Plasmid-based mutation detection system in transgenic fish

• US 6,472,583 B1
• Filed: 10/26/1999
• Issued: 10/29/2002
• Est. Priority Date: 10/26/1998
• Status: Expired due to Term

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Winn, Richard N.
• Primary Examiner(s): Nguyen, Dave T.
• Assistant Examiner(s): Shukla, Ram R.
• Application Number:
  US09/427,218
• Time in Patent Office: 1,099 Days
• Field of Search: 800/20, 800/3, 800/21, 800/25
• US Class Current: 800/3
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/40 Fish
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

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.

Claims

1. 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.

2. The method of claim 1 wherein the extracting step yields extracted DNA comprising plasmid DNA and chromosomal DNA.

3. The method of claim 2 further comprising cleaving the extracted DNA with a restriction endonuclease to yield at least one DNA fragment comprising the plasmid-derived mutation target nucleic acid sequence and a multiplicity of DNA fragments comprising chromosomal DNA.

4. The method of claim 3 further comprising separating the DNA fragment comprising the plasmid-derived mutation target nucleic acid sequence from the multiplicity of DNA fragments comprising chromosomal DNA to yield isolated plasmid-derived DNA comprising the mutation target nucleic acid sequence.

5. The method of claim 4 wherein the assayable mutation target nucleic acid sequence comprises a lacZ gene comprising a lacZ operator.

6. The method of claim 5 wherein the step of separating the DNA fragment comprising the plasmid-derived mutation target nucleic acid sequence from the multiplicity of DNA fragments comprising chromosomal DNA comprises:

contacting the DNA fragments with an affinity support comprising a lacZ operator binding material to bind the DNA fragment comprising the plasmid-derived mutation target nucleic acid sequence; and

eluting the bound DNA fragment from the support.

7. The method of claim 6 wherein the contacting step is performed after the cleaving step.

8. The method of claim 1 wherein the providing step comprises providing a transgenic fish that has been or is suspected of having been exposed to a mutagen.

9. The method of claim 6 wherein the contacting step and the cleaving step are performed simultaneously.

10. The method of claim 1 further comprising exposing the transgenic fish to a mutagen prior to extracting the DNA comprising the mutation target nucleic acid sequence.

11. The method of claim 10 wherein the mutagen is selected from the group consisting of a chemical, a radioisotope and electromagnetic radiation.

12. The method of claim 1 wherein the DNA is extracted from an organ or tissue of the transgenic fish.

13. The method of claim 12 further comprising analyzing the mutation.

14. The method of claim 13 wherein the step of analyzing the mutation comprises determining a tissue-specific or organ specific mutation frequency.

15. The method of claim 1 wherein the fish is selected from the group consisting of a medaka and a fundulus.

16. The method of claim 1 wherein the assayable mutation target nucleic acid sequence comprises a lacZ gene comprising a lacZ operator.

17. The method of claim 16 wherein the step of detecting the presence of a mutation in the mutation target nucleic acid sequence comprises:

transforming a host restriction-negative, lacZ⁻

galE⁻

bacterial host with the recovered DNA comprising the lacZ gene;

culturing the transformed bacterial host on a lactose-containing or lactose analogue-containing medium; and

selectively detecting a transformed bacterial host that comprises a mutation in the lacZ gene, wherein growth of the bacterial host is indicative of the existence of said mutation.

18. The method of claim 17 comprising, prior to transforming the bacterial host, ligating the recovered DNA to yield a circular DNA.

19. The method of claim 1 wherein the assayable mutation target nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of a lacI gene sequence, a lacZ gene sequence and a lac promoter sequence.

20. The method of claim 1 wherein the step of detecting the presence of a mutation in the mutation target nucleic acid sequence comprises performing a bioassay.

21. The method of claim 1 further comprising analyzing the mutation in the mutation target nucleic acid sequence.

22. The method of claim 21 wherein the step of analyzing the mutation comprises determining the nucleic acid sequence of at least a portion of the mutation target nucleic acid sequence.

23. 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.

27. The method of claim 23 wherein the fish is selected from the group consisting of a medaka and a fundulus.

28. The method of claim 23 wherein the assayable mutation target nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of a lacI gene sequence, a lacZ gene sequence and a lac promoter sequence.

29. The method of claim 23 further comprising analyzing the mutation in the mutation target nucleic acid sequence.

30. The method of claim 29 wherein the step of analyzing the mutation comprises determining the nucleic acid sequence of at least a portion of the mutation target nucleic acid sequence.

31. The method of claim 29 wherein analyzing the mutation comprises determining a mutation spectrum of the suspected mutagen.

32. The method of claim 23 wherein the assayable mutation target nucleic acid sequence comprises a lacZ gene comprising a lacZ operator.

24. The method of wherein 23 wherein the DNA is extracted from an organ or tissue of the transgenic fish.

25. The method of claim 24 furthers comprising analyzing the mutation.

26. The method of claim 25 wherein the step of analyzing the mutation comprises determining a tissue-specific or organ specific mutation frequency.