Gene silencing methods

Details Gene silencing methods Gene silencing methods

2000-03-22

2003-03-11

Yucel, Remy (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C536S023720, C536S024500

Reexamination Certificate

active

06531647

ABSTRACT:

TECHNICAL FIELD
The present invention relates to methods and materials for controlling gene silencing in plants, and various processes and products employing these methods and materials.
PRIOR ART
Co-suppression and Anti-sense Suppression of Endogenous Genes
It is known that stably-integrated transgenes (referred to as ‘STgenes’ or ‘intGENES’ below) which may be constitutively expressed may be used to suppress homologous endogenous (‘HEgenes’) plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes. Subsequently it has been described how many, if not all plant genes can be “silenced” by transgenes. Gene silencing requires sequence homology between the transgene and the gene that becomes silenced (Matzke, M. A. and Matzke, A. J. M. (1995),
Trends in Genetics,
11: 1-3). This sequence homology may involve promoter regions or coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J. M. (1993)
Annu. Rev. Plant Physiol. Plant Mol. Biol.,
44: 53-76, Vaucheret, H. (1993)
C. R. Acad. Sci. Paris,
316: 1471-1483, Vaucheret, H. (1994),
C. R. Acad. Sci. Paris,
317: 310-323, Baulcombe, D. C. and English, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180, Park, Y-D., et al (1996),
Plant J.,
9: 183-194).
When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. In at least one example the coding sequence transgene was constructed without a promoter (Van Blokland, R., et al (1994),
Plant J.,
6: 861-877).
Co-suppression of Transgenes
It has also become clear that gene silencing (gs) can account for some characteristics of transgenic plants that are not easily reconciled with conventional understanding of genetics. For example the wide variation in STgene expression between sibling lines with a Stgene construct is due in part to gene silencing: low expressers are those with a high level of gene silencing whereas high expressers are those in which gene silencing is absent or induced late in plant development. In this case there is no requirement for there to be an HEgene corresponding to the STgene (see e.g., Elamayan & Vaucheret (1996) Plant J., 9: 787-797.
Viral Resistance
In addition to observations of STgenes, gs has also been implicated in virus resistance. In these cases various factors including ectopic DNA interactions
6
, DNA methylation
7
, transgene expression level
8
and double stranded RNA
9
have been proposed as initiators of gene silencing.
Additionally in non-transgenic plants, it has been demonstrated that leaves which develop subsequently to systematic spread of a virus in a plant contain lower levels of virus than do symptomatic leaves. This resistance may be similar in nature to transgene-induced gene silencing (see e.g. Ratcliff et al (1997)
Science,
276: 1558-1560).
Cytoplasmically Replicating Viral Constructs
Biosource Technologies, in WO 95/34668, have suggested the use of genetic constructions based on RNA viruses which replicate in the cytoplasm of cells to provide inhibitory RNA, either anti-sense or sense (“co-suppressor”) RNA. The constructs were used to inhibit a particular HEgene (phytoene desaturase). Cells were transfected with the cytoplasmically-replicating genetic constructions in which the RNA encoding region is specific for the gene of interest. The hybrid viruses spread throughout the plant, including the non-inoculated upper leaves (as verified by transmission electron microscopy). System-wide gene silencing was reported following transfection.
GB patent application 9703146.2, and PCT/GB98/00442, both filed in the name of John Innes Centre Innovations Limited, are hereby incorporated by reference. These applications, which were not published prior to the claimed priority date of the present application, discuss various constructs (‘amplicons’) which are intended to be stably integrated into the plant genome, and to generate cytoplasmically replicating constructs which are capable of eliciting gene silencing.
Silencing in Animals
Fire et al (1998) Nature 391: 806-811 (not published prior to the claimed priority date of the present application) discusses the use of RNA, particularly double-stranded RNA, to achieve silencing of endogenous genes and GFP-transgenes in nematodes. The demonstrated interference effect was apparently able to cross cell-boundaries.
Applications for Gene-silencing
In principle there is an enormous practical potential of gs for crop improvement. It is possible to silence genes conferring unwanted traits in the plant by transformation with transgene constructs containing elements of these genes. Examples of this type of application include gs of ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; gs of genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of F
1
hybrids; gs of genes involved in lignin biosynthesis to improve the quality of paper pulp made from vegetative tissue of the plant; gene silencing of genes involved in flower pigment production to produce novel flower colours; gene silencing of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by gene silencing of genes required for toxin production.
Gene silencing is also useful for investigating gene function in that it can be used to impose an intermediate or a null phenotype for a particular gene, which can provide information about the function of that gene in vivo.
A major complication in the practical exploitation of this phenomenon to date is the unpredictable and low occurrence of gene silencing. Therefore, it has not been realistic to attempt gene silencing in plants that are difficult to transform and for which it is difficult to produce many transformants. Similarly, it would be difficult to activate (and deactivate) gene silencing against several different traits or against several viruses in the same plant. Even with plants that are easy to transform the need to generate multiple lines limits the ease of exploitation of gene silencing.
INVENTION
The present inventors have now demonstrated a novel means of providing consistent, controlled, systemic gene silencing within a system, particularly a mature plant, which may (but is preferably not) a transgenic plant. This novel approach is clearly distinct from previously described approaches to gene silencing, for example, transwitch and antisense technologies, in that it describes a multicomponent system in which there is the potential to regulate the gene silencing spatially and optionally temporally.
The current invention is also distinct from the virus-induced gene silencing described previously by Biosource Technologies. In the current invention there is no absolute requirement that the transgenes conferring the gene silencing or their transcripts are able to replicate using viral components or through mechanisms that resemble virus replication, although in certain advantageous embodiments they may do so. Importantly, the systemic silencing of the invention does not require that the transgenes or their transcripts use virus-derived mechanisms to move between cells (e.g. ‘movement proteins’ as they are termed in the art).
These movement proteins are encoded by most (probably nearly all) plant viruses. Movement proteins are normally recognised by mutation analysis of a viral genome. Mutation of a movement protein gene affects the ability of a virus to spread in the infected plant but does not affect the ability of the virus to replicate. Examples of viral movement proteins identified in this way include the 30 kDa protein of tobacco mosaic virus (Deom et al., 1987), the 25 kDa, 12 kDa and 8 kDa triple gene block proteins of potato virus X (
FIG. 1C
) (Angell

Affiliated with

Also associated with

Rating

Gene silencing methods does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Gene silencing methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Gene silencing methods will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3048598