Xenopus tropicalis Mutation Resource

Overview

Assigning functions to genes identified in large-scale sequencing efforts is fundamental to understanding vertebrate development, health and disease. Gain-of-function strategies, such as mRNA injection and transgenic mis-expression, identify activities of which gene products are capable, but for which they are not necessarily responsible during development. Loss-of-function strategies, such as analysis of mutants and morpholino antisense "knockdowns", identify roles for which particular genes are required.

In a collaborative effort with the laboratory of Dr. Lyle Zimmerman at the National Institute for Medical Research and Dr. Richard Harland at the University of California we are developing the amphibian Xenopus tropicalis as a model in which vertebrate gene function can be approached with combinations of loss-of-function, gain-of-function, and embryological techniques. Another member of the genus, X. laevis, has been a highly productive model system in cell and developmental biology due to the ease of molecular and surgical manipulations of its embryos, but its complex tetraploid genome and relatively long generation time have precluded genetic approaches.

X. tropicalis, by contrast, is a true diploid with a generation time as short as three months. Unlike zebrafish, its genomic organisation seems not to have diverged significantly from that of most vertebrates, and genetic and antisense strategies are likely to reveal novel functions. Its relatively short lifecycle facilitates multi-generation transgenic approaches, and adapting embryological and molecular techniques and probes from X. laevis to the smaller X. tropicalis embryo is usually straightforward. Recessive phenotypes may be readily uncovered, stable transgenic lines can be bred more quickly, and pseudogenes or alleles, which complicate promoter analysis and gene knockdowns, are less commonly encountered.

A panel of X. tropicalis mutations in a large sample of known genes would be useful for a wide range of studies of gene function in vertebrate development, especially to mesh emerging genomic resources with the strengths of Xenopus' embryology and amenability to gain-of-function approaches.(See review Carruthers and Stemple [16427790])

Mutation Resource Pipeline

Mutation Resource Pipeline

Mutation Resource Pipeline

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Our first goal is to generate a stable Xenopus tropicalis reverse genetic resource (Xenopus tropicalis Mutant Resource) from which carriers of mutations in the vast majority of genes can be efficiently recovered for phenotypic analysis. This resource will be derived from non-mosaic F2 progeny of in vitro mutagenized sperm, maintained in the form of parallel libraries of genomic DNA (for screening) and both frozen testes and living animals (from which to recover identified carriers) (See Mutation Resource Pipeline Schema).

We have developed an invitro sperm ENU mutagenesis protocol that we will use to generate a mutant library of 6000 F2 individuals.

From this library we will carry out reverse genetic screenings through a sequence based TILLING method (see review Stemple DL, 2005 [14726927]) and will characterize mutants by a morphological phenotype description and an expression microarray analysis.

We will screen the genomic DNA library by re-sequencing amplified exons of 175 selected genes requested by the Xenopus community and publicly document all confirmed base-changes predicted to be mutagenic. We will make these alleles available to the research community either as living animals or frozen sperm. The F3 carriers of specific nonsense alleles for each gene will be recovered from the living or frozen sperm libraries and raised to maturity. Sibling crosses of identified F3 carriers then provide homozygous mutant progeny for subsequent analysis via a systematic phenotyping pipeline.

Footnotes

  • Genetic and genomic prospects for Xenopus tropicalis research.

    Carruthers S and Stemple DL

    Seminars in cell & developmental biology 2006;17;1;146-53

  • TILLING--a high-throughput harvest for functional genomics.

    Stemple DL

    Nature reviews. Genetics 2004;5;2;145-50

* quick link - http://q.sanger.ac.uk/mfo3kwa7