Modified Gene Silencing

Abstract

This invention relates to methods of controlling gene expression or gene suppression in eukaryotic cells. One aspect of this invention includes modifying the degree of silencing of a target gene by use of a modified suppression element. Another aspect includes providing a eukaryotic cell having a desired phenotype resulting from transcription in the eukaryotic cell of a modified suppression element. Also provided are transgenic eukaryotic cells, transgenic plant cells, plants, and seeds containing modified suppression elements, and useful derivatives of such transgenic plant cells, plants, or seeds, such as food or feed products.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts a method for mapping silencing efficacy of a suppression element, useful in designing or selecting a modified suppression element, as described in Example 1.

FIG. 2 depicts results of the experiments described in Example 2 as a plot of 18:1 fatty acid levels as a function of size of the suppression element.

FIG. 3 depicts results of the experiments described in Example 2, as a plot of 18:1 fatty acid levels as a function of size of the suppression element.

Claims

1. A method of modifying the degree of silencing of a target gene, comprising transcribing in a eukaryotic cell a modified suppression element, thereby obtaining a modified degree of silencing of said target gene, relative to a reference degree of silencing obtained through the transcription in said eukaryotic cell of a reference suppression element that corresponds to said target gene.

2. The method of claim 1, wherein said target gene is endogenous to said eukaryotic cell.

3. The method of claim 1, wherein said target gene is exogenous to said eukaryotic cell.

4. The method of claim 1, wherein said target gene comprises one contiguous nucleotide sequence.

5. The method of claim 1, wherein said target gene comprises non-contiguous nucleotide sequences.

6. The method of claim 1, wherein said eukaryotic cell is selected from an animal cell and a plant cell.

7. The method of claim 1, wherein said modified and reference suppression elements transcribe to at least partially double-stranded RNA.

8. The method of claim 1, wherein said modified and reference suppression elements comprise at least one of: (a) DNA that comprises at least one anti-sense DNA segment that is anti-sense to at least one segment of said target gene;(b) DNA that comprises multiple copies of at least one anti-sense DNA segment that is anti-sense to at least one segment of said target gene;(c) DNA that comprises at least one sense DNA segment that is at least one segment of said target gene;(d) DNA that comprises multiple copies of at least one sense DNA segment that is at least one segment of said target gene;(e) DNA that transcribes to RNA for suppressing said target gene by forming double-stranded RNA and comprises at least one anti-sense DNA segment that is anti-sense to at least one segment of said target gene and at least one sense DNA segment that is at least one segment of said target gene;(f) DNA that transcribes to RNA for suppressing said target gene by forming a single double-stranded RNA and comprises multiple serial anti-sense DNA segments that are anti-sense to at least one segment of said target gene and multiple serial sense DNA segments that are at least one segment of said target gene;(g) DNA that transcribes to RNA for suppressing said target gene by forming multiple double strands of RNA and comprises multiple anti-sense DNA segments that are anti-sense to at least one segment of said target gene and multiple sense DNA segments that are at least one segment of said target gene, and wherein said multiple anti-sense DNA segments and said multiple sense DNA segments are arranged in a series of inverted repeats;(h) DNA that comprises nucleotides derived from a plant miRNA;(i) DNA that comprises nucleotides of a siRNA;(j) DNA that transcribes to an RNA aptamer capable of binding to a ligand; and(k) DNA that transcribes to an RNA aptamer capable of binding to a ligand, and DNA that transcribes to regulatory RNA capable of regulating expression of said target gene, wherein said regulation is dependent on the conformation of said regulatory RNA, and said conformation of said regulatory RNA is allosterically affected by the binding state of said RNA aptamer

9. The method of claim 1, wherein said modified degree of silencing is increased silencing, relative to said reference degree of silencing

10. The method of claim 1, wherein said modified degree of silencing is decreased silencing, relative to said reference degree of silencing

11. The method of claim 1, wherein said modified suppression element comprises at least one fragment of said reference suppression element.

12. The method of claim 11, wherein said at least one fragment is obtained by truncation of said reference suppression element.

13. The method of claim 11, wherein said at least one fragment comprises at least about 1% of said reference suppression element.

14. The method of claim 1, wherein said modified suppression element is selected by mapping the silencing efficacy for the target gene.

15. The method of claim 1, wherein said modified suppression element is further selected to avoid silencing of non-target genes.

16. The method of claim 1, wherein said modified suppression element is further selected to avoid generation of undesirable polypeptides.

17. A method of providing a eukaryotic cell having a desired phenotype resulting from transcription in said eukaryotic cell of a modified suppression element, comprising: (a) providing a range of modified suppression elements, wherein each modified suppression element comprises a fragment of a reference suppression element;(b) separately introducing each of said modified suppression elements into a eukaryotic cell, thereby producing a plurality of transgenic eukaryotic cells;(c) transcribing in each of said transgenic eukaryotic cells the modified suppression element therein introduced, and observing the resulting phenotype resulting from said transcribing; and(d) selecting from said plurality of transgenic eukaryotic cells at least one eukaryotic cell having said desired phenotype.

18. The method of claim 17, wherein said eukaryotic cell comprises a transgenic plant cell.