Vectors comprising CpG islands without position effect varigation and having increased expression
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07H-021/02
C07H-021/04
A01N-065/00
A01N-043/04
A61K-031/70
출원번호
UP-0117960
(2002-04-05)
등록번호
US-7812148
(2010-11-01)
우선권정보
GB-0109335.0(2001-04-17)
발명자
/ 주소
Crombie, Robert Lachlan
Williams, Steven Geraint
출원인 / 주소
Millipore Corporation
대리인 / 주소
O'Connor, Cozen
인용정보
피인용 횟수 :
1인용 특허 :
8
초록▼
Polynucleotides and vectors comprising an expressible nucleic acid flanked by a 5′ extended methylation-free CpG island and a 3′ selectable marker element are disclosed. Such polynucleotides and vectors provide a means for obtaining high levels of expression of the flanked expressible
Polynucleotides and vectors comprising an expressible nucleic acid flanked by a 5′ extended methylation-free CpG island and a 3′ selectable marker element are disclosed. Such polynucleotides and vectors provide a means for obtaining high levels of expression of the flanked expressible nucleic acid. Preferred embodiments include combinations of 5′ extended methylation-free CpG islands and 3′ antibiotic resistance genes.
대표청구항▼
What is claimed is: 1. An isolated vector comprising polynucleotide, wherein the polynucleotide comprises: a. an extended methylation-free CpG island encompassing dual, divergently transcribed promoters, wherein the CpG island 1. comprises a DNA sequence associated with the 5′ end of ubiquit
What is claimed is: 1. An isolated vector comprising polynucleotide, wherein the polynucleotide comprises: a. an extended methylation-free CpG island encompassing dual, divergently transcribed promoters, wherein the CpG island 1. comprises a DNA sequence associated with the 5′ end of ubiquitously expressed genes; and/or 2. extends across a region encompassing more than one transcriptional start site; and/or 3. extends more than 500 bp; b. an expressible nucleic acid terminated by a polyadenylation signal, and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene, wherein when the vector is linearized and integrated into a chromosome, both the extended methylation-free CpG island and the selectable marker gene are operably linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 2000 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 2. An isolated vector comprising polynucleotide, wherein the polynucleotide comprises: a. an extended methylation-free CpG island encompassing dual, divergently transcribed promoters, wherein the CpG island 1. comprises a DNA sequence associated with the 5′ end of ubiquitously expressed genes; and/or 2. extends across a region encompassing more than one transcriptional start site; and/or 3. extends more than 500 bp; b. an expressible nucleic acid terminated by a polyadenylation signal, and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene, wherein when the vector is linearized and integrated into a chromosome, both the extended methylation-free CpG island and the selectable marker gene are operably linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1500 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 3. An isolated vector comprising polynucleotide, wherein the polynucleotide comprises: a. an extended methylation-free CpG island encompassing dual, divergently transcribed promoters, wherein the CpG island 1. comprises a DNA sequence associated with the 5′ end of ubiquitously expressed genes; and/or 2. extends across a region encompassing more than one transcriptional start site; and/or 3. extends more than 500 bp; b. an expressible nucleic acid terminated by a polyadenylation signal, and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene, wherein when the vector is linearized and integrated into a chromosome, both the extended methylation-free CpG island and the selectable marker gene are operably linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1000 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 4. An isolated vector comprising polynucleotide, wherein the polynucleotide comprises: a. an extended methylation-free CpG island encompassing dual, divergently transcribed promoters, wherein the CpG island 1. comprises a DNA sequence associated with the 5′ end of ubiquitously expressed genes; and/or 2. extends across a region encompassing more than one transcriptional start site; and/or 3. extends more than 500 bp; b. an expressible nucleic acid terminated by a polyadenylation signal, and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene, wherein when the vector is linearized and integrated into a chromosome, both the extended methylation-free CpG island and the selectable marker gene are operably linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 500 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 5. The isolated vector of any one of claims 1-4, wherein the antibiotic resistance gene is from a Streptomyces species. 