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Researchers use glowing frogs to investigate eye genetics

Remember when you were young and couldn't sleep at night because you were afraid of glowing, green-eyed creatures? Perhaps your parents told you that they didn't exist to ease your troubled mind. They were wrong.

Researchers in Gilmer Hall are combining a gene from a frog that controls where a protein is active with a green florescent protein known as GFP from a jellyfish. This unique biological blending creates frogs with eyes -- and nervous systems -- that literally glow a bright, neon green.

For the past three years, University Biology Prof. Robert M. Grainger and his troop of researchers have engineered green-eyed Xenopus tropicalis frogs to understand how the different parts of the amphibian embryo develop and work in conjunction with each other.

With embryos "you start out with a simple structure and you end up with something very complex," Grainger said. "And it's amazing that at least most of the time it works out right."

Grainger's lab is one of the first to use X. tropicalis for genetic studies.

Funded by the National Institutes of Health, Grainger's research specifically investigates how genes within the eye of a frog function in its development.

The experiment

Evolutionarily speaking, organisms often share a common bond: for instance, Pax6 is a gene present in every animal species from the frog to the human. The function of Pax6 is to assist in the formation of the eye. Mutations in this gene can cause aniridia, which is an absence of the iris and the leading genetic cause of blindness in humans.

In general, while one component of a gene codes for the production of specific proteins, another component can control where the protein gets expressed.

For Grainger's experiments, the control region (from the frog) directs the gene's activity to the eye. The GFP (from the jellyfish) is the marker that indicates to researchers where the gene is expressed in the frog by illuminating the particular cells with a striking glow.

"Seeing the frogs' eyes glow is a spectacular sight, truly a marvel of modern science," said David Z. Rose, a fourth-year College student and assistant in Grainger's lab.

The frogs' eyes glow because of a little genetic magic. Frog sperm DNA is cut in specific places, using restriction enzymes. Next, the sperm is combined with the GFP of the jellyfish DNA. The sperm carrying the new DNA is injected into a female's egg cells, which incorporate the DNA permanently into the genome. The frogs thereafter can pass on their glowing features to their offspring.

Armed with this technique, scientists can understand the functions of the Pax6 gene. And with more restriction enzymes, researchers can chop Pax6 into increasingly smaller pieces until the gene no longer functions. This genetic slicing allows scientists to determine the key region responsible for controlling gene expression.

"This is a very important lesson in biology, that genes are not active everywhere but they are only active in certain parts in the body, and this green protein tells you that in a very dramatic sort of way," post doctoral researcher Nicolas Hirsch said.

A new research tool

The X. tropicalis frog is a fairly new addition to the group of animals commonly used in genetic testing, which for decades has included flies and mice. X. tropicalis is smaller than the previously used X. laevis frog, whose four sets of chromosomes (known as tetraploidy) posed many obstacles for geneticists. In contrast, X. tropicalis has two copies of its genes, which makes it more conducive to research -- and mutation. It also develops much faster than X. laevis, reaching sexual maturity in under five months. Like X. laevis, it can be induced to lay eggs at any time with the injection of specific hormones. This gives researchers a relatively constant supply of eggs instead of having to wait for the frogs to lay a fresh batch each spring.

The transparency of X. tropicalis tadpoles further facilitates the scientists' studies.

"I think the greatest advantage is that [X. tropicalis] combines two areas of science: genetics with embryology," Hirsch said. "Being able to watch the gene expression in the living embryo is a powerful tool for research."

Grainger agreed, adding that the large eggs and large embryos lead some people to believe they are the best tools to study early development.

Where it's all leading

Valuable information about human genetically based diseases can emerge from Grainger's work. After all, frogs develop in similar ways to humans and allow for testing that would not be acceptable in humans. Conceivably, they might engineer frog embryos with the same deficiencies as humans and try to treat them for the disease, Grainger said.

He said he plans to develop the frogs as an educational and research tool for the science world at large. He added he hopes to see the frog made available to high school and college students.

"This is a dramatic example of gene expression," Grainger said.

"Another goal is making it known to the scientific community that we have these animals and they are available for people to use in their research," Hirsch said.

What's in the future for transgenic X. tropicalis?

"Frogs with green brains or green gills or green muscles," Hirsch said. "We could study the development of lots of different tissues."

"Florescence is spooky," Grainger said. "Once people see something like this they never forget it"

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