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Aim 1. We will carry out a pilot mutagenesis with gamma irradiation.
We will establish conditions for mutagenesis; and screen F3 embryos examining morphology and molecular markers to identify early defects in the neural plate, mesoderm, and selected organs.
Aim 2 Evaluation of mutants using candidate genes.
We expect that many mutants will correspond initially to mutants isolated in the zebrafish, or may arise from mutations in obvious candidate genes. We will screen directly by polymerase chain reaction for mutations in those genes. Rescue of these mutants with injected mRNA, or phenocopy of the mutants using morpholino oligonucleotides will provide support for gene identification.
Aim 3. A resource of deletions will be extremely valuable for future work. Amphibian lampbrush chromosomes provide a means to examine cytology at high resolution. When we find deletions in genes (from aim 2), we will obtain genomic probes from the region, and examine the size of the deletion by exploiting in situ hybridization to lampbrush chromosomes from heterozygous animals.
Aim 4. In the X. tropicalis transgenesis technique, sperm nuclei are manipulated in vitro to facilitate recombination in sperm chromatin. We will expand the repertoire of in vitro reactions in sperm chromatin by exploiting the advances in biochemistry of both site-specific and homologous recombination. Specifically, we will assess the possibility of using well-defined transposons for insertional mutagenesis, and we will utilize key recombination proteins to facilitate homologous recombination events that result in targeted insertions.