6. The isolated vector of claim 5, wherein the antibiotic resistance gene is a puromycin resistance gene. 7. The isolated vector of claim 6, wherein the puromycin resistance gene is the puromycin N-acetyl transferase gene from Streptomyces alboniger. 8. The isolated vector of claim 7, wherein the puromycin resistance gene is a modified puromycin N-acetyl transferase gene from Streptomyces alboniger. 9. The isolated vector of claim 8, comprising SEQ ID NO: 3. 10. The isolated vector of claim 5, wherein the antibiotic resistance gene is a neomycin resistance gene. 11. The isolated vector of claim 10, wherein the neomycin resistance gene is the aminoglycoside phosphotransferase gene from Streptomyces fradiae. 12. The isolated vector of claim 5, wherein the antibiotic resistance gene is a hygromycin resistance gene. 13. The isolated vector of claim 12, wherein the antibiotic resistance gene is the hygromycin phosphotransferase gene from Streptomyces hygroscopicus. 14. The isolated vector of claim 5, wherein the antibiotic resistance gene is a bleomycin resistance gene. 15. The isolated vector of claim 14, wherein the bleomycin resistance gene is the bleomycin binding protein from Streptomyces verticillus. 16. The isolated vector of claim 14, wherein the bleomycin resistance gene is the bleomycin N-acetyltransferase gene from Streptomyces verticillus. 17. The isolated vector of claim 5, wherein the antibiotic resistance gene is a blasticidin resistance gene. 18. The isolated vector of claim 17, wherein the blasticidin resistance gene is the blasticidin S-acetyltransferase gene from Streptomyces verticillum. 19. The isolated vector of any one of claims 1-4, wherein the antibiotic resistance gene is the aminocyclitol phosphotransferase from Escherichia coli. 20. The isolated vector of any one of claims 1-4, wherein the antibiotic resistance gene is the neomycin phosphotransferase gene from transposon Tn5. 21. The isolated vector of any one of claims 1-4, wherein the vector is a plasmid. 22. The isolated vector of any one of claims 1-4, wherein the expressible nucleic acid encodes a recombinant protein for expression in an in vitro cell culture system. 23. The isolated vector of any one of claims 1-4 comprising nucleotides 1-10551 of SEQ ID NO: 1. 24. The isolated vector of any one of claims 1-4 comprising nucleotides 1-13547 of SEQ ID NO: 2. 25. A method for obtaining expression of an expressible nucleic acid comprising transfecting the isolated vector of any one of claims 1-4 into a host cell. 26. A method for obtaining a desired gene product comprising expressing the expressible nucleic acid from the isolated vector of any one of claims 1-4 in a host cell, wherein the expressible nucleic acid encodes the desired gene product and recovering the desired gene product. 27. The isolated vector of any one of claims 1-4, wherein the expressible nucleic acid is operably linked to a cytomegalovirus immediate/early promoter. 28. The isolated vector of any one of claims 1-4, wherein the expressible nucleic acid is contained within a multiple cloning site and the multiple cloning site is further operably linked to a promoter. 29. The isolated vector of claim 28, wherein said promoter is a cytomegalovirus immediate/early promoter. 30. The vector CET710. 31. The vector CET720. 32. A vector comprising nucleotides 1-12041 of SEQ ID NO:9. 33. The vector CET1010. 34. A vector comprising nucleotides 1-11646 of SEQ ID NO:10. 35. The vector CET1020. 36. A vector comprising nucleotides 1-9027 of SEQ ID NO:11. 37. The vector CET1030. 38. A vector comprising nucleotides 1-12221 of SEQ ID NO:12. 39. The vector CET1110. 40. A vector comprising nucleotides 1-11828 of SEQ ID NO:13. 41. The vector CET1120. 42. A vector comprising nucleotides 1-9209 of SEQ ID NO:14. 43. The vector CET1130. 44. An isolated vector comprising polynucleotide comprising: a. an extended methylation-free CpG island selected from the group consisting of: an extended methylation-free CpG island comprising an 8 Kb DNA fragment spanning the human hnRNPA2 gene; an extended methylation-free CpG island comprising an 8 Kb fragment spanning the murine hnRNPA2 gene; an extended methylation-free CpG island comprising nucleotides 1-7898 of SEQ ID NO: 15; and an extended methylation free island comprising a 2.0 kb DNA fragment spanning the human β-actin CpG island/promoter region and a 1.8 kb DNA fragment spanning the human PDCD2 CpG island/promoter region; and combinations thereof; b. an expressible nucleic acid terminated by a polyadenylation