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Modified gene-silencing RNA and uses thereof

a technology of rna and genes, applied in the field of modified genesilencing rna, can solve the problems of hammering the intact maintenance of these nucleic acids

Inactive Publication Date: 2009-06-25
COMMONWEALTH SCI & IND RES ORG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0048]The invention further provides chimeric sense RNA molecules or chimeric DNA molecules encoding such chimeric sense RNA molecules for reduction of expression of a target gene in a cell of a eukaryotic organism in cooperation with a chimeric antisense RNA molecule. In this embodiment of the invention, the chimeric sense RNA molecule comprises a sense target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides having at least about 94% sequence identity to the nucleotide of the target gene, operably linked to a largely double-stranded RNA region.

Problems solved by technology

Indeed, the chimeric nucleic dsRNA molecules or the genes encoding those RNA molecules contain large, more or less perfect inverted repeat structures, and such structures tend to hamper the intact maintenance of these nucleic acids in intermediate prokaryotic cloning hosts.

Method used

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  • Modified gene-silencing RNA and uses thereof

Examples

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Effect test

example 1

Construction of the Different Plant Lines Containing Different Chimeric Genes Used

[0162]As an example target gene to downregulate the expression using the various constructs, the EIN2 gene from Arabidopsis thaliana was chosen. The downregulation of the expression of the EIN2 gene can easily be visualized by germinating seeds on MS-ACC medium (containing aminocyclopropane-1-carboxylic acid (ACC)) and incubating either in the dark or in light.

[0163]Dark-grown EIN2 silenced seedlings grown in the dark have a longer hypocotyl and a more developed root system compared to wildtype (“wt”) seedlings, whereas EIN2 silenced seedlings grown in light can be differentiated from the wt seedlings by their larger cotyledon size (see FIG. 3).

[0164]The EIN2 nucleotide sequence to be used in the different constructs in sense or antisense orientation was amplified by PCR using oligonucleotide primers with a nucleotide sequence as represented in SEQ ID NO: 12 and 13 using genomic DNA (nucleotide sequenc...

example 2

Analysis of Expression of the EIN2 Gene in Transgenic Arabidopsis Lines Comprising the Different Chimeric Genes of Example 1

[0172]The chimeric constructs represented in FIG. 2 were introduced into Agrobacterium tumefaciens using conventional methods and the resulting Agrobacterium strains were used to introduce the chimeric genes into Arabidopsis ecotype Landsberg erecta through the dipping method. Transgenic lines were selected on 15 mg / L hygromycin or 50 mg / L kanamycin as the selective agent. T1 opr F1 seed was collected and assayed for EIN2 silencing.

[0173]To this end, the seed was plated on MS medium containing 50 μM ACC. The plates were sealed tightly with parafilm and kept either under light or in the dark. Silencing was scored by looking at the size of roots and cotyledons (incubation in the light) or by looking at the size of roots or hypocotyls (incubation in the dark). In EIN2 silenced lines, the roots or hypocotyls are significantly longer, and the cotyledons are signific...

example 3

Analysis of Expression of the EIN2 Gene in Arabidopsis Lines Obtained by Crossing of the Transgenic Arabidopsis Lines Comprising the Different Chimeric Genes of Example 1

[0175]By cross-pollination between the Arabidopsis lines MBW353, MBW355, MBW359, and MBW360 new lines were obtained simultaneously containing sense and antisense EIN2 constructs. These new lines were analyzed in a similar way as described in Example 2. The results are summarized in Table 2. Plants wherein at least one of the transgenes contained a PSTVd sequence were very efficiently silenced.

TABLE 2Summary of the efficiency of EIN2 silencing inA. thaliana plants comprising different combinationof sense and antisense EIN2 constructs.ShortN° of linesN° of linesFrequency ofLinedescriptiontestedsilencedsilencingMBW353 ×EIN2 sense7228.5%MBW360Native intronsAndEIN2 antisenseMBW353 ×EIN2 sense3 100%MBW359Native intronsAndEIN2 antisense3PSTVdPdk intronMBW355 ×EIN2 sense54  80%MBW360Native intronsPSTVdAndEIN2 antisenseMBW35...

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Abstract

Methods and means for efficiently downregulating the expression of any gene of interest in eukaryotic cells and organisms are provided. To this end, the invention provides modified antisense and sense RNA molecules, chimeric genes encoding such modified antisense or sense RNA molecules and eukaryotic organisms such as plants, animals or fungi, yeast or molds, comprising the modified antisense and / or sense RNA molecules or the encoding chimeric genes.

Description

FIELD OF THE INVENTION[0001]The present invention relates to methods for efficiently downregulating the expression of any gene of interest in eukaryotic cells and organisms. To this end, the invention provides modified antisense and sense RNA molecules, chimeric genes encoding such modified antisense or sense RNA molecules, and eukaryotic organisms such as plants, animals or fungi, yeasts or molds, comprising the modified antisense and / or sense RNA molecules and / or the chimeric genes encoding those RNA molecules.BACKGROUND ART[0002]Recently, it has been shown that introduction of double-stranded RNA (dsRNA) also called interfering RNA (RNAi), or hairpin RNA is an effective trigger for the induction of gene silencing in a large number of eukaryotic organisms, including animals, fungi, and plants.[0003]Both the qualitative level of dsRNA-mediated gene silencing (i.e., the level of gene silencing within an organism) and the quantitative level (i.e., the number of organisms showing a si...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12Q1/68C12N15/82A61K31/7105C12N15/11C12N5/04C12N1/19C12N5/08A01H5/00C12N1/15C12N5/10C12N15/09
CPCA01K2217/05C12N15/111C12N15/8218C12N2330/30C12N2310/111C12N2310/14C12N2310/53C12N15/8249
Inventor WANG, MING-BOWATERHOUSE, PETER
Owner COMMONWEALTH SCI & IND RES ORG
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