Claims
- 1. A transgenic non-human animal whose germ cells and somatic cells contain an exogenous nucleic acid molecule which is transcribed to produce a target pre-mRNA molecule comprising:
i) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (ii) at least one intron sequence or consensus splice site, wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of the pre-trans-splicing molecule.
- 2. A transgenic mouse whose germ cells and somatic cells contain a exogenous nucleic acid molecule which is transcribed to produce a target pre-mRNA molecule comprising:
(i) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (it) at least one intron sequence or consensus splice site wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of the pre-trans-splicing molecule.
- 3. The transgenic animal of claim 1, wherein the target pre-mRNA further comprises nucleotide sequences encoding a reporter molecule.
- 4. The transgenic mouse of claim 2, wherein the target pre-mRNA further comprises nucleotide sequences encoding a reporter molecule.
- 5. The transgenic animal of claim 1, wherein the target pre-mRNA contains a 3′ or 5′ consensus sequences required for spliceosomal mediated splicing.
- 6. The transgenic mouse of claim 2, wherein the target pre-mRNA contains 3′ or 5′ consensus sequences required for spliceosomal mediated splicing.
- 7. The transgenic animal of claim 1, further comprising a exogenous nucleic acid molecule encoding a pre-trans-splicing molecule.
- 8. The transgenic mouse of claim 2, further comprising a exogenous nucleic acid molecule encoding a pre-trans-splicing molecule.
- 9. The transgenic animal of claim 7 wherein the exogenous nucleic acid molecule is an expression vector.
- 10. The transgenic mouse of claim 8 wherein the exogenous nucleic acid molecule is an expression vector.
- 11. The transgenic animal of claim 7 wherein the expression vector is a viral vector.
- 12. The transgenic mouse of claim 8 wherein the expression vector is a viral vector.
- 13. The transgenic animal of claim 11 wherein the viral vector is an adenovirus vector.
- 14. The transgenic mouse of claim 12 wherein the viral vector is an adenovirus vector.
- 15. The transgeneic animal of claim 3 wherein the reporter molecule is an enzyme.
- 16. The transgenic mouse of claim 4 wherein the reporter molecule is an enzyme.
- 17. The transgenic animal of claim 3 wherein the reporter molecule is a bioluminescent or chemoluminescent molecule.
- 18. The transgenic mouse of claim 4 wherein the reporter molecule is a bioluminescent or chemoluminescent molecule.
- 19. The transgenic animal of claim 15 wherein the enzyme is β-galactosidase.
- 20. The transgenic mouse of claim 16 wherein the enzyme is β-galactosidase.
- 21. The transgenic animal of claim 1 wherein the target pre-mRNA molecule is encoded by pCUBT4.2.
- 22. The transgenic mouse of claim 2 wherein the target pre-mRNA molecule is encoded by pCUBT4.2.
- 23. A method of producing the transgenic animal of claim 1, wherein said method comprises:
i) introducing an exogenous nucleic acid molecule into an embryonic stem cell, wherein said nucleic acid molecule is transcribed to form a target pre-mRNA molecule comprising:
(a) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (b) at least one intron sequence or consensus splice site
wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of a pre-trans-splicing molecule; ii) injecting the embryonic stem cell into a blastocyst, iii) transplanting said blastocyst into the reproductive tract of an animal, iv) allowing said blastocyst to develop into an animal whose genome contains said exogenous nucleic acid molecule, and v) screening said animal of step (iv) to identify a transgenic animal whose genome comprises said selectable marker.
- 24. A method of producing the mouse of claim 2, wherein said method comprises:
i) introducing an exogenous nucleic acid molecule into an embryonic stem cell, wherein said nucleic acid molecule is transcribed to form a target pre-mRNA molecule comprising:
(a) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (b) at least one intron sequence or consensus splice site, and
wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of the pre-trans-splicing molecule; ii) injecting the embryonic stem cell into a blastocyst, iii) transplanting said blastocyst into the reproductive tract of a mouse, iv) allowing said blastocyst to develop into a mouse whose genome contains said exogenous DNA construct, and v) screening said mouse of step (iv) to identify a transgenic mouse whose genome comprises said selectable marker.
