Xenopus Breakout Group


Xenopus laevis 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 libraries.

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.

  • EST Database. 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.

  • Full length 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.

  • Microarrays. 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.

  • Model Organism 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.

  • X. tropicalis 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.

  • Training and 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.

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