Methods and Means for Obtaining Modified Phenotypes

Abstract

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.

1 . (canceled) 
     
     
         2 . A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising the step of introducing a chimeric DNA comprising the following operably linked parts:
 a) a promoter, operative in the plant cell;   b) a DNA region, which when transcribed, yields an RNA molecule comprising   an RNA region capable of forming an artificial hairpin RNA structure   comprising two annealing RNA sequences wherein one of the annealing RNA sequences of the hairpin RNA structure comprises a sense sequence that is identical to at least 100 consecutive nucleotides of a nucleotide sequence of the nucleic acid of interest and wherein the second of said annealing RNA sequences comprises an antisense sequence with is identical to at least 100 consecutive nucleotides of the complement of at least part of the nucleotide sequence of the nucleic acid of interest and wherein the sense and antisense sequences of the artificial hairpin structure are not naturally occurring in one RNA molecule or the sense and antisense sequences are separated by a spacer region with is heterologous with respect to the target gene; and   c) a DNA region involved in transcription termination and polyadenylation.   
     
     
         3 - 12 . (canceled) 
     
     
         13 . A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell comprising the step of introducing into the plant cell a chimeric RNA molecule comprising at least one RNA region with a nucleotide sequence comprising
 i. a sense nucleotide sequence of at least 15 consecutive nucleotides having 100% sequence identity with at least part of the nucleotide sequence of the nucleic acid of interest; and   ii. an antisense nucleotide sequence including at least 15 consecutive nucleotides, having about 100% sequence identity with the complement of said at least 15 consecutive nucleotides of said sense nucleotide sequence;   wherein said RNA region is capable of forming an artificial hairpin RNA structure with a double stranded RNA stem by base-pairing between the sense and antisense nucleotide sequences such that said at least 15 consecutive nucleotides of the sense sequence basepair with said at least 15 consecutive nucleotides of the antisense sequence.   
     
     
         14 - 20 . (canceled) 
     
     
         21 . A method for obtaining a phenotype associated with the reduction of expression of a nucleic acid of interest in a plant cell, said method comprising
 a. selecting within said nucleotide sequence of interest, a target sequence of at least 15 consecutive nucleotides;   b. designing a sense nucleotide sequence corresponding to the length of the selected target sequence and which has a sequence identity of about 100% with said selected target sequence;   c. designing an antisense nucleotide sequence which:
 i) has a sequence identity of about 100% with the complement of said at least 15 consecutive nucleotides of said sense nucleotide sequence; 
 ii) comprises a stretch of at least about 15 consecutive nucleotides with 100% sequence identity to the complement of a part of said sense nucleotide sequence; 
   d. introducing an RNA molecule comprising both said sense and antisense nucleotide sequences into a plant cell comprising the nucleic acid of interest; and   e. observing the phenotype by a suitable method.   
     
     
         22 . (canceled) 
     
     
         23 . A plant cell, comprising a nucleic acid of interest, which is normally capable of being phenotypically expressed, further comprising a chimeric RNA molecule comprising at least one RNA region with a nucleotide sequence comprising
 i. a sense nucleotide sequence of at least 15 consecutive nucleotides having 100% sequence identity with at least part of the nucleotide sequence of the nucleic acid of interest; and   ii. an antisense nucleotide sequence including at least 15 consecutive nucleotides, having about 100% sequence identity with the complement of said at least 15 consecutive nucleotides of said sense nucleotide sequence;   wherein said RNA is capable of forming an artificial hairpin RNA with a double stranded RNA region by base-pairing between the sense and antisense nucleotide sequences such that said at least 15 consecutive nucleotides of the sense sequence basepair with said at least 15 consecutive nucleotides of the antisense sequence.   
     
     
         24 - 25 . (canceled) 
     
     
         26 . A plant comprising the plant cell of  claim 23 . 
     