signal; and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene and wherein when the vector is linearized and integrated into a chromosome, both the CpG island and the selectable marker gene are operably-linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free CpG island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 2000 bp of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 45. An isolated vector comprising a polynucleotide comprising: a. an extended methylation-free CpG island selected from the group consisting of: an extended methylation-free CpG island comprising an 8 Kb DNA fragment spanning the human hnRNPA2 gene; an extended methylation-free CpG island comprising an 8 Kb fragment spanning the murine hnRNPA2 gene; an extended methylation-free CpG island comprising nucleotides 1-7898 of SEQ ID NO: 15; and an extended methylation free island comprising a 2.0 kb DNA fragment spanning the human β-actin CpG island/promoter region and a 1.8 kb DNA fragment spanning the human PDCD2 CpG island/promoter region; and combinations thereof; b. an expressible nucleic acid terminated by a polyadenylation signal; and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene and wherein when the vector is linearized and integrated into a chromosome, both the CpG island and the selectable marker gene are operably-linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free CpG island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1500 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 46. An isolated vector comprising a polynucleotide comprising: a. an extended methylation-free CpG island selected from the group consisting of: an extended methylation-free CpG island comprising an 8 Kb DNA fragment spanning the human hnRNPA2 gene; an extended methylation-free CpG island comprising an 8 Kb fragment spanning the murine hnRNPA2 gene; an extended methylation-free CpG island comprising nucleotides 1-7898 of SEQ ID NO: 15; and an extended methylation free island comprising a 2.0 kb DNA fragment spanning the human β-actin CpG island/promoter region and a 1.8 kb DNA fragment spanning the human PDCD2 CpG island/promoter region; and combinations thereof; b. an expressible nucleic acid terminated by a polyadenylation signal; and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene and wherein when the vector is linearized and integrated into a chromosome, both the CpG island and the selectable marker gene are operably-linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free CpG island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1000 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid. 47. An isolated vector comprising a polynucleotide comprising: a. an extended methylation-free CpG island selected from the group consisting of: an extended methylation-free CpG island comprising an 8 Kb DNA fragment spanning the human hnRNPA2 gene; an extended methylation-free CpG island comprising an 8 Kb fragment spanning the murine hnRNPA2 gene; an extended methylation-free CpG island comprising nucleotides 1-7898 of SEQ ID NO: 15; and an extended methylation free island comprising a 2.0 kb DNA fragment spanning the human β-actin CpG island/promoter region and a 1.8 kb DNA fragment spanning the human PDCD2 CpG island/promoter region; and combinations thereof; b. an expressible nucleic acid terminated by a polyadenylation signal; and c. a selectable marker gene operably linked to a promoter, wherein the selectable marker gene is an antibiotic resistance gene and wherein when the vector is linearized and integrated into a chromosome, both the CpG island and the selectable marker gene are operably-linked to the expressible nucleic acid, and the components are positioned in the order: extended methylation-free CpG island, expressible nucleic acid, selectable marker gene, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and wherein the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 500 by of the proximal end of the selectable marker gene, and wherein the expression of the expressible nucleic acid is increased relative to the expression in the presence of the extended methylation-free CpG island alone or when the selectable marker is 5′ of the expressible nucleic acid.
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이 특허에 인용된 특허 (8)
Chung Jay H. (Bethesda MD) Felsenfeld Gary (Chevy Chase MD), DNA sequence which acts as a chromatin insulator element to protect expressed genes from cis-acting regulatory sequences.
Palmiter Richard (Seattle WA) Sandgren Eric P. (Philadelphia PA) Brinster Ralph L. (Gladwyne PA), Method of preparing recombinant proteins in transgenic animals containing metallothionein gene elements that bestow tiss.
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