sciences (Training Grant in Environmental Toxicology T32-ES007020)Massachusetts Institute of Technology. Center for Environmental Health Sciences (P30-ES002109)National Institute of Environmental Health Sciences (5-UO1-ES016045)National Institute of Environmental Health Sciences (1-R21-ES019498)National Institute of Environmental Health Sciences (R44-ES021116

Engineering a single cell microarray platform for high throughput DNA damage and repair analysis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.DNA damage contributes to cancer, aging, and heritable diseases. Ironically, DNA damaging agents are also commonly used in current cancer treatment. We therefore need robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. The single cell gel electrophoresis (comet) assay has become a standard method for DNA damage analysis, however, it is not well suited for use in clinical and epidemiological settings due to issues of low throughput, poor reproducibility, and a laborious image analysis requirement. To overcome these limitations, we applied microfabrication techniques to engineer an arrayed cell comet platform that maximizes the number of analyzable cells and provides spatial encoding for automated imaging and analysis. Additionally, we developed complementary software that eliminates the inherent bias of manual analysis by automatically selecting comets from the defined array. In its 96-well format, the so-called CometChip integrates with high throughput screening technologies, further increasing throughput and removing user error. This improved approach enables multiple cell types, chemical conditions, and repair time points to be assayed in a single gel with improved reproducibility and processing speed, while maintaining the simple protocol and versatility of the comet assay to assess a wide range of DNA damage. Using the CometChip, we evaluated a variety of DNA damaging agents, revealing repair profiles that can be used to gain insight into biological mechanisms of damage sensitivities. We confirmed the ability of the CometChip to identify deficiencies in four major DNA repair pathways, supporting the use of the assay in determining pathway sensitivities that may be useful in guiding treatment strategies that more selectively target cancerous cells and reduce side-effects. We also used the platform to evaluate potential inhibitors of DNA repair, which are emerging as promising adjuvants in cancer management. Taken together, the CometChip enables high throughput genotoxic evaluation of chemical exposures, discovery of novel chemotherapeutic strategies, and measurement of DNA repair kinetics for identification of susceptible populations and disease prevention. The CometChip is a significant advancement in DNA damage and repair technology, providing high throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis.by David McGregor Weingeist.Ph.D

Single-cell analysis tools for drug discovery and development

The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed

Interaction of Bestrophin-1 and Ca2+ Channel β-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits