Claims
- 1. A method of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell, said method comprising:
introducing (i) a circular targeting construct, comprising a first recombination site and the polynucleotide sequence of interest, and (ii) a site-specific recombinase into the eucaryotic cell, wherein the genome of said cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by the site-specific recombinase, maintaining the cell under conditions that allow recombination between said first and second recombination sites, wherein the recombination is mediated by the site-specific recombinase and the result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the eucaryotic cell.
- 2. The method of claim 1, wherein the site-specific recombinase is selected from the group consisting of Cre recombinase, Cre-like recombinase, Flp recombinase, and R recombinase.
- 3. The method of claim 2, wherein the recombinase normally facilitates recombination between two recombination sites, wherein said sites are essentially the same, and where the sites are designated recombinase-mediated-recombination sites (RMRS).
- 4. The method of claim 3, wherein the RMRS comprises a first DNA sequence (RMRS5′), a core region A, and a second DNA sequence (RMRS3′) in the relative order RMRS5′-core region A-RMRS3′.
- 5. The method of claim 4, wherein said RMRS5′ and RNRS3′ comprise palidromic sequences.
- 6. The method of claim 5, wherein RMRS5′ and RNRS3′ comprise palidromic sequences of approximately 10-20 base pairs, and the core region comprises approximately 3-15 base pairs.
- 7. The method of claim 4, wherein said RMRS is a loxp site and the recombinase is Cre.
- 8. The method of claim 4, wherein said RMRS is a FRT site and the recombinase is FLP.
- 9. The method of claim 4, wherein (i) the second recombination site is a pseudo-RMRS site, and said second recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, and (ii) said first recombination site is a hybrid-recombination site comprising RMRS5′-core region B-RMRS3′.
- 10. The method of claim 4, wherein (i) the second recombination site is a pseudo-RMRS site, and said second recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, and (ii) said first recombination site comprises attT5′-core region B-attT3′.
- 11. The method of claim 1, wherein the site-specific recombinase is a recombinase encoded by a phage selected from the group consisting of φC31, TP901-1, and R4.
- 12. The method of claim 11, wherein the recombinase normally facilitates recombination between a bacterial genomic recombination site (attB) and a phage genomic recombination site (attP).
- 13. The method of claim 12, wherein (i) the second recombination site comprises a pseudo-attP site, and (ii) said first recombination site comprises the attB site.
- 14. The method of claim 13, wherein said recombinase is encoded by φC31.
- 15. The method of claim 12, wherein (i) the second recombination site comprises a pseudo-attB site, and (ii) said first recombination site comprises the attP site.
- 16. The method of claim 15, wherein said recombinase is encoded by φC31.
- 17. The method of claim 15, wherein said recombinase is encoded by phage R4.
- 18. The method of claim 15, wherein said recombinase is encoded by phage TP901-1.
- 19. The method of claim 12, wherein (i) attB comprises a first DNA sequence (attB5′), a bacterial core region, and a second DNA sequence (attB3′) in the relative order attB5′-bacterial core region-attB3′, (ii) attP comprises a first DNA sequence (attP5′), a phage core region, and a second DNA sequence (attP3′) in the relative order attP5′-phage core region-attP3′, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, said recombination-product sites comprising the relative order attB5′-recombination-product site-attP3′ and attP5′-recombination-product site-attB3.
- 20. The method of claim 19, wherein (i) the second recombination site is a pseudo-attP site, and said second recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, (ii) said first recombination site is an attB site comprising attB5′-bacterial core region-attB3′, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, said recombination-product sites comprising the relative order attT5′-recombination-product site-attB3′{polynucleotide of interest}attB5′-recombination-product site-attT3′.
- 21. The method of claim 19, wherein (i) the second recombination site is a pseudo-attB site, and said second recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, (ii) said first recombination site is an attP site comprising attP5′-phage core region-attP3′, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, said recombination-product sites comprising the relative order attT5′-recombination-product site-attP3′{polynucleotide of interest}attP5′-recombination-product site-attT3′.
- 22. The method of claim 1, wherein said circular targeting construct further comprises a bacterial origin of replication.
- 23. The method of claim 1, wherein said circular targeting construct further comprises a selectable marker.
