Gene silencing
AssigneeE.I. du Pont de Nemours and Company
InventorMilo Aukerman
Life of this patent
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Abstract
The invention provides methods and compositions useful in target sequence suppression and target sequence validation. The invention provides polynucleotide constructs useful for gene silencing, as well as cells, plants and seeds comprising the polynucleotides. The invention also provides a method for using microRNA to silence a target sequence.
Claims76
A method of inhibiting expression of a target sequence in a cell comprising:
- (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising in the following order:
- (i) at least about 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO:
- 3,
- (ii) a first oligonucleotide of 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,
- (iii) at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO:
- 3,
- (iv) the second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and
- (v) at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO:3;
- wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and
- (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.
An isolated polynucleotide comprising in the following order at least 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO:
- 3, a first oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO:
- 3, a second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and the second oligonucleotide is substantially complementary to the target sequence, and at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO:
- 3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.
A method of inhibiting expression of a target sequence in a cell comprising:
- (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, wherein the polynucleotide comprises in the following order:
- (i) nucleotides 1-38 of SEQ ID NO:
- 3,
- (ii) a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,
- (iii) nucleotides 60-106 of SEQ ID NO:
- 3,
- (iv) a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and
- (v) nucleotides 128-159 of SEQ ID NO:3;
- wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and
- (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.
An isolated polynucleotide comprising in the following order nucleotides 1-38 of SEQ ID NO:
- 3, a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, nucleotides 60-106 of SEQ ID NO:
- 3, a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a miRNA, and the second oligonucleotide is substantially complementary to the target sequence, and nucleotides 128-159 of SEQ ID NO:3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.
A method of inhibiting expression of a target sequence in a cell comprising:
- (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant miRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a miRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin;
- and
- (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.
A method of inhibiting expression of a target sequence in a cell comprising:
- (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant miR172 miRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a miRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin;
- and
- (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.
A method of inhibiting expression of a target sequence in a cell comprising:
- (a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified Arabidopsis miR172 miRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a miRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin;
- and
- (b) expressing the nucleic acid construct for a time sufficient to produce the miRNA, wherein the miRNA inhibits expression of the target sequence.
An isolated polynucleotide comprising a polynucleotide which encodes a modified plant miRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a miRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.
An isolated polynucleotide comprising a polynucleotide encoding a modified plant miR172 miRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a miRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.
An isolated polynucleotide comprising polynucleotide encoding a modified Arabidopsis miR172 miRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a miRNA which is substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.
Dependencies are inferred from the claim text.