Gene chip engineers

Peter Hoyt checks the operation of the Packard multiprobe liquid-handling system and mechanical gripper. This state-of-the-art robotic tool is used to prepare samples (e.g., cloned DNA) for use on microarrays to analyze gene expression.

What does a gene do in a mouse, fish, or some other organism? One technology that allows biologists to spy on gene activity is the microarray, a microscope glass slide dotted with an orderly array of DNA sequences.

Mitch Doktycz, Peter Hoyt, and their colleagues in the Life Sciences Division (LSD) specialize in designing and using microarrays, or gene chips, to help determine which genes are expressed as a result of specific diseases or exposures to environmental toxins. They also have developed technologies to speed up and reduce the costs of preparing DNA probes for gene chips, as well as genetic material from mice, fish, and bacteria.

"We look at hundreds to thousands of samples of biological liquids simultaneously on our microarrays," Doktycz says. "We are evaluating expressed messenger RNA (mRNA) isolated from various mouse tissues, including skin, liver, lung, brain, muscle, kidney, fat, heart, pancreas, spleen, gut, and testes.

In collaboration with Ed Michaud's group in LSD (see Complex Biological Systems in Mice), Doktycz's group has analyzed gene expression in samples collected from mice afflicted with skin disease to figure out which genes are altered in the diseased mice compared with equivalent genes in normal mice. A gene that is altered produces abnormally high or low levels of mRNA, which eventually results in altered levels of protein.

Doktycz is also collaborating with ORNL's Russ Knapp and Ed Michaud to determine the genes that are altered in mouse models of obesity and diabetes. Specifically, these experiments use gene chips to determine how new anti-diabetic drugs treat the disease and affect expression patterns of these genes.

Working with Mark Greeley of ORNL's Environmental Sciences Division (ESD), Doktycz and his colleagues have developed a "zebrafish tox-chip microarray." It is used to determine which genes are turned on in zebrafish embryos exposed to hormone-mimicking chemicals.

Besides applying microarrays to gene expression and genome studies, these ORNL researchers are developing more economical ways to prepare samples—extracting mRNA from cells for gene expression studies and attaching various DNA probes to microarrays.

"When we complete development of automated sample processing, hundreds of tissue samples will be processed in an afternoon," Doktycz says. "Using conventional techniques, it can take three to four days to analyze 12 samples. With our method, 96 samples can be prepared in parallel in just a few hours. This sample productivity is needed because we can make more than 100 gene chips in less than a day. Currently, we can print several thousand DNA spots on each gene chip."

Hoyt has developed an inexpensive, high-throughput liquid-handling method of extracting mRNA from tissues. A snippet of mouse skin or other tissue is broken up into cells by simultaneously homogenizing the samples. After the cells are placed in a microtiter plate, mRNA is isolated from them using an automated procedure. Hoyt is now beta testing a new Packard Bioscience robotic instrument for "walk-away" automated processing of mRNA samples. The same instrument is used to prepare the complementary DNA test samples placed on the gene chip (where they will pair with the matching mRNA samples).

Doktyz has been making a mark in the field of spotting technologies. He recently helped devise a commercial inkjet technology for dispensing microscopic drops of biological fluids at high speeds, a technology that could hasten the development of new therapeutic drugs. He worked on this project with Rheodyne, a California company that makes high-end valves, under a cooperative research and development agreement. The resulting "hybrid valve" is now produced commercially by Innovadyne Technologies, Inc., a Rheodyne spin-off company.

Gene chips and related technologies are revolutionizing biological studies. To help meet the need for faster, better, and cheaper ways to spy on genes, Doktycz and his colleagues are being ingenious.