IN VIVO GENOME-WIDE MUTAGENESIS

• US 20100178696A1
• Filed: 02/08/2008
• Published: 07/15/2010
• Est. Priority Date: 02/09/2007
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Wu, Sen, Capecchi, Mario R.
• Granted Patent: US 8,546,135 B2
• Time in Patent Office: Days
• US Class Current: 435/320.100
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

Disclosed herein are compositions and methods for deleting or duplicating DNA in a mammalian genome. Also disclosed are compositions and methods for generating a random genome-wide chromosome rearrangement. Also disclosed are compositions and methods for streamlined construction of gene targeting vectors.

Claims

1. A gene-trap vector comprising a piggyBac transposon substantially lacking the splice donor sites.

2. The gene-trap vector of claim 1, wherein the vector can comprise an insert of at least 16 kb.

3. The gene-trap vector of claim 1, wherein the vector substantially lacks cryptic RNA splicing.

4. The gene-trap vector of claim 1, wherein vector comprises a 3′

transposon terminal sequence from a piggyBac transposon comprising the 3′

inverted terminal repeat (3′

ITR) and less than 933, 391, or 292 nucleic acids from the adjacent internal sequence.

5. The gene-trap vector of claim 2, wherein the 3′

ITR comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO;

95.

6. The gene-trap vector of claim 5, wherein the 3′

ITR and adjacent internal sequence consist essentially of the nucleic acid sequence SEQ ID NO;

238.

7. The gene-trap vector of claim 2, wherein the 3′

transposon terminal sequence consist essentially nucleic acids 1-453 of the sequence SEQ ID NO;

238.

8. The gene-trap vector of claim 2, wherein the 3′

transposon terminal sequence consist essentially nucleic acids 1-354 of the sequence SEQ ID NO;

238.

9. The gene-trap vector of claim 2, wherein the 3′

transposon terminal sequence does not comprise one or more of the splice donor sites represented by nucleic acids 78-79, 466-467, 475-476, 479-480, 860-861, or 944-945 of SEQ ID NO;

238.

10. The gene-trap vector of claim 1, wherein the vector comprises a 5′

transposon terminal sequence from a piggyBac transposon comprising the 5′

inverted terminal repeat (5′

ITR) and less than 643 or 294 nucleic acids from the adjacent internal sequence.

11. The gene-trap vector of claim 10, wherein the 5′

ITR comprises the nucleic acid sequence SEQ ID NO;

96.

12. The gene-trap vector of claim 10, wherein the 5′

ITR comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO;

96.

13. The gene-trap vector of claim 10, wherein the 5′

transposon terminal sequence consist essentially of the nucleic acid sequence SEQ ID NO;

239.

14. The gene-trap vector of claim 10, wherein the 5′

transposon terminal sequence consist essentially of nucleic acids 352-680 of the sequence SEQ ID NO;

239.

15. A gene-trap vector comprising the formula:

3′

TR-lox-SA-R¹-lox-5′

TR

wherein 3′

TR and 5′

TR are piggyBac 3′ and

5′

transposon terminal sequences comprising 3′ and

5′

inverted terminal repeats (ITR), respectively;

wherein the lox sites are in the same orientation;

wherein SA is a splice acceptor; and

wherein R¹ is a first reporter sequence linked to SA.

16. The gene-trap vector of claim 15, wherein the 3′

ITR comprises the nucleic acid sequence SEQ ID NO;

95.

17. The gene-trap vector of claim 15, wherein the 3′

ITR comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO;

95.

18. The gene-trap vector of claim 17, wherein the 3′

transposon terminal sequence comprise less than 933 nucleic acids from the internal sequence from a piggyBac transposon.

19. The gene-trap vector of claim 17, wherein the 3′

transposon terminal sequence comprises less than 454 nucleic acids from the internal sequence from a piggyBac transposon.

20. The gene-trap vector of claim 17, wherein the 3′

transposon terminal sequence comprises less than 355 nucleic acids from the internal sequence from a piggyBac transposon.

21. The gene-trap vector of claim 15, wherein the 3′

transposon terminal sequence consist essentially of the nucleic acid sequence SEQ ID NO;

238.

22. The gene-trap vector of claim 15, wherein the 3′

transposon terminal sequence consist essentially of nucleic acids 1-453 of the sequence SEQ ID NO;

238.

23. The gene-trap vector of claim 15, wherein the 3′

transposon terminal sequence consist essentially of nucleic acids 1-354 of the sequence SEQ ID NO;

238.

24. The gene-trap vector of claim 15, wherein the 3′

transposon terminal sequence does not comprise one or more of the splice donor sites represented by nucleic acids 78-79, 466-467, 475-476, 479-480, 860-861, or 944-945 of SEQ ID NO;

238.

25. The gene-trap vector of claim 15, wherein the 5′

ITR comprises the nucleic acid sequence SEQ ID NO;

96.

26. The gene-trap vector of claim 15, wherein the 5′

ITR comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO;

96.

27. The gene-trap vector of claim 15, wherein the 5′

transposon terminal sequence comprises less than 643 nucleic acids from the 5′

internal sequence from a piggyBac transposon.

28. The gene-trap vector of claim 15, wherein the 5′

transposon terminal sequence comprises less than 294 nucleic acids from the 5′

internal sequence from a piggyBac transposon.

29. The gene-trap vector of claim 15, wherein the 5′

transposon terminal sequence consist essentially of the nucleic acid sequence SEQ ID NO;

239.

30. The gene-trap vector of claim 15, wherein the 5′

transposon terminal sequence consist essentially of nucleic acids 352-680 of the sequence SEQ ID NO;

239.

31. The gene-trap vector of claim 15, comprising the formula:

3′

TR-lox-SA-R¹-X-R²-lox-X-5′

TR

wherein R² is a second reporter sequence functionally linked to an expression control sequence, and wherein X is a recombination site.

32. The gene-trap vector of claim 15, comprising the formula:

3′

TR-lox-SA R¹-X-R²-X-lox-5′

TR

wherein R² is a second reporter sequence functionally linked to an expression control sequence, and wherein X is a recombination site.

33. The gene-trap vector of claim 31, wherein X is FRT

34. The gene-trap vector of claim 31, wherein X is attB or attP.

54. The gene-trap vector of claim 32, wherein X is FRT

55. The gene-trap vector of claim 32, wherein X is attB or attP.

35. A gene-trap vector comprising the nucleic acid sequence set forth in SEQ ID NO:

105, SEQ ID NO;

106, or SEQ ID NO;

107.

36. A gene-trap vector comprising the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

105, SEQ ID NO;

106, or SEQ ID NO;

107.

37-53. -53. (canceled)