     
         27 - 38 . (canceled) 
     
     
         39 . The method of  claim 13 , wherein said RNA molecule further comprises a spacer nucleotide sequence located between said sense and said antisense nucleotide sequences. 
     
     
         40 . The method of  claim 39 , wherein said spacer nucleotide sequence has a length between 4 and 200 nucleotides. 
     
     
         41 . The method of  claim 13 , wherein said nucleic acid of interest is a gene integrated in the genome of said plant cell. 
     
     
         42 . The method of  claim 41 , wherein said gene is an endogenous gene. 
     
     
         43 . The method of  claim 41 , wherein said gene is a foreign transgene. 
     
     
         44 . The method of  claim 13 , wherein said nucleic acid of interest is comprised in the genome of an infecting virus. 
     
     
         45 . The method of  claim 44 , wherein said infecting virus is an RNA virus. 
     
     
         46 . The method of  claim 13 , wherein said plant cell is comprised within a plant. 
     
     
         47 . The method of  claim 13 , wherein said sense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with at least 20 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 20 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         48 . The method of  claim 13 , wherein said sense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with at least 50 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 50 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         49 . The method of  claim 13 , wherein said sense nucleotide sequence includes at least 100 consecutive nucleotides having between 95% and 100% sequence identity with at least 100 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 100 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 100 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         50 . The method of  claim 13 , wherein said RNA molecule is generated by transcription from a template DNA which comprises two copies of a DNA region, each copy comprising the sense nucleotide sequence, and the two copies being in an inverted repeat orientation and being under control of a promoter. 
     
     
         51 . The method of  claim 50 , wherein said transcription is an in vitro transcription method. 
     
     
         52 . The method of  claim 21 , wherein said RNA molecule is generated by transcription from a template DNA which comprises two copies of a DNA region, each copy comprising the selected target sequence, the two copies being in an inverted repeat orientation and being under control of a promoter. 
     
     
         53 . The method of  claim 52 , wherein said transcription is an in vitro transcription method. 
     
     
         54 . The method of  claim 13 , wherein said introducing of the chimeric RNA molecule into said plant cell is liposome-mediated. 
     
     
         55 . The method of  claim 21 , wherein said introducing of the RNA molecule into said plant cell is liposome-mediated. 
     
     
         56 . The plant cell of  claim 23 , wherein said RNA molecule further comprises a spacer nucleotide sequence located between said sense and said antisense nucleotide sequences. 
     
     
         57 . The plant cell of  claim 56 , wherein said spacer nucleotide sequence has a length between 4 and 200 nucleotides. 
     
     
         58 . The plant cell of  claim 23 , wherein said sense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with at least 20 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 20 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         59 . The plant cell of  claim 23 , wherein said sense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with at least 50 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 50 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         60 . The plant cell of  claim 23 , wherein said sense nucleotide sequence includes at least 100 consecutive nucleotides having between 95% and 100% sequence identity with at least 100 consecutive nucleotides of said part of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 100 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 100 consecutive nucleotides of said sense nucleotide sequence. 
     
     
         61 . The plant cell of  claim 23 , wherein said nucleic acid of interest is a gene integrated in the genome of said plant cell. 
     
     
         62 . The plant cell of  claim 61 , wherein said gene is an endogenous gene. 
     
     
         63 . The plant cell of  claim 62 , wherein said gene is a foreign transgene. 
     
     
         64 . The plant cell of  claim 23 , wherein said nucleic acid of interest is comprised in the genome of an infecting virus. 
     
     
         65 . The plant cell of  claim 64 , wherein said infecting virus is an RNA virus. 
     
     
         66 . The plant cell of  claim 23 , wherein said RNA molecule is generated by transcription from a template DNA which comprises two copies of a DNA region, each copy comprising the sense nucleotide sequence, and the two copies being in an inverted repeat orientation and being under control of a promoter. 
     
     
         67 . The plant cell of  claim 66 , wherein said transcription is an in vitro transcription method.