By Sean Henahan, Access Excellence

A transgenic tadpole created with the new technique

BOSTON, MA (10/7/96) The development of a "no frills" method for generating transgenic frogs could make "Xenopus laevis" the prince among lab animals used for studying genetic development, report Harvard researchers. The discovery appears to offer several practical advantages over the use of transgenic "knock-out" mice.

Also known as the African clawed frog, X. laevis has become the animal model of choice among embryologists. The eggs are large, one millimeter in diameter, making them easy to work with in culture. However, until now, researchers have had little success in creating genetically manipulated frog embryos.

"Earlier methods of injecting nucleic acids into frog eggs were crude and only partially successful at expressing the introduced genes. 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," said Kristen Kroll, a researcher in the Department of Cell Biology at Harvard Medical School.

Working with colleague Enrique Amaya, Dr. Kroll developed a remarkably simple solution to the problem. The method involves combining sperm nuclei and the DNA construct in an Eppendorf tube and let it stand for five minutes. Enzymes are then added to mark "nicks" into the nuclei's chromosomes, which allow the DNA construct to insert itself into the sperm DNA. An extract is also added 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."

The researchers were surprised to find that the frog 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, in contrast, 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 should give a big boost to researchers interested in the earliest embryonic stages of the life cycle. For example, Kroll and Amaya have already 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.

This research appeared in the October 1996 issue of the journal "Development".