Plasmid-based mutation detection system in transgenic fish
AssigneeUniversity of Georgia Research Foundation, Inc.
InventorRichard N. Winn
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Abstract
The present invention provides transgenic fish whose somatic and germ cells contain a genomically integrated plasmid containing a heterologous mutation target nucleic acid sequence that is detectable via bioassay in a bacterial cell into which the target nucleic acid has been introduced. The frequency and character of mutations in the mutatable target nucleic acid sequence following exposure of the transgenic fish to one or more potentially mutagenic agents can thus be evaluated.
Claims32
A method for detecting mutations in the DNA of a transenic fish comprising:
- providing a transgenic fish, wherein the DNA of the transgenic fish comprises multiple concatemeric genomically integrated copies of a plasmid comprising an assayable mutation target nucleic acid sequence;
- gently disaggregating at least a portion of the fish to yield disaggregated fish material;
- digesting the disaggregated fish material with a proteinase for a period of no longer than about 1-½ hours at a temperature of about 37° C.;
- extracting DNA comprising the mutation target nucleic acid sequence from the disaggregated fish material sufficient to detect a mutation in the mutation target nucleic acid sequence;
- and
- detecting the presence of a mutation in the mutation target nucleic acid sequence.
A method for evaluating the mutagenicity of a suspected mutagen comprising:
- exposing a transgenic fish to a suspected mutagen, wherein the DNA of the transgenic fish comprises multiple concatemeric genomically integrate copies of a plasmid comprising an assayable mutation target nucleic acid sequence;
- gently disaggregating at least a portion of the fish to yield disaggregated fish material;
- digesting the disaggregated fish material with a proteinase for a period of no longer than about 1-½ hours at a temperature of about 37° C.;
- extracting DNA comprising the mutation target nucleic acid sequence from the disaggregated fish material sufficient to detect a mutation in the mutation target nucleic acid sequence;
- and
- detecting the presence of a mutation in the mutation target nucleic acid sequence.
The method of wherein 23 wherein the DNA is extracted from an organ or tissue of the transgenic fish.
Dependencies are inferred from the claim text.