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 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.