Claims
- 1. A method for identifying a transformed cell which has undergone site-specific homologous recombination utilizing a PPS vector comprising:
a) transforming cells with a PPS vector designed to undergo site-specific homologous recombination wherein the vector comprises:
a first DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome; a second DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination; a third DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome and is different from the first DNA sequence; and a fourth DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to a cellular endogenous genomic sequence and therefore incapable of undergoing site-specific homologous recombination; b) propagating cells to select for or enrich for those which have been transformed with said PPS vector by selecting for the presence of the positive selectable marker gene product of said second DNA sequence; and c) separating cells which have said second DNA sequence encoding a positive selectable marker from cells which have said fourth DNA sequence encoding a positive selectable marker.
- 2. The method of claim 1, further comprising d) characterizing the genomic DNA of said cells carrying the second DNA sequence encoding a positive selectable marker but not carrying the fourth DNA sequence encoding a positive selectable marker for the site-specific homologous recombination events which allow for modification of the cellular target DNA
- 3. The method of claim 1 wherein said PPS vector includes positive selectable markers detectable by addition of antibiotics to cell cultures.
- 4. The method of claim 1 wherein said PPS vector includes positive selectable markers which may be detected by fluorescence light emission.
- 5. The method of claim 1 wherein said positive selectable markers allow for the separation of cells containing DNA encoding one marker from cells containing DNA encoding another or both markers.
- 6. The method of claim 1 wherein said cells are capable of homologous recombination.
- 7. The method of claim 1 wherein said cells are from a multicellular organism.
- 8. The method of claim 1 wherein said cells are from plants.
- 9. The method of claim 1 wherein said cells have undergone multiple rounds of site-specific homologous recombination for the purposes of multiple modifications of the endogenous cellular genome.
- 10. The method of claim 1 wherein said cells may be utilized to create a multicellular organism.
- 11. The method of claim 1 wherein said cells are embryonic stem cells.
- 12. An isolated PPS vector for site-specific homologous recombination in cells capable of undergoing homologous recombination, the vector comprising:
a first DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a second DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, and encodes a positive selectable marker capable of allowing for the identification of cells containing said positive selectable marker, a third DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a fourth DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, and encodes a positive selectable marker which allows for the separation of cells containing said positive selectable marker from cells not containing said positive selectable marker wherein the organization of said PPS vector in 5′ to 3′ orientation comprises: the first DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, the second DNA sequence which encodes a positive selectable marker, the third DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, and the fourth DNA sequence which encodes a positive selectable marker; wherein the vector is capable of undergoing site-specific homologous recombination resulting in modification of cellular endogenous target genomic DNA sequences.
- 13. The PPS vector of claim 12 wherein said cellular endogenous genomic target DNA is comprised of exons and introns.
- 14. The PPS vector of claim 13 wherein said vector contains all or portions of exons and introns which are substantially homologous to cellular target genomic DNA sequences.
- 15. The PPS vector of claim 12 wherein said vector contains all or portions of regulatory elements which are substantially homologous to cellular target genomic DNA sequences.
- 16. The PPS vector of claim 12 wherein said vector contains alterations in sequences which are substantially homologous to cellular target genomic DNA sequences including deletions, substitutions, additions or point mutations.
- 17. The PPS vector of claim 12 wherein said positive selection marker encoded by said fourth DNA sequence is selected from DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED or HcRED.
- 18. The PPS vector of claim 12 wherein said positive selection marker encoded by said second DNA sequence is selected from a group of DNA sequences encoding resistance markers including neo, puro, blasticidin, bleomycin, zeocin or hygro.
- 19. The PPS vector of claim 12 wherein said positive selection marker encoded by said second DNA sequence is selected from a group of DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED and HcRED.
- 20. The PPS vector of claim 12 wherein said vector may include an additional fifth DNA sequence which is nonhomologous to cellular endogenous genomic DNA sequences, is positioned external to said first and third DNA sequences on the opposite side containing said fourth DNA sequence which encodes a positive selectable marker and encodes a positive selectable marker.
- 21. The PPS vector of claim 12 wherein said fourth and fifth DNA sequences may encode positive selectable markers which allow for the separation of cells containing DNA encoding said selectable markers from cells which do not contain DNA encoding said selectable markers.
- 22. The PPS vector of claim 12 wherein said vector has lengths of homology for said first and third DNA sequences which are between about 50 bp and 50,000 base pairs.
- 23. The PPS vector of claim 12 wherein said vector results in the modification of cellular endogenous genomic target DNA sequences.
- 24. The PPS vector of claim 12 wherein said vector introduces exogenous regulatory elements into the cellular endogenous genomic target DNA sequences.
- 25. An enriched population of cells generated through a method according to claim 1 wherein said cells have undergone site-specific homologous recombination.
- 26. A non-human transgenic animal generated by the method of claim 1 wherein said animal has been generated from cells which have undergone site-specific homologous recombination.
- 27. A transgenic plant generated by the method of claim 1 wherein said plant has been generated from cells which have undergone site-specific homologous recombination.