- 25. A method of producing the transgenic animal of claim 1, wherein said method comprises:
i) introducing an exogenous nucleic acid molecule into a fertilized egg, wherein said nucleic acid molecule is transcribed to form a target pre-mRNA molecule comprising:
(a) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (b) at least one intron sequence or consensus splice site, and
wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of the pre-trans-splicing molecule; ii) transplanting said fertilized egg into the animal, iii) allowing said fertilized egg to develop into an animal whose genome contains said exogenous nucleic acid molecule, and iv) screening said animal of step (iii) to identify a transgenic animal whose genome comprises said exogenous nucleic acid.
- 26. A method of producing the transgenic mouse of claim 2, wherein said method comprises:
i) introducing an exogenous nucleic acid molecule into a fertilized egg, wherein said nucleic acid molecule is transcribed to form a target pre-mRNA molecule comprising:
(a) one or more target binding domains that target binding of the target pre-mRNA molecule to a pre-trans-splicing molecule; and (b) at least one intron sequence or consensus splice site, and wherein said target pre-mRNA molecule is a substrate for a trans-splicing reaction in the presence of the pre-trans-splicing molecule; ii) transplanting said fertilized egg into a mouse, iii) allowing said fertilized egg to develop into mouse whose genome contains said exogenous nucleic acid molecule, and iv) screening said mouse of step (iii) to identify a transgenic mouse whose genome comprises said exogenous nucleic acid.
- 27. The method of claim 23, 24, 25 or 26 wherein the exogenous nucleic acid molecule is pCUBt4.2
- 28. A method of producing a chimeric RNA molecule in the transgenic animal of claim 1 comprising contacting the target pre-mRNA molecule expressed within the cells of the animal with a pre-trans-splicing molecule recognized by nuclear splicing components wherein said exogenous nucleic acid molecule comprises:
(i) one or more target binding domains that target binding of the nucleic acid molecule to the target pre-mRNA expressed within a cell; (ii) a 3′ splice region comprising a branchpoint and a 3′ splice acceptor site or a 5′ splice site; and (iii) nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 29. A method of producing a chimeric RNA molecule in the transgenic mouse of claim 2 comprising contacting the target pre-mRNA molecule expressed within the cells of the animal with a pre-trans-splicing molecule recognized by nuclear splicing components wherein said exogenous nucleic acid molecule comprises:
(i) one or more target binding domains that target binding of the nucleic acid molecule to the target pre-mRNA expressed within a cell; (ii) a 3′ splice region comprising a branchpoint and a 3′ splice acceptor site or 5′ splice site; and (iii) nucleotide sequence to be trans-spliced to the target pre-mRNA;
wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 30. The method of claim 28 or 29, wherein the pre-trans-splicing molecule is PTM 24.
- 31. The method of claim 28 or 29 wherein the pre-trans-splicing molecule is encoded by the adenovirus vector Ad LacZPTM-24.
- 32. The method of claim 28 or 29 wherein said target pre-mRNA comprises a nucleotide sequence encoding a reporter molecule.
- 33. A method for testing the ability of a pre-trans-splicing molecule to mediate a trans-splicing reaction comprising;
(i) contacting the pre-target mRNA expressed in the transgenic animal of claim 1 with the pre-trans-splicing molecule wherein a portion of the pre-trans-splicing trans-splicing molecule is spliced to a portion of the pre-target mRNA to form a chimeric mRNA; and (ii) detecting the presence of the chimeric mRNA molecule.
- 34. A method for testing the ability of a pre-trans-splicing molecule to mediate a trans-splicing reaction comprising;
(i) contacting the pre-target mRNA expressed in the transgenic mouse of claim 2 with the pre-trans-splicing molecule wherein a portion of the pre-trans-splicing molecule is spliced to a portion of the pre-target mRNA to form a chimeric mRNA; and (ii) detecting the presence of the chimeric mRNA molecule.
- 35. The method of claim 33 or 34 wherein the pre-trans-splicing molecule is encoded by the adenovirus vector Ad.LacZPTM 24.
- 36. The method of claim 33 or 34 wherein the chimeric mRNA is detected.
- 37. The method of claim 33 or 34 wherein a reporter molecule encoded by the chimeric mRNA is detected.
- 38. The method of claim 33 or 34 wherein the reporter molecule is an enzyme.
- 39. The claim of claim 33 or 34 wherein the reporter molecule is a chemiluminescent or bioluminescent molecule.
- 40. A recombinant adenovirus vector capable of expressing PTM 24.
- 41. The recombinant adenovirus Ad.LacZPTM24 as depicted in FIG. 3.
Government Interests
[0001] The present invention involves subject matter developed under NIH Grant No. 2R44DK56526.