dna DNA damage is known to drive mutations and cell toxicity, both of which promote cancer and aging. Recent results also suggest that DNA damage may modulate disease progression following infection. Research in the Engelward laboratory centers on the interplay between DNA damage and its downstream consequences, with the goal of understanding the underlying mechanisms that drive cell toxicity, mutagenesis, and ultimately disease.

research_1Progress in science depends on having the appropriate technology to ask the important questions. Recognizing that homology directed repair modulates disease susceptibility, this laboratory was the first to create a ‘recombomouse’ model in which rare recombinant cell fluoresce. This gave rise to many studies of environmental and genetic factors that modulate the risk of large-scale sequence rearrangements.

research_2With an interest in focusing on common human conditions, we turned our attention to inflammation. By inducing inflammation in the recombomice, we have shown that repeated exposure to inflammation induces genetic rearrangements. Furthermore, recent studies are aimed at understanding the impact of inflammation on susceptibility to other exogenous exposures, such as chemicals in our food or in pollution.

research_4The interplay between DNA repair pathways can sometimes be a major driver of sequence instability. We have exploited genetically engineered mice that carry defects in specific DNA repair genes in order to learn about how one DNA pathway impacts another. Interestingly, these studies show that many spontaneous sequence rearrangements are caused by inter-pathway interactions.

research_5While studies using mouse models have been very informative, we also wanted to develop better ways to study DNA damage and repair in humans. To study DNA damage and repair in human cells, an approach was envisioned for high throughput analysis of DNA damage in human cells. In collaboration with the laboratory of Dr. S, Bhatia (MIT HST EECS), a traditional DNA repair assay called the ‘comet assay’ was modernized by exploiting microfabrication techniques. The resulting DNA damage and repair platform provides better reproducibility and greatly increased throughput. It is our hope that this tool can be used for many applications, including studies of environmental epidemiology.

research_6It is increasingly apparent that microbes influence our health in many ways. For example, pathogenic microbes can promote cancer. To learn more about the underlying mechanism of pathogenicity, we are exploring the potential for pathogenic microbes to damage DNA. Interestingly, we have found that S. pneumoniae can induce DNA damage in human cells, and we have contributed to work showing that colibactin, an E. coli pathogenicity factor, is a DNA crosslinking agent. In addition to understanding pathogenicity, our team is also working on understanding the impact of beneficial microbes on cancer risk.

Taken together, work in this laboratory is at the interface between biological engineering and environmental health, with the goals of developing novel technologies, applying these technologies to accelerate basic research, and ultimately using our understanding of disease processes to inform disease prevention.

CometChip: Microarray for DNA Damage

stamp_bigFor decades, researchers have measured DNA damage using the single cell gel electrophoresis assay, or comet assay. Originally developed by Singh et al in 1988, the basis for the assay is that damaged DNA migrates more readily than undamaged DNA when electrophoresed. Although effectively used in thousands of publications, the assay nevertheless suffers from sample-to-sample variation and it is highly laborious.

Increasing amounts of DNA damage are visible in the comets below. The nucleoid from a normal cell is on the left, whereas the nucleoid from a cell exposed to DNA damage is on the right.

Increasing amounts of DNA damage are visible in the comets below. The nucleoid from a normal cell is on the left, whereas the nucleoid from a cell exposed to DNA damage is on the right.

To increase reproducibility and throughput, we collaborated with the Bhatia laboratory (MIT HST EECS). Using microfabrication, we developed a method for making an array of microwells in agarose. A single cell (or groups of cells) can be loaded into each well.

Traditionally, 100 comets are analyzed for each sample, and it had been necessary to image each comet individually, which was highly laborious. By creating a cell microarray, comets share the same focal plane, making it possible to capture 100 comets in just one, saving hours in labor. In addition, in-house software enables automated comet analysis, which reduces bias and increases throughput.

The platform is useful for studies of single cells or clusters of cells, and it has been shown to be effective for analysis of DNA damage in a broad range of mammalian cell types, from human primary cells to human cancer cells.

  • Increased Throughput
  • Permits a broader range of cell concentrations
  • High consistency and efficiency
  • Lower inter- and intra- experimental noise
  • Detects many classes of DNA lesions
  • Detects multiple repair pathways
  • Can be used to measure repair capacity in human cells
  • The CometChip is now commercially available through Trevigen.




MicroColonyChip: Cell Survival Quantitation

We have recently developed the MicroColonyChip, a new cell survival platform that uses a novel metric: colony size (Cell Reports, 2019).

The ‘gold standard’ for quantification of cytotoxicity is the colony forming assay. This assay requires that colonies grow long enough to form visible colonies that are then counted manually. We have created a new approach that enables miniaturization and automation. Instead of waiting for visible colonies, microcolonies are grown in tiny micron scale wells in agarose. The resulting microcolony array is then imaged and the size distribution of the colonies is derived using automated image analysis software, greatly reducing labor associated with colony counting using the gold standard assay. Results from the MicroColonyChip are indistinguishable from results using the gold standard colony forming assay, which demonstrates that the MicroColonyChip is exquisitely sensitive.

The MicroColonyChip assay is also as sensitive as the Cell TiterGlo assay, yet is fully automated and robust to artifacts. For example, depending on the number of cells used to seed the Cell TiterGlo assay, results are highly variable. In contrast, the MicroColonyChip gives rise to consistent results over orders of magnitude differences in seeding density. Furthermore, Cell TiterGlo is a measure of metabolic activity, whereas the MicroColonyChip is measuring the actual number of cells, making the assay resistant to artifacts caused by conditions that impact cellular metabolism.

RaDR Mice: Fluorescence Detection of Mutant Cells

pancreasHomologous recombination is critical for repairing double strand breaks (DSBs), but it likely evolved primarily as amechanism for repairing broken replication forks. DNA replication forks can break down when they encounter DNA lesions.

forkIn order to detect homologous recombination in adult animals, we genetically engineered a DNA substrate wherein recombination between two non-functional copies of enhanced yellow fluorescent protein (EYFP) gives rise to fluorescent cells.
Rare recombinant cells can be seen in the pancreatic tissue of the fluorescent yellow direct repeat (FYDR).

A limitation of FYDR mice is that many tissues do not express the construct, so fluorescence cannot be detected. We therefore targeted a recombination reporter to the Rosa26 locus, which is known to be highly expressed.

rosa2pos-neg-fydr-w-text2Recombinant cells can be detected in almost all tissues of the Rosa26 direct repeat (RADR) mice.

The FYDR mice have been used extensively. Most recently, we have used these mice to probe the relationship between cell division and recombination in vivo, and to explore the impact of inflammation on recombination susceptibility.


We are exploring the relationships between specific DNA lesions (such as 3-methyladenine, shown here) and homologous recombination events.

Origin of Mutant Cells

Origin of Mutant Cells