TRANSGENESIS, MUTATION AND THE TROPICALIS GENOME PROJECT
In the decades after Hogben's brief sojourn in Cape Town, Xenopus had proven
to be a real boon to developmental biologists. In large part, the contributions
were due to development of assays for inductive events, tissue/tissue or cell/interactions
that are essential for forming the embryos. The most influential of these was
the animal cap assay for mesodermal induction developed by Nieuwkoop (he of
the "Normal Table") and the "Keller sandwich" for neural
induction (Ray Keller). These bioassays facilitated the screening of mRNAs
for roles in tissue formation and were responsible for the identification of
a number of important genes such as veg1 (Doug Melton) and noggin (Richard
Harland). While these assays were great for gene discovery, interfering with
gene function was more difficult. If the gene of interest needed a partner
to produce its effect (a dimerization partner in a receptor, for example),
its function could be interfered with by introducing an inactive form of the
gene (dominant negative). The gene might also be introduced in the wrong tissue
(ectopic) or at the wrong time (heterochronic) and developmental deviations
produced. All of these assays were transient, the construct (an RNA or cDNA)
could be introduced into a blastomere easily by injection (the cells are very
large) but, at some point, the resultant protein would be too dilute, due to
subsequent cell divisions, to have any effect. (Variants of this approach such
as antisense message or RNAi have similar drawbacks). For the developmental
biologists, most of whom focus on very early events such as pattern formation,
these obstacles were relatively minor. For us though, because sexual differentiation
occurs so late in development, transient transfection was close to useless.
A real breakthrough in this field was the development of a method to create transgenic Xenopus by Kris Kroll and Enrique Amaya (1996). Kris, a graduate student in John Gerhardt's lab, had settled on this very ambitious project for her thesis and she was joined in the effort by Enrique, a post-doc in the lab. The method that they developed involved collecting sperm nuclei, decondensing them, hitting the preparation with a mild dose of restriction enzyme and providing the gene of choice (usually in frame with a marker such as GFP or Bgalactosidase). The nuclei are then injected into eggs; those that get one nucleus continue to develop into embryos while the rest fail to gastrulate.
The method has been successfully adopted and modified by a number of labs
(including ours) and has been applied with very good success.
Figure 5 Green fluorescent protein (GFP) driven off the promoter for the crystalline protein specific (mostly) to lens; photos of transgenic Xenopus from the Grainger lab.
Integration into the germ line permits the establishment of various lines of transgenic frogs.
We are using these methods to determine how the androgen receptor functions in sexual differentiation. For example, knock downs of endogenous androgen receptor expression (using transgenic animals expressing dominant negative receptors) should clarify the ways in which androgen exerts its various effects on development (cell survival, proliferation and specification). Because we intend to use genetic approaches as well, we are also focusing on X. tropicalis.