Enhancement of virus induced gene silencing (VIGS) through viral-based expression of inverted-repeats

• US 20040053225A1
• Filed: 09/12/2003
• Published: 03/18/2004
• Est. Priority Date: 09/13/2002
• Status: Abandoned Application

• Current Assignee: Large Scale Biology Corporation
• Original Assignee: Large Scale Biology Corporation
• Inventors: Lacomme, Christophe, Pogue, Gregory P.
• Application Number:
  US10/660,860
• Publication Number:
  US 20040053225A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 2320/12 in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

Virus-based expression vectors carrying sequences corresponding to endogenous host genes trigger silencing through a homology-dependent RNA degradation mechanism. Virus-induced gene silencing (VIGS), is useful as a reverse-genetic tool for use in functional genomic programs for loss-of-function transient assays-based screening. Described herein is an approach to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production.

Claims

1. A DNA construct containing an infectious clone of an RNA virus containing a hairpin sequence corresponding to a gene encoded within the nucleus of the target host, said hairpin sequence being a sequence fragment of greater than 20 bp in length where the sequence fragment in the sense orientation is followed by different sequence fragment, derived from the first in the reverse complement orientation, with:

no intervening sequence;

or an intervening sequence of no greater than the collective length of the two sequence fragments comprising the hairpin.

2. A cytoplasmic inhibition of nuclear gene expression resulting from hairpin RNA expression from an RNA virus genome in accordance with claim 1.

3. A plant host experiencing cytoplasmic inhibition of gene expression following infection with an RNA virus genome containing a hairpin nucleotide sequence in accordance with claim 1.

4. An animal host experiencing cytoplasmic inhibition of gene expression following infection with an RNA virus genome containing a hairpin nucleotide sequence in accordance with claim 1.

5. A method for determining nuclear gene function through a process of cytoplasmic inhibition of gene expression following infection with an RNA virus genome containing a hairpin nucleotide sequence in accordance with claim 1.

6. A hairpin sequence vector in accordance with claim 1 comprising a tobacco mosaic virus.

7. A hairpin sequence vector in accordance with claim 1 comprising a barley striped mosaic virus genome.

8. A virus vector for cytoplasmic gene silencing applications, in accordance with claim 1, comprising a very short, yet highly active gene silencing inducer, such as said hairpin sequence, wherein said virus vector exhibits improved genetic stability.

9. A vector as specified in claim 1, that contains a 20-30 nucleotide hairpin sequence for infection of mammalian cells and delivery of hairpin RNA to the cytosol for cytoplasmic gene inhibition.

10. A vector as specified in claim 9, derived from the alphavirus, rubivirus virus families.