US2011165580A1PendingUtilityA1
Response Element Regions
Est. expiryApr 22, 2024(expired)· nominal 20-yr term from priority
G01N 33/5041G01N 33/5023G01N 33/6863
38
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Claims
Abstract
Response element regions, DNA constructs comprising response element regions, host cells comprising response element regions, and methods of using response element regions are provided.
Claims
exact text as granted — not AI-modified1 . An isolated nucleic acid comprising a response element region comprising: (i) the sequence GTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i).
2 . An isolated nucleic acid comprising a response element region comprising:
(a) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); (b) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); (c) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTT CCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC or (ii) a sequence complementary to the sequence in (i); or (d) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTT CCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); wherein Y, X, and Z are each independently selected from a nucleic acid sequence of 0 to 23 nucleotides.
3 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-
Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACC
wherein Y, X, and Z are each a nucleic acid sequence of 0 nucleotides.
4 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-
Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACC
wherein Y, X, and Z are each a nucleic acid sequence of 8 nucleotides.
5 . The isolated nucleic acid of claim 4 , wherein Y, X, and Z are each the nucleic acid sequence GCCGTACC.
6 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-
Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACC
wherein Y is a nucleic acid sequence of 8 nucleotides, X is a nucleic acid sequence of 10 nucleotides, and Z is a nucleic acid sequence of 16 nucleotides.
7 . The isolated nucleic acid of claim 6 , wherein Y is the nucleic acid sequence GCCGTACC, X is the nucleic acid sequence TACCGGTCTG, and Z is the nucleic acid sequence ACCGGCCTAGTGCGTC.
8 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC
wherein Y and X are each a nucleic acid sequence of 0 nucleotides.
9 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC
wherein Y and X are each a nucleic acid sequence of 8 nucleotides.
10 . The isolated nucleic acid of claim 9 , wherein Y and X are each the nucleic acid sequence GCCGTACC.
11 . The isolated nucleic acid of claim 2 , comprising the sequence:
GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCGT
CATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCGTCATTT
CCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTTCCAGG
AAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC
wherein Y is a nucleic acid sequence of 8 nucleotides and X is a nucleic acid sequence of 10 nucleotides.
12 . The isolated nucleic acid of claim 11 , wherein Y is the nucleic acid sequence GCCGTACC and X is the nucleic acid sequence TACCGGTCTG.
13 . A vector comprising a promoter and nucleic acid comprising a response element region comprising: (i) the sequence GTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i).
14 . A vector comprising a promoter and nucleic acid comprising a response element region comprising:
(a) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); (b) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); (c) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTT CCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC or (ii) a sequence complementary to the sequence in (i); or (d) (i) the sequence GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAA TCACCGTCATTTCCAGGAAATCACC-Y-GTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACC-X-GTCATTT CCAGGAAATCACCGTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATC ACC-Z-GTCATTTCCAGGAAATCACCGTCATTTCCAGGAAATCACCG TCATTTCCAGGAAATCACC or (ii) a sequence complementary to the sequence in (i); wherein Y, X, and Z are each independently selected from a nucleic acid sequence of 0 to 23 nucleotides.
15 . The vector of claim 14 , further comprising a reporter nucleic acid, wherein the response element region is operably linked to the promoter and the promoter is operably linked to the reporter nucleic acid.
16 . The vector of claim 15 , wherein the promoter is a TK promoter and the reporter nucleic acid is a nucleic acid that encodes luciferase.
17 . The vector of claim 15 , wherein the promoter is a SV40 promoter and the reporter nucleic acid is a nucleic acid that encodes luciferase.
18 . A host cell comprising a vector of any one of claims 13 to 17 .
19 . The host cell of claim 18 , wherein the host cell is a signaling molecule-responsive host cell.
20 . The host cell of claim 18 , wherein host cell is responsive to at least one signaling molecule selected from G-CSF, EPO, and IL-3.
