Research objectives and content
Objectives. The Amphibian embryo is one of the best systems for elucidating the mechanisms of early development. While the advantages of this system are numerous, one major disadvantage is that it has not been possible to manipulate the system at a genetic level. The aim of this proposal is to bring genetics to the frog, Xenopus. To this end we propose to develop a method for performing insertional mutagenesis in the frog embryo. Background and Significance. Our understanding of the molecular mechanisms of induction and patterning of the early vertebrate embryo has largely come from experiments in the frog. Indeed. the amphibian embryo is large and accessible allowing its manipulation with accuracy and ease. In addition, one can missexpress genes of interest in the early embryo by microinjecting in vitro transcribed RNAs. Recently Dr. Amaya's group at the Wellcome/CRC Institute in Cambridge has improved our ability to manipulate gene expression by developing an efficient method of generating transgenic frog embryos Briefly, this method involves integrating DNA into isolated sperm nuclei in vitro followed by their transplantation into eggs Because DNA integration occurs before fertilisation, the resulting transgenic embryos are not chimeric and breeding is not required. This technique allow gene function studies in the frog using genetic approaches and one can now monitor the temporal and spacial expression of the gene of interest in the embryo with high precision. Although useful. these methods have the disadvantage that they involve missexpression and/or gain of function experiments. The alternative loss of function experiments in the frog are usually restricted to the expression of dominant negative mutants which are rarely specific to one gene product. Therefore, there is a well justified need to develop strategies for producing null mutations of endogenous genes in the frog.
Training content (objective, benefit and expected impact)
Specific Aims. The overall aim of this proposal is to determine whether the transgenic technique developed in Dr Amaya's laboratory can be adopted for an insertional mutagenesis scheme in the frog. First we plan to determine whether a gene trap and/or enhancer trap approach can be used in Xenopus to identify genes with developmentally regulated expression patterns. This technique will enable us to mutagenise the genome, find genes with interesting patterns of expression and easily clone the 'tagged' gene. These are major advantages over the zebrafish genetic screens, where positional cloning of the mutated genes is long and tedious. Gene/enhancer trap experiments have been successfully used in mice and flies, and are currently performed by other investigators at the Wellcome/CRC Institute. Given the ease to manipulate the amphibian embryo, we feel that in the short term, such methods will lead to the identification of new genes implicated in early vertebrate embryogenesis. Then, we aim to determine whether such insertions have resulted in recessive null mutations which give phenotypes in homozygous mutant animals. We plan to perform the gene trap and/or enhancer trap experiments in Xenopus tropicalis, which is diploid, rather than the more commonly used Xenopus laevis.
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