Methods and Means for Obtaining Modified Phenotypes

Patent metadata

Field	Value
Publication number	US-2016355833-A1
Application number	US-201615212142-A
Country	US
Kind code	A1
Filing date	Jul 15, 2016
Priority date	Aug 13, 1999
Publication date	Dec 8, 2016
Grant date	—

Abstract

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated, target-specific RNA is provided by transcription of a chimeric gene comprising a promoter and a DNA region encoding the target-specific RNA.

First claim

1 . (canceled)

2 . A chimeric DNA comprising a promoter operably linked to a target specific DNA region which encodes an unpolyadenylated hairpin RNA, wherein the target specific DNA region comprises a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein the target specific antisense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of a complement of a part of an RNA molecule transcribed or produced from a nucleic acid of interest in an animal cell, wherein the target specific sense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of the part of the RNA molecule transcribed or produced from the nucleic acid of interest, wherein the target specific sense nucleotide sequence and the target specific antisense nucleotide sequence are separated and linked by a spacer sequence, such that the unpolyadenylated hairpin RNA resulting from transcription of the target specific DNA region comprises the spacer sequence located between sense and antisense nucleotide sequences in the unpolyadenylated hairpin RNA, and the sense and antisense nucleotide sequences are complementary to each other so as to form the unpolyadenylated hairpin RNA.

3 . The chimeric DNA of claim 2 , wherein said target specific sense nucleotide sequence corresponds to a translated region of the nucleic acid of interest.

4 . The chimeric DNA of claim 2 , wherein the target specific sense nucleotide sequence corresponds to an untranslated region of the RNA molecule produced from the nucleic acid of interest.

5 . The chimeric DNA of claim 2 , wherein the promoter is recognized by a eukaryotic RNA polymerase I or III and the DNA further comprises a terminator for the polymerase I or III.

6 . The chimeric DNA of claim 2 , wherein the nucleic acid of interest is a gene incorporated in the genome of the animal cell.

7 . The chimeric DNA of claim 2 , wherein the nucleic acid of interest is an endogenous gene of the animal cell.

8 . The chimeric DNA of claim 2 , wherein the nucleic acid of interest is a viral nucleic acid.

9 . The chimeric DNA of claim 2 , wherein the unpolyadenylated hairpin RNA lacks a 5′ cap structure.

10 . The chimeric DNA of claim 2 , wherein the spacer sequence has a length of from 4 to 200 basepairs.

11 . The chimeric DNA of claim 2 , wherein the promoter is a constitutive promoter.

12 . The chimeric DNA of claim 2 , wherein the promoter is an inducible promoter.

13 . The chimeric DNA of claim 2 , wherein the promoter is recognized by a single subunit RNA polymerase from a bacteriophage.

14 . The chimeric DNA of claim 2 , wherein the promoter is recognized by a eukaryotic RNA polymerase III and the DNA further comprises a terminator for the polymerase III.

15 . The chimeric DNA of claim 2 , wherein the target specific DNA region which is transcribed to form the unpolyadenylated hairpin RNA lacks a DNA region involved in 3′ end formation and polyadenylation.

16 . The chimeric DNA of claim 15 , wherein the target specific DNA region comprises a 3′ end formation signal and lacks a polyadenylation signal.

17 . An animal cell in culture comprising a chimeric DNA which comprises a promoter operably linked to a target specific DNA region which encodes an unpolyadenylated hairpin RNA, wherein the target specific DNA region comprises a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein the target specific antisense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of a complement of a part of an RNA molecule transcribed or produced from a nucleic acid of interest in the animal cell, wherein the target specific sense nucleotide sequence comprises 20 consecutive nucleotides in a sequence identical to the sequence of the part of the RNA molecule transcribed or produced from the nucleic acid of interest, wherein the target specific sense nucleotide sequence and the target specific antisense nucleotide sequence are separated and linked by a spacer sequence, such that the unpolyadenylated hairpin RNA resulting from transcription of the target specific DNA region comprises the spacer sequence located between sense and antisense nucleotide sequences in the unpolyadenylated hairpin RNA, and wherein the sense and antisense nucleotide sequences are complementary to each other so as to form the unpolyadenylated hairpin RNA.

18 . The animal cell in culture of claim 17 , wherein the unpolyadenylated RNA is expressed in the cell.

Assignees

• Commw Scient Ind Res Org

Inventors

• Waterhouse Peter Michael
• Wang Ming-Bo

Classifications

• C12N15/8218 Primary

  Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS] · CPC title

• C12N9/2405

  Glucanases · CPC title

• C12Y302/01031

  Beta-glucuronidase (3.2.1.31) · CPC title

• C12N15/8247

  involving modified lipid metabolism, e.g. seed oil composition · CPC title

• C12N15/8283

  for virus resistance · CPC title