- 24. The method of claim 23, wherein said selectable marker provides for either positive or negative selection.
- 25. The method of claim 1, wherein said polynucleotide sequence of interest comprises a transcriptional promoter sequence.
- 26. The method of claim 1, wherein said polynucleotide sequence of interest comprises at least one expression cassette.
- 27. The method of claim 26, wherein said expression cassette comprises a promoter operably linked to a polynucleotide sequence that encodes a product.
- 28. The method of claim 27, wherein said product is an RNA molecule.
- 29. The method of claim 27, wherein said product is a polypeptide.
- 30. The method of claim 1, wherein the site-specific recombinase is introduced into the cell as a polypeptide.
- 31. The method of claim 1, wherein the site-specific recombinase is introduced into the cell as a polynucleotide encoding the recombinase.
- 32. The method of claim 31, wherein an expression cassette comprises the polynucleotide encoding the recombinase.
- 33. The method of claim 32, wherein the expression cassette is carried on a transient expression vector.
- 34. The method of claim 32, that further comprises introducing the site-specific recombinase into the cell as a polypeptide.
- 35. The method of claim 1, wherein said recombinase is introduced into the cell before introducing the circular targeting construct.
- 36. The method of claim 1, wherein said recombinase is introduced into the cell concurrently with introducing the circular targeting construct.
- 37. The method of claim 1, wherein said recombinase is introduced into the cell after introducing the circular targeting construct.
- 38. A vector for site-specific integration of a polynucleotide sequence into the genome of a eucaryotic cell, said vector comprising,
(i) a circular backbone vector, (ii) a polynucleotide of interest operably linked to a eucaryotic promoter, and (iii) a first recombination site, wherein the genome of said cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by a site-specific recombinase.
- 39. The vector of claim 38, wherein said site-specific recombinase is derived from a bacteriophage.
- 40. The vector of claim 38, wherein said circular backbone vector is a procaryotic or eucaryotic vector.
- 41. The vector of claim 38, wherein said polynucleotide of interest operably linked to a eucaryotic promoter further comprises additional control elements.
- 42. The vector of claim 38, wherein the site-specific recombinase is selected from the group consisting of Cre recombinase, Cre-like recombinase, Flp recombinase, and R recombinase.
- 43. The vector of claim 39, wherein the site-specific recombinase is a recombinase encoded by a phage selected from the group consisting of φC31, TP901-1, and R4.
- 44. The vector of claim 43, wherein the site-specific recombinase is encoded by phage φC31.
- 45. The vector of claim 39, wherein the recombinase normally facilitates recombination between a bacterial genomic recombination site (attB) and a phage genomic recombination site (attP).
- 46. The vector of claim 45, wherein said first recombination site is either attB or attP.
- 47. The vector of claim 46, wherein said recombinase is the site-specific recombinase encoded by phage φC31.
- 48. The vector of claim 38, wherein said circular backbone vector further comprises a bacterial origin of replication.
- 49. The vector of claim 38, wherein said circular backbone vector further comprises a selectable marker.
- 50. The vector of claim 49, wherein said selectable marker provides for either positive or negative selection.
- 51. A kit for site-specific integration of a polynucleotide sequence into the genome of a eucaryotic cell, said kit comprising,
(i) a vector of claim 38, and (ii) a site-specific recombinase.
- 52. The kit of claim 51, wherein the site-specific recombinase is provided as a polypeptide composition.
- 53. The kit of claim 51, wherein the site-specific recombinase is provided as a polynucleotide encoding the recombinase.
- 54. The kit of claim 51, wherein the site-specific recombinase is provided as both a polypeptide and a polynucleotide encoding the recombinase.
- 55. A eucaryotic cell having a modified genome, said modified genome comprising an integrated polynucleotide sequence of interest whose integration was mediated by a recombinase and wherein said integration was into a recombination site native to the eucaryotic cell genome and said integration created a recombination-product site comprising said polynucleotide sequence.
- 56. The cell of claim 55, wherein said recombination-site product comprises the components attT5′-recombination-product site-attB3′ and attB5′-recombination-product site-attT3′, wherein (i) the native recombination site is a pseudo-attP site, and said native recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, (ii) said integrated polynucleotide sequence comprises a first recombination site comprising an attB site comprising attB5′-bacterial core region-attB3′, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, said recombination-product sites comprising the relative order attT5′-recombination-product site-attB3′{polynucleotide of interest}attB5′-recombination-product site-attT3′.