21 . The host cell of claim 20 , wherein host cell is responsive to at least one signaling molecule selected from recombinant methionyl human granulocyte colony-stimulting factor, epoetin alfa, and darbepoetin alfa.
22 . A method for determining the activity of a test composition comprising a signaling molecule, comprising
a) contacting the test composition with a signaling molecule-responsive host cell comprising the vector of claim 15 under conditions in which the reporter nucleic acid expresses a reporter protein in response to the signaling molecule; and b) detecting the reporter protein to determine the activity of the test composition.
23 . The method of claim 22 , further comprising comparing the level of detected reporter protein expression in (b) with the level of reporter protein expressed by a signaling molecule-responsive host cell comprising the vector of claim 15 in the absence of the signaling molecule.
24 . The method of claim 22 , further comprising comparing the level of detected reporter protein expression in (b) with the level of reporter protein expressed by a signaling molecule-responsive host cell comprising the vector of claim 15 in the presence of a standard composition comprising the signaling molecule.
25 . The method of claim 24 , further comprising calculating the relative potency of the test composition, wherein the relative potency is calculated by dividing the signaling molecule concentration of the standard composition that gives a level of reporter protein expression of U by the signaling molecule concentration of the test composition that gives a level of reporter protein expression of U.
26 . The method of any of claims 22 to 25 , wherein the promoter is a TK promoter and the reporter nucleic acid encodes luciferase.
27 . The method of any of claims 22 to 25 , wherein the promoter is a SV40 promoter and the reporter nucleic acid encodes luciferase.
28 . The method of any of claims 22 to 25 , wherein host cell is responsive to at least one signaling molecule selected from G-CSF-like molecule, erythropoietic product, and IL-3.
29 . The method of claim 28 , wherein host cell is responsive to at least one signaling molecule selected from recombinant methionyl human granulocyte colony-stimulting factor, epoetin alfa, and darbepoetin alfa.
30 . A method for determining whether a test compound has activity of a given signaling molecule, comprising
a) contacting the test compound with a signaling molecule-responsive host cell comprising the vector of claim 15 under conditions in which the reporter nucleic acid expresses a reporter protein in response to compounds that have the activity of the given signaling molecule; b) detecting the reporter protein; c) comparing the level of detected reporter protein expression in (b) with the level of detected reporter protein expressed by a signaling molecule-reponsive host cell comprising the vector of claim 15 in the absence of the test compound to determine whether the test compound has the activity of the given signaling molecule.
31 . The method of claim 30 , wherein the promoter is a TK promoter and the reporter nucleic acid encodes luciferase.
32 . The method of claim 30 , wherein the promoter is a SV40 promoter and the reporter nucleic acid encodes luciferase.
33 . The method of claim 30 , wherein the given signaling molecule is selected from G-CSF-like molecule, erythropoietic product, and IL-3.
34 . The method of claim 33 , wherein the given signaling molecule is selected from recombinant methionyl human granulocyte colony-stimulting factor, epoetin alfa, and darbepoetin alfa.
35 . A method for determining whether a test compound has activity of a given signaling molecule, comprising
a) contacting the test compound with a signaling molecule-responsive host cell comprising the vector of claim 15 under conditions in which the reporter nucleic acid expresses a reporter protein in response to compounds that have the activity of the given signaling molecule; b) detecting the reporter protein; c) comparing the level of detected reporter protein expression in (b) with the level of detected reporter protein expressed by a signaling molecule-responsive host cell comprising the vector of claim 15 in the presence of the given signaling molecule, but in the absence of the test compound, to determine whether the test compound has the activity of the given signaling molecule.
36 . The method of claim 35 , wherein the promoter is a TK promoter and the reporter nucleic acid encodes luciferase.
37 . The method of claim 35 , wherein the promoter is a SV40 promoter and the reporter nucleic acid encodes luciferase.
38 . The method of claim 35 , wherein the given signaling molecule is selected from G-CSF-like molecule, erythropoietic product, and IL-3.
