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US 2014/0196160 A1Published application
Methods and Means for Monitoring and Modulating Gene Silencing
AssigneeCommonwealth Scientific and Industrial Research Organisation
InventorsPeter Waterhouse, Susan Wesley, Chris Helliwell
Life of this patent
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Abstract
Methods and means are provided for monitoring and modulating reduction of gene expression in eukaryotic organisms, using double stranded RNA comprising, in addition to the dsRNA region comprising nucleotide sequences homologous to the target gene, additional dsRNA regions designed to down regulate a second gene or which are unrelated to the target gene.
Claims33
A method for monitoring the reduction of the expression of a target gene in a cell of a eukaryotic organism, said method comprising the steps of
- a) providing the eukaryotic cell with a dsRNA comprising a first region, a second region, a third region and a fourth region
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to a nucleotide sequence complementary to 19 consecutive nucleotides from said sense nucleotide region of the target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region:
- iv) said third region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of a second gene present in said eukaryotic cell and which is different from said target gene;
- v) said fourth region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of said 19 consecutive nucleotides from said sense nucleotide region of said second gene;
- vi) said third and fourth region being capable of forming a double stranded RNA region;
- and
- b) monitoring the reduction of the expression of said target gene by analyzing the reduction in expression of said second gene.
6 more elements
An dsRNA comprising a first region, a second region, a third region and a fourth region
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to a nucleotide sequence complementary to 19 consecutive nucleotides from said sense nucleotide region of the target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region:
- iv) said third region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of a second gene present in said eukaryotic cell and which is different from said target gene;
- v) said fourth region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of said 19 consecutive nucleotides from said sense nucleotide region of said second gene;
- and
- vi) said third and fourth region being capable of forming a double stranded RNA region.
4 more elements
A DNA molecule for measuring the reduction of expression of a target gene in a cell of a eukaryotic organism comprising
- a) a promoter region which functions in said eukaryotic cell;
- operably linked to
- b) a DNA region which when transcribed yields a dsRNA molecule, said dsRNA comprising a first, second, third and fourth region;
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to a nucleotide sequence complementary to 19 consecutive nucleotides from said sense nucleotide region of the target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region:
- iv) said third region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of a second gene present in said eukaryotic cell and which is different from said target gene;
- v) said fourth region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of said 19 consecutive nucleotides from said sense nucleotide region of said second gene;
- vi) said third and fourth region being capable of forming a double stranded RNA region;
- and
- c) a transcription termination and polyadenylation region which functions in said eukaryotic organism, wherein said second gene is an endogenous gene of said eukaryotic organism or a transgene stably integrated into the genome of cells of said eukaryotic organism.
8 more elements
A method for identifying, within a population of dsRNA-mediated gene-silenced eukaryotic organisms, the organisms with the desired degree of silencing of a target gene comprising:
- a) providing cells of said eukaryotic organisms with a dsRNA comprising a first region, a second region, a third region and a fourth region
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to a nucleotide sequence complementary to 19 consecutive nucleotides from said sense nucleotide region of the target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region:
- iv) said third region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from a sense nucleotide region of a second gene present in said eukaryotic cell and which is different from said target gene;
- v) said fourth region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of said 19 consecutive nucleotides from said sense nucleotide region of said second gene;
- vi) said third and fourth region being capable of forming a double stranded RNA region;
- and
- b) identifying said organism with said desired degree of silencing of said target gene, by selecting said organisms with the desired degree of silencing of said second gene.
6 more elements
A method for modulating the reduction of the expression of a target gene in a cell of a eukaryotic organism, comprising the steps of
- a) providing the eukaryotic cell with a dsRNA comprising a first region, a second region, a third region and a fourth region,
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from the sense nucleotide sequence of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of 19 consecutive nucleotides from said sense nucleotide sequence of said target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region;
- iv) said third region and said fourth region comprising complementary nucleotide sequences which have a sequence identity of less than 50% to the nucleotide sequence of said target gene and which are capable of forming a double stranded RNA,
- wherein said target gene is an endogenous gene in said eukaryotic cell or a transgene stably integrated in the genome of said eukaryotic cell.
3 more elements
An RNA molecule for modulating the expression of a target gene in a cell of a eukaryotic organism, comprising a first region, a second region, a third region and a fourth region,
- i) said first region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to 19 consecutive nucleotides from the sense nucleotide sequence of said target gene;
- ii) said second region comprising a nucleotide sequence of at least 19 consecutive nucleotides having at least 94% sequence identity to the complement of 19 consecutive nucleotides from said sense nucleotide sequence of said target gene;
- iii) said first region and said second region being capable of forming a double stranded RNA region;
- iv) said third region and said fourth region comprising complementary nucleotide sequences which have a sequence identity of less than 50% to the nucleotide sequence of said target gene and which are capable of forming a double stranded RNA,
- wherein said target gene is an endogenous gene in said eukaryotic cell or a transgene, stably integrated in the genome of said eukaryotic cell.
2 more elements
Dependencies are inferred from the claim text.