- 57. The cell of claim 55, wherein said recombination-site product comprises the components attT5′-recombination-product site-attB3′ and attB5′-recombination-product site-attT3′, wherein (i) the native recombination site is a pseudo-attB site, and said native recombination site comprises a first DNA sequence (attT5′), a core region B, and a second DNA sequence (attT3′) in the relative order attT5′-core region B-attT3′, (ii) said integrated polynucleotide sequence comprises a first recombination site comprising an attP site comprising attP5′-phage core region-attP3′, and (iii) wherein the recombinase meditates production of recombination-product sites that can no longer act as a substrate for the recombinase, said recombination-product sites comprising the relative order attT5′-recombination-product site-attP3′{polynucleotide of interest}attP5′-recombination-product site-attT3′.
- 58. A transgenic animal comprising at least one cell of claim 55.
- 59. A transgenic plant comprising at least one cell of claim 55.
- 60. A method of treating a disorder in a subject in need of such treatment, said method comprising:
site-specifically integrating a polynucleotide sequence of interest in a genome of at least one cell of the subject, where said site-specific integration of the polynucleotide sequence of interest is performed as described in claim 1, wherein said polynucleotide facilitates production of a product that treats said disorder in the subject.
- 61. The method of claim 60, wherein said site-specific integration is carried out in vivo in the subject.
- 62. The method of claim 60, wherein said site-specific integration is carried out ex vivo in cells and the cells are introduced into the subject.
- 63. A method of modifying a genome of a cell, said method comprising
inserting an attB or an attP recombination site into the genome of a cell, wherein (i) said recombination site is recognized by a recombinase, and (ii) said cell normally does not comprise the attB or attP site, to provide a modified genome containing an attB or an attP site.
- 64. The method of claim 63, wherein said cell is a eucaryotic cell.
- 65. The method of claim 63, wherein said inserting is carried out by transforming the cell with a polynucleotide containing the recombination site under conditions such that the polynucleotide is inserted into the genome.
- 66. The method of claim 63, further comprising
introducing (i) a circular targeting construct, comprising an attP recombination site and a polynucleotide sequence of interest, and (ii) a site-specific recombinase into the eucaryotic cell, wherein the genome of said cell comprises an attB recombination site and recombination between the attP and attB recombination sites is facilitated by the site-specific recombinase, maintaining the cell under conditions that allow recombination between said attP and attB recombination sites, wherein the recombination is mediated by the site-specific recombinase and the result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the cell.
- 67. The method of claim 63, further comprising introducing (i) a circular targeting construct, comprising an attB recombination site and a polynucleotide sequence of interest, and (ii) a site-specific recombinase into the eucaryotic cell, wherein the genome of said cell comprises an attP recombination site and recombination between the attB and attP recombination sites is facilitated by the site-specific recombinase,
maintaining the cell under conditions that allow recombination between said attB and attP recombination sites, wherein the recombination is mediated by the site-specific recombinase and the result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the cell.
- 68. The method of claim 63, wherein the site-specific recombinase is a recombinase encoded by a phage selected from the group consisting of φC31, TP901-1, and R4.
- 69. An expression cassette, comprising
a polynucleotide encoding a site-specific recombinase, wherein (i) the recombinase is encoded by a phage selected from the group consisting of φC31, TP901-1, and R4, and (ii) the recombinase is operably linked to a eucaryotic promoter.
- 70. The expression cassette of claim 69, further comprising a backbone vector that is a procaryotic or eucaryotic vector.
- 71. The expression cassette of claim 69, wherein said recombinase operably linked to a eucaryotic promoter further comprises additional control elements.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to U.S. Provisional Patent Application Serial No. 60/097,166, filed Aug. 19, 1998, from which priority is claimed under 35 USC §119(e)(1), and which application is incorporated herein by reference in its entirety.
Government Interests
[0002] This invention was made with support under NIH Grant R01 DK51834 from the National Institutes of Health, U.S. Department of Health and Human Services. Accordingly, the United States Government may have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60097166 |
Aug 1998 |
US |