39 . The method of claim 38 , wherein the given signaling molecule is selected from recombinant methionyl human granulocyte colony-stimulting factor, epoetin alfa, and darbepoetin alfa.
40 . A method for determining whether a test compound impacts the activity of a signaling molecule, comprising
a) contacting the test compound with a signaling molecule-responsive host cell comprising the vector of claim 15 in the presence of the signaling molecule under conditions in which the reporter nucleic acid expresses a reporter protein in response to the signaling molecule; b) detecting the reporter protein; c) comparing the level of detected reporter protein expression in (b) with the level of detected reporter protein expressed by a signaling molecule-reponsive host cell comprising the vector of claim 15 in the presence of the signaling molecule, but in the absence of the test compound, to determine whether the test compound impacts the activity of the signaling molecule.
41 . The method of claim 40 , wherein the promoter is a TK promoter and the reporter nucleic acid encodes luciferase.
42 . The method of claim 40 , wherein the promoter is a SV40 promoter and the reporter nucleic acid encodes luciferase.
43 . The method of claim 40 , wherein host cell is responsive to at least one signaling molecule selected from G-CSF, EPO, and IL-3.
44 . The method of claim 43 , wherein host cell is responsive to at least one signaling molecule selected from G-CSF, epoetin alfa, and darbepoetin alfa.
45 . A method of producing a polypeptide from an ex vivo mammalian system, comprising producing the polypeptide, testing the polypeptide with the host cell of claim 18 , and determining the amount of protein produced and/or activity of the protein produced by the ex vivo system.
46 . A response element region comprising more than one response element sequences comprising the sequence GTCATTTCCAGGAAATCACC wherein the center region of at least two response element sequences are spatially oriented to be in the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the tenth and eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC.
47 . A response element region comprising more than one response element sequence core regions comprising the sequence TTCCAGGAA wherein the center region of at least two response element sequence core regions are spatially oriented to be in the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the fifth and sixth nucleotides AG of the sequence TTCCAGGAA.
48 . A response element region comprising at least two series of more than one response element sequences comprising the sequence GTCATTTCCAGGAAATCACC;
wherein each series of more than response element sequences are linked together by a sequence of approximately eight nucleotides, wherein, within a first series of the response element sequences, each center region of the response element sequences are spatially oriented to be in approximately the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the tenth and eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC; wherein, within a second series of the response element sequences, each center region of the response element sequences are spatially oriented to be in approximately the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the tenth and eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC; and wherein the center region of the response element sequences of the second series of the response element sequences are spatially oriented to be approximately 72 to 86 degrees from the center region of the first series of the response element sequences as determined from the y and z axis relative to the center axis of the double-helical DNA as the x axis.
49 . A response element region comprising at least two series of more than one response element sequences comprising the sequence GTCATTTCCAGGAAATCACC;
wherein each series of more than one response element sequences are linked together by a sequence of approximately eight nucleotides, wherein, within a first series of the response element sequences, each center region of the response element sequences are spatially oriented to be in approximately the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the tenth and eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC; wherein, within a second series of the response element sequences, each center region of the response element sequences are spatially oriented to be in approximately the same location (on the y and z axis) plus or minus 36 degrees, relative to the center axis of the double-helical DNA (x-axis), wherein the center region is the tenth and eleventh nucleotides AG of the sequence GTCATTTCCAGGAAATCACC; and wherein the center region of the response element sequences of the second series of the response element sequences are spatially oriented to be approximately 144 to 180 degrees from the center region of the first series of the response element sequences as determined from the y and z axis relative to the center axis of the double-helical DNA as the x axis.
50 . The isolated nucleic acid of claim 2 , wherein Y, X, and/or Z are independently selected from a sequence that is capable of binding to at least one transcription factor selected from _NFAT, AP-1, CRE, NFκB, and a member of the STAT protein family.Join the waitlist — get patent alerts
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