Gene expression
AssigneeWoodcock Washburn LLP
InventorsRobert Lachlan Crombie, Steven Geraint Williams
Life of this patent
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Abstract
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.
Claims74
An isolated polynucleotide comprising:
- a. an extended methylation-free CpG island,
- b. an expressible nucleic acid terminated by a polyadenylation signal, and
- c. a selectable marker operably linked to a promoter,
- wherein both the CpG island and the selectable marker 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, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 2000 bp of the proximal end of the selectable marker.
An isolated polynucleotide comprising:
- a. an extended methylation-free CpG island,
- b. an expressible nucleic acid terminated by a polyadenylation signal, and
- c. a selectable marker operably linked to a promoter,
- wherein both the CpG island and the selectable marker 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, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1500 bp of the proximal end of the selectable marker.
An isolated polynucleotide comprising:
- a. an extended methylation-free CpG island,
- b. an expressible nucleic acid terminated by a polyadenylation signal, and
- c. a selectable marker operably linked to a promoter,
- wherein both the CpG island and the selectable marker 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, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 1000 bp of the proximal end of the selectable marker.
An isolated polynucleotide comprising:
- a. an extended methylation-free CpG island,
- b. an expressible nucleic acid terminated by a polyadenylation signal, and
- c. a selectable marker operably linked to a promoter,
- wherein both the CpG island and the selectable marker 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, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 500 bp of the proximal end of the selectable marker.
A vector comprising:
- a. an extended methylation-free CpG island,
- b. a multiple cloning site, and
- c. an antibiotic resistance gene from a Streptomyces species,
- wherein both the CpG island and the antibiotic resistance gene are operably-linked to the expressible nucleic acid, and the components are positioned in the order:
- extended methylation-free CpG island, multiple cloning site, selectable marker, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the multiple cloning site is within 2000 bp of the proximal end of the antibiotic resistance gene.
The vector CET710.
The vector CET720.
The vector CET740.
The vector CET760.
The vector CET780.
The vector CET820.
The vector CET823.
The vector comprising nucleotides 1-12041 of SEQ ID NO:
- 9.
The vector CET1010.
The vector comprising nucleotides 1-11646 of SEQ ID NO:
- 10.
The vector CET1020.
The vector comprising nucleotides 1-9027 of SEQ ID NO:
- 11.
The vector CET1030.
The vector comprising nucleotides 1-12221 of SEQ ID NO:
- 12.
The vector CET1110.
The vector comprising nucleotides 1-11828 of SEQ ID NO:
- 13.
The vector CET1120.
The vector comprising nucleotides 1-9209 of SEQ ID NO:
- 14.
The vector CET1130.
A method for obtaining a desired gene product comprising expressing the polynucleotide of 1-4 in a host cell and recovering the desired gene product.
A non-human transgenic animal comprising an artificially introduced extended methylation-free CpG island element and an artificially introduced selectable marker, wherein both the CpG island and the selectable marker 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, in the 5′ to 3′ orientation with respect to the sense strand of the expressible nucleic acid, and the polyadenylation signal at the 3′ end of the expressible nucleic acid is within 2000 bp of the proximal end of the selectable marker.
Dependencies are inferred from the claim text.