is a unique resource for two critical vertebrate biological areas:
early embryonic development and cell biology. In the former, X. laevis
has led the way in establishing the mechanisms of early fate decisions,
patterning of the basic body plan, and organogenesis. Contributions in
cell biology and biochemistry include seminal work on chromosome replication,
chromatin and nuclear assembly, cell cycle components, cytoskeletal elements,
and signaling pathways. Information amassed from these studies provides
a strong underpinning for future work, and, although X. laevis
is superb for characterizing the activities of particular genes, only
a tiny fraction have as yet been assayed. A major goal now is to examine
the expressed genome in the context of the biological phenomena mentioned
above using genomic technology, specifically ESTs and full-length cDNA
In recent years, Xenopus
tropicalis has emerged as a complementary system in which to combine
genetic approaches with the established strengths of the X. laevis
system. New strategies will be feasible when genetic variants
are examined in an embryological context, e.g. by making genetic chimeras,
and generation of stable transgenic reporter lines in the short-generation
X. tropicalis will increase the feasibility of many embryological
assays. Since the degree of sequence similarity and functional interchangeability
is high, X. tropicalis studies will also benefit from the
X. laevis EST and cDNA cloning experiments.
In preparation for
this workshop, the opinions of the Xenopus community were canvassed
and the proposals below are based on this information together with discussions
at this workshop.
The highest priority, one which is ready to be undertaken immediately,
is the generation of an X. laevis EST database that should
consist of 500,000 clones (estimated to be at least half of the expressed
sequence complexity). Initially this should capitalize on existing
cDNA libraries and should be complemented with normalized libraries
from selected stages. A small EST database in X. tropicalis
(50,000 clones) will provide a beginning for genomic research in this
organism. Estimated cost: $5,500,000 over two years.
cDNA Sequences. Full length sequenced, unique cDNAs from X.
laevis eggs and embryos are needed for functional studies (e.g.
expression cloning strategies). Estimated cost for preparation, sequencing
and arraying 50,000 full length cDNAs is $15,000,000 over three years.
This rapidly evolving technology promises major advances. The
utility of Xenopus for developmental studies, e.g. explantation,
induction, and overexpression assays, make array technology especially
valuable in this system. Array technology will become applicable as
EST/cDNA sequences come on line. Therefore, funding to produce and
make available Xenopus chips to the community should begin
one year after EST/cDNA sequencing is initiated. Estimated cost: $1,000,000
over three years.
Database. There is an immediate need for expansion of the present
Xenopus database, the Xenopus Molecular Marker Resource
(XMMR). The expanded database should encompass the existing data,
more comprehensive information concerning gene expression patterns,
additional fate maps and anatomical atlases of embryonic stages, and
information generated from the EST database and cDNA libraries. Sequences
should be organized along the lines of databases for other organisms
so that data is easily retrievable. Initial requirements include computational
facilities and a skilled data manager; as the sequence database matures,
a bioinformatics professional will become essential. Estimated cost:
$400,000 per year.
PAC and BAC
Libraries. The high efficiency transgenesis procedure recently
developed for Xenopus creates the need for large insert libraries
for cloning and analysis of genomic sequences, specifically for promoter
analysis of the X. tropicalis, diploid genome. Similar libraries
in X. laevis will enable comparison of putative control elements.
Estimated cost: $1,000,000.
genomic resources. Creation and preliminary characterization
of radiation hybrid panels are required in anticipation of genetic
screens and transgenic insertions. Additional support is needed for
pilot genetic studies, including chemical and insertional mutagenesis.
Estimated cost: $150,000 for the RHP project and $300,000 a year for
pilot genetic studies.
Stock Centers. Resource centers are urgently needed for broader
dissemination of new technology and for animal stocks. In the case
of new technologies this should take the form of training centers
in host labs experienced in transgenesis or antisense ablation. For
animal stocks, including transgenic and genetically altered lines,
one stock center is initially required with the expectation that this
will expand as more permanent lines are established. Estimated cost:
for training centers, $100,000 per year per host lab by supplementation
of existing grants; for a stock center, $500,000-$1,000,000 for construction
and $100,000 per year for operating costs.