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Frog Is Prince of New Technology

Harvard Medical School

Simple but elegant

Harvard Researcher Devises Powerful, Low-Tech Method of Generating Transgenic Frogs, Which Is Quickly Becoming Major Tool for Developmental Studies

BOSTON-Simple but elegant.

No, this is not about the latest collection from Calvin Klein. It's about the latest creation by Kristen Kroll. The postdoctoral fellow in the Department of Cell Biology at Harvard Medical School has designed an easy, no-frills method of producing transgenic frogs by the hundreds, inexpensively and overnight

The new technology, to be published in the October Development, will open up the study of frog embryology to modern genetic manipulation similar to that widely used in so-called transgenic and knock-out mice The not-so-elegant creature Xenopus laevis, or African clawed frog, has been a favorite of embryologists for decades. At one millimeter in diameter, its eggs are huge by embryologists' standards. They are easily accessible, and they readily develop in a culture dish under the observing eye of the scientist. For this reason, Xenopus has yielded the bulk of knowledge scientists have been able to glean from traditional tissue grafting experiments since the heyday of embryology in the 1920s and 130s.

Despite all that, this species has stubbornly resisted attempts at creating genetically manipulated embryos that would allow the unraveling of the molecular pathways that turn the fertilized egg into a highly organized tadpole. Earlier methods of injecting nucleic acids into frog eggs were crude and only partially successful at expressing the introduced genes, says Kroll. "We really needed the means to achieve stable integration of introduced DNA in the embryo and to fine-tune the time and place of expression," she says.

As a first step, Kroll harked back to the 1950s, when embryologists had first transplanted cell nuclei into frog eggs looking to see if early cells lost chromosomes during differentiation into specialized cells. Though creating transgenic frogs was not on those researchers' minds, their method seemed like a good starting point. After three years, Kroll, then a graduate student at the University of California at Berkeley, had established a method that in fact yielded transgenic frogs, though not nearly as efficiently and reliably as she wished.

At that time Enrique Amaya, co-author of the upcoming paper, joined her at UC Berkeley. Together, Kroll and Amaya worked out a deceptively simple recipe.

They combine sperm nuclei and the DNA construct in an Eppendorf tube and let it stand for five minutes. Then they add a hint of enzyme to chew tiny nicks into the nuclei's chromosomes, which allow the DNA construct to insert itself into the sperm DNA. To further nudge the insertion along, they also add an extract made from cells in interphase, a certain period of the cell division cycle in which the cell's chromatin is comparatively loose and therefore poised to absorb DNA. After 10 more minutes of incubation, Kroll and Amaya simply transfer the mix into a glass capillary and inject it into freshly harvested frog eggs.

Kroll can treat up to 500 eggs in an hour and of those, 10 to 20 percent develop into embryos. "It is trivial to do these injections," she says. "All you need to do is show up in the lab, fix up your mix, and shoot it in. The next day you can analyze your embryos, and study how the introduced gene affects their development."

Surprisingly, these embryos express the inserted DNA in all of their cells, or, if the researcher chooses, in all cells of the tissues targeted for expression. Transgenic mouse embryos often express the transgenic DNA only in some cells of the desired tissues and then have to be bred over several months to yield a homogeneously expressing mouse line.

Kroll's procedure requires only basic equipment found in every embryology lab, costing about $1,000. That may make transgenic frogs the least expensive of all genetically transformed animals today. By comparison, the $100,000 price tag for equipment needed to create transgenic mice and the cost of housing and breeding mice raises the average cost for each transgenic mouse line to roughly $10,000.

The transgenic frogs will open the doors to a wide range of studies, especially of the earliest embryonic stages. As a first example, Kroll and Amaya created embryos in which they genetically destroyed the function of the receptor for a common signaling molecule, fibroblast growth factor (FGF). When analyzing those embryos, they learned that the FGF receptor is critical during an early embryonic involution process called gastrulation. Without the functioning receptor, the embryos failed to develop muscle and connective tissue, which normally arise from the mesoderm. By disabling the FGF receptor at later times during development, Kroll and Amaya were able to pinpoint the exact developmental stage when the signaling pathway involving the FGF receptor helps form the mesoderm and its derivative tissue.

With her transgenics in hand, Kroll is beginning to ask more specific questions about the molecular pathways of tissue and organ development. But already it is clear, she says, that the technique "offers invaluable advantages, even though at first it seemed so wild and woolly."

Editors, please note: Vivid color prints of genetically altered embryos and a photograph of Kris Kroll are available on request.

Enrique Amaya now is at the Wellcome/CRC Institute in Cambridge, UK.

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