Bacteriophage-based transgenic fish for mutation detection

• US 6,307,121 B1
• Filed: 05/28/1999
• Issued: 10/23/2001
• Est. Priority Date: 05/31/1998
• 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): Clark, Deborah J. R.
• Assistant Examiner(s): CHEN, SHIN LIN
• Application Number:
  US09/322,725
• Time in Patent Office: 879 Days
• Field of Search: 800/3, 800/20, 800/21, 800/25, 800/18, 536/23.1, 536/23.5, 536/23.7
• 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 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The present invention provides transgenic fish whose somatic and germ cells contain a genomically integrated bacteriophage lambda-derived transgene construct. The transgene construct can include an excisable test nucleic acid sequence containing a heterologous mutation target nucleic acid sequence that is detectable via bioassay in a bacterial cell into which the test nucleic acid has been introduced. The frequency of mutations in the mutation target nucleic acid sequence following exposure of the transgenic fish to one or more potentially mutagenic agents can thus be evaluated.

Claims

1. A transgenic fish whose genomic DNA comprises a recoverable assayable mutation target nucleic acid sequence operably linked to a bacteriophage lambda-derived transgene construct;

wherein the mutation target nucleic acid sequence is recoverable from the transgenic fish, transferable into a bacterial host, and assayable in the bacterial host for the presence of a mutation.

2. The transgenic fish of claim 1 wherein the assayable mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the lacI gene, the lacZ gene, the lac promoter sequence, the cII gene, the cII mRNA ribosome binding site and the cII protein-activated P_RE promoter sequence.

3. The transgenic fish of claim 2 wherein the assayable mutation target sequence comprises the lacI gene.

4. The transgenic fish of claim 2 wherein the assayable mutation target sequence comprises the cII gene.

5. The transgenic fish of claim 1 wherein the assayable mutation target sequence comprises the cII region of bacteriophage lambda.

6. The transgenic fish of claim 1 wherein the bacteriophage lambda-derived transgenic construct comprises at least one cos site.

7. The transgenic fish of claim 1 wherein said fish is a teleost fish.

8. The transgenic fish of claim 7 wherein said fish is selected from the group consisting of a medaka, a zebrafish, a mummichog, a killifish, a channel catfish, a common carp and a trout.

9. The transgenic fish of claim 1 wherein said fish is sterile.

10. The transgenic fish of claim 1 that is hemizygous for the mutation target nucleic acid sequence.

11. The transgenic fish of claim 1 that is homozygous for the mutation target nucleic acid sequence.

12. A method for detecting mutations in the DNA of a transgenic fish comprising:

providing a transgenic fish whose genomic DNA comprises a recoverable assayable mutation target nucleic acid sequence operably linked to a bacteriophage lambda-derived transgene construct;

recovering genomic DNA comprising the mutation target nucleic acid operably linked to the bacteriophage lambda-derived transgene construct;

transferring the genomic DNA into a bacterial host; and

bioassaying in the bacterial host for the presence of a mutation in the mutation target nucleic acid sequence.

13. The method of claim 12 wherein the recovering step comprises extracting the genomic DNA from the fish to yield extracted genomic DNA comprising bacteriophage lambda-derived DNA comprising the assayable mutation target nucleic acid sequence and chromosomal DNA.

14. The method of claim 13 wherein the recovering step further comprises packaging the bacteriophage lambda-derived DNA to yield packaged phage.

15. The method of claim 14 wherein the transferring step comprises introducing the packaged phage into a bacterial host.

16. The method of claim 15 wherein the mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the cII gene, the cII mRNA ribosome binding site and the cII protein-activated P_RE promoter sequence of bacteriophage lambda.

17. The method of claim 16 wherein the bacterial host comprises an hfl⁻

E. coli strain.

18. The method of claim 15 wherein the mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the lacI gene, the lacZ gene and the lac promoter sequence.

19. The method of claim 16 wherein the bacterial host comprises an E. coli strain that produces a polypeptide that complements the polypeptide encoded by the alacZ region of the lacZ gene to form active β

-galactosidase.

20. The method of claim 12 wherein the providing step comprises providing a transgenic fish that has been or is suspected of having been exposed to a mutagen.

21. The method of claim 12 further comprising exposing the transgenic fish to a mutagen prior to recovering the DNA comprising the mutation target nucleic acid sequence.

22. The method of claim 21 wherein the mutagen is selected from the group consisting of a chemical, a radioisotope and electromagnetic radiation.

23. The method of claim 22 further comprising analyzing the mutation in the mutation target nucleic acid sequence.

24. The method of claim 23 wherein analyzing the mutation comprises determining the nucleic acid sequence of the mutation target nucleic acid sequence.

25. A method for evaluating the mutagenicity of a suspected mutagen comprising:

exposing a transgenic fish to a suspected mutagen, wherein the genomic DNA of the transgenic fish comprises a recoverable assayable mutation target nucleic acid sequence operably linked to a bacteriophage lambda-derived transgene construct;

recovering genomic DNA comprising the mutation target nucleic acid sequence operably linked to the bacteriophage lambda-derived transgene construct;

transferring the genomic DNA into a bacterial host; and

bioassaying in the bacterial host for the presence of a mutation in the mutation target nucleic acid sequence.

26. The method of claim 25 wherein the mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the lacI gene, the lacZ gene, the lac promoter sequence, the cII gene, the cII mRNA ribosome binding site and the cII protein-activated P_RE promoter sequence.

27. The method of claim 25 further comprising analyzing the mutation in the mutation target nucleic acid sequence.

28. The method of claim 27 wherein analyzing the mutation comprises determining the nucleic acid sequence of the mutation target nucleic acid sequence.

29. The method of claim 27 wherein analyzing the mutation comprises determining a mutation spectrum of the suspected mutagen.

30. A method for making a transgenic fish for mutagenesis detection, said method comprising microinjecting heterologous DNA into a one-cell fish embryo, and incubating the microinjected embryo such that the embryo develops into a fish, wherein the heterologous DNA comprises a recoverable assayable mutation target nucleic acid sequence operably linked to a bacteriophage lambda-derived transgene construct, the mutation target nucleic acid sequence being capable of being recovered, transferred into a bacterial host, and bioassayed in the bacterial host for the presence of a mutation.

31. The method of claim 30 wherein the mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the laci gene, the lacZ gene, the lac promoter sequence, the cII gene, the cII mRNA ribosome binding site and the cII protein-activated P_RE promoter sequence.

32. The method of claim 30 wherein the heterologous DNA is microinjected through the micropyle within about 5 minutes following fertilization.

33. A mutagenesis assay system comprising:

a transgenic fish whose genomic DNA comprises a recoverable assayable mutation target nucleic acid sequence operably linked to a bacteriophage lambda-derived transgene construct; and

a bacterial cell bioassay system for use in detecting the presence of a mutation in the target nucleic acid sequence that has been recovered from the transgenic fish after exposure to a potential mutagen, the bacterial cell bioassay system comprising a bacterial cell into which the target nucleic acid sequence has been introduced.

34. The mutagenesis assay system of claim 33 wherein the mutation target nucleic acid sequence comprises at least one nucleic acid sequence selected from the group consisting of the lacI gene, the lacZ gene, the lac promoter sequence, the cII gene, the cII mRNA ribosome binding site and the cII protein-activated P_RE promoter sequence.

35. The mutagenesis assay system of claim 33 wherein the transgenic fish is sterile.