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Mutagenesis

There are currently multiple preliminary efforts to mutagenized the X. tropicalis genome.

The Harland Lab is using gamma ray as a mutagen. X. tropicalis sperm is exposed to a cesium gamma ray source. This mutagenized sperm is used to fertilize eggs which will produce founder frogs. Founder frogs will be raised to maturity and mature females isolated. Haploids from these animals will be screened for developmental defects using in situ hybridization of various tissue specific markers. These phenotypes will then be confirmed in the diploid state. (Mutagenesis strategy and our progress...)

The Grainger Lab is attempting to uncover natural occuring mutations in wild caught frogs by "instant" inbreeding using late pressure gynogenetic diploids. They have also initiated chemical mutagenesis with ENU.

Lyle Zimmerman and Derek Stemple are using ENU mutagenesis. They have mutagenized adult male frogs and then are raising F1 females from these mutagenized animals. Using early pressure gynogenesis (which supresses extrusion of the polar body), they intend to screen progeny of these F1 females.

In addition, Derek Stemple is developing a reverse genetic strategy. His lab is sequencing particular genes to identify mutations induced by ENU mutagenesis. In collaboration with Lyle Zimmerman, they have isolated frozen sperm from P0 males and are sequencing particular loci. Once desirable mutations are found, they will thaw and fertilize eggs with the appropriate sperm and then isolate the mutant.

Paul Mead is using an insertional mutagenesis strategy to mutagenize the frog genome. By injecting the Sleeping Beauty transposon and transposase RNA, his group has been able to produce transgenics that express ubiquitously or in a tissue specific manner. Enhancer and gene trap strategies are being developed. Additionally by driving expression of the transposase in the male germline, they hope to take advantage of transposon "hopping" to produce large numbers of animals with random insertions of transposon.