- 28. A method for identifying a transformed cell which has undergone site-specific homologous recombination comprising:
a) propagating cells so that they are capable of undergoing transformation with exogenous DNA and b) transforming cells with a first DNA vector designed to undergo site-specific homologous recombination, the vector comprising:
a first DNA sequence which is substantially homologous to endogenous genomic DNA sequences present within the host genome, a second DNA sequence which encodes a positive selection characteristic in said cells yet is nonhomologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination, a third DNA sequence which is substantially homologous to endogenous genomic DNA sequences present within the host genome and is different from the first DNA sequence, wherein the vector is capable of undergoing site-specific homologous recombination in cells through strand exchange between the first DNA sequence with endogenous target sequences and the third DNA sequence with endogenous target DNA sequences; wherein the organization of the DNA sequences in the vector in 5′ to 3′ orientation comprises: the first DNA sequence which is substantially homologous to target DNA sequences, the second DNA second which encodes a positive selectable marker, the third DNA sequence which is substantially homologous to target DNA sequences. c) transforming said cells with a second DNA vector either simultaneously or sequentially in relation to transformation of the first vector, the second vector comprising:
a DNA sequence which encodes a unique selection characteristic in said cells yet is nonhomologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination. d) propagating cells to select for or enrich for those which have been successfully transformed with the first DNA vector by selecting for the presence of the positive selectable marker gene product of the first vector, and e) separating cells which have sequences encoding a positive selectable marker of the first DNA vector from cells which have DNA sequences encoding a unique selectable marker from said second DNA vector.
- 29. The method of claim 28, further comprising f) characterizing the genomic DNA of said cells carrying the DNA sequence encoding a positive selectable marker of the first vector but not carrying DNA sequences encoding the unique selectable marker of the second DNA vector for the site-specific homologous recombination events which allow for modification of the cellular target DNA.
- 30. The method of claim 28 wherein said positive selection characteristic in said first DNA vectors allows for the separation of cells containing DNA encoding one marker from cells containing DNA encoding another or both markers.
- 31. The method of claim 28 wherein said cells are capable of homologous recombination.
- 32. The method of claim 28 wherein said cells are from a multicellular organism.
- 33. The method of claim 28 wherein said cells are from plants.
- 34. The method of claim 28 wherein said cells have undergone multiple rounds of site-specific homologous recombination for the purposes of multiple modifications of the endogenous cellular genome.
- 35. The method of claim 28 wherein said cells may be utilized to create a multicellular organism.
- 36. The method of claim 28 wherein said cells are embryonic stem cells.
- 37. The first DNA vector according to claim 28 for site-specific homologous recombination in cells capable of undergoing homologous recombination, the vector comprising:
a first DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, a second DNA sequence which is nonhomologous to cellular endogenous genomic sequences, is not capable of undergoing homologous recombination in said cells, and encodes a positive selectable marker capable of allowing for the identification of cells containing said positive selectable marker, a third DNA sequence which is substantially homologous to cellular endogenous genomic sequences and is capable of undergoing homologous recombination in said cells, wherein the organization of said first DNA vector in 5′ to 3′ orientation comprises: the first DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences, the second DNA sequence which encodes a positive selectable marker, the third DNA sequence which is substantially homologous to cellular endogenous genomic DNA sequences; wherein the vector is capable of undergoing site-specific homologous recombination resulting in modification of cellular endogenous target genomic DNA sequences.
- 38. The first DNA vector of claim 28 wherein said cellular endogenous genomic target DNA is comprised of exons and introns.
- 39. The first DNA vector of claim 28 wherein said vector contains all or portions of exons and introns which are substantially homologous to cellular target genomic DNA sequences.
- 40. The first DNA vector of claim 28 wherein said vector contains all or portions of regulatory elements which are substantially homologous to cellular target genomic DNA sequences.
- 41. The first DNA vector of claim 28 wherein said vector contains alterations in sequences which are substantially homologous to cellular target genomic DNA sequences including deletions, substitutions, additions or point mutations.
- 42. The first DNA vector of claim 28 wherein said second DNA sequence encodes a positive selection marker is selected from DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED or HcRED.
- 43. The first DNA vector of claim 28 wherein said second DNA sequence encodes a positive selection marker is selected from DNA sequences encoding resistance markers including neo, puro, blasticidin, bleomycin, zeocin or hygro.
- 44. The second DNA vector of claim 28 wherein said DNA sequence which encodes a unique selection characteristic in said cells is selected from DNA sequences encoding fluorescent proteins including GFP, CFP, YFP, RFP, dsRED or HcRED.
- 45. The second DNA vector of claim 28 wherein said DNA sequence which encodes a unique selection characteristic in said cells is selected from DNA sequences encoding negative selection marker proteins including Hprt, gpt, HSV-tk, Diphtheria toxin, ricin toxin or cytosine deaminase.
- 46. The first DNA vector of claim 28 wherein said vector has lengths of homology for said first and third DNA sequences which are between about 50 bp and 50,000 base pairs.
- 47. The first DNA vector of claim 28 wherein said vector results in the modification of cellular endogenous genomic target DNA sequences.
- 48. The first DNA vector of claim 28 wherein said vector introduces exogenous regulatory elements into the cellular endogenous genomic target DNA sequences.
- 49. An enriched populations of cells generated by the method of claim 28 wherein said cells have undergone site-specific homologous recombination.
- 50. A transgenic non-human animal generated by the method of claim 28 wherein said animals have been generated from cells which have undergone site-specific homologous recombination.
- 51. A transgenic plant generated by the method of claim 28 wherein said animals have been generated from cells which have undergone site-specific homologous recombination.
RELATED APPLICATION DATA
[0001] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 60/300,953 filed on Jun. 26, 2001, which is incorporated by reference herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60300953 |
Jun 2001 |
US |