In vivo Assembly of Transcription Units

Patent metadata

Field	Value
Publication number	US-2017191074-A1
Application number	US-201715466669-A
Country	US
Kind code	A1
Filing date	Mar 22, 2017
Priority date	Feb 13, 2004
Publication date	Jul 6, 2017
Grant date	—

Abstract

Methods of providing gene suppression DNA in a eukaryotic organism comprising introducing a first DNA segment and at least one second DNA segment into the genome of the organism. One of the DNA segments contains a promoter and a transcribable DNA. Another DNA segment contains at least part of the transcribable DNA. When inserted in tandem, the DNA segments are assembled in vivo forming a recombinant transcription unit. RNA transcribed from the transcription unit can form double-stranded RNA.

First claim

What is claimed is:

1 . A method of in vivo assembling of a recombinant transcription unit for gene suppression,
said method comprising introducing into a chromosome in a cell a first segment of DNA and a distinct second segment of DNA, each comprising a part of a recombinant transcription unit for gene suppression; wherein said first segment of DNA comprises a promoter DNA and transcribable DNA from at least one gene targeted for suppression in either a sense or an anti-sense orientation; wherein said second segment of DNA comprises at least a part of said transcribable DNA; wherein, when inserted in tandem into a chromosome in a cell, said first and second segments of DNA can form an assembled transcription unit comprising said promoter DNA operably linked to transcribable DNA from the first segment of DNA linked to transcribable DNA from the second segment of DNA and wherein RNA transcribed from said assemble transcription unit is capable of forming double-stranded RNA; and selecting a transgenic cell in which said double-stranded RNA is produced.

2 . A method of claim 1 wherein at least one of said first and second segments of DNA further comprises a selectable marker transcription unit.

3 . A method of claim 1 wherein said first and second segments of DNA are introduced from a common plasmid.

4 . A method of claim 1 wherein said first and second of said segments of DNA are introduced from separate plasmids.

5 . A method of claim 3 further comprising introducing a third segment of DNA comprising a selectable marker transcription unit.

6 . A method of claim 5 wherein said third segment of DNA is introduced from a second plasmid.

7 . A method of claim 1 wherein said first segment of DNA comprises a promoter DNA and DNA from at least one gene targeted for suppression in an anti-sense orientation with respect to transcription initiated by said promoter.

8 . A method of claim 3 wherein said first and second segments of DNA are T-DNAs which are co-integrated into the genome of an eukaryotic organism by Agrobacterium tumefaciens -mediated transformation using a plasmid vector comprising said T-DNAs, wherein said plasmid vector comprises
(a) a first T-DNA comprising, between left and right T-DNA borders, a promoter which is operable in said eukaryotic organism operably linked to DNA from at least one gene targeted for suppression;
(b) a second T-DNA comprising, between left and right T-DNA borders, at least part of said DNA from at least one gene targeted for suppression.

9 . A method of claim 8 wherein said DNA from one or more genes targeted for suppression in said first T-DNA is adjacent to a right T-DNA border and wherein said at least part of said DNA from one or more genes targeted for suppression in said second T-DNA is located adjacent to a right T-DNA border.

10 . A method of claim 8 wherein said organism is a plant.

11 . A method of claim 8 wherein in each of said T-DNAs there is common DNA of identical sequence adjacent to one of said T-DNA borders, wherein said common DNA is oriented in each T-DNA to enable annealing of T-strands produced from the two T-DNAs, whereby said borders are eliminated from the assembled transcription unit.

12 . A method of claim 8 wherein DNA from at least one gene targeted for suppression in said first T-DNA is in an anti-sense orientation with respect to transcription initiated by said promoter.

13 . A plasmid vector for Agrobacterium tumefaciens -mediated, tandem co-integration of two T-DNAs into the genome of a plant for in vivo assembly of a gene suppression transcription unit, wherein said plasmid vector comprises
(a) a first T-DNA comprising in series a promoter which is operable in said plant, DNA from at least one gene targeted for suppression and a T-DNA border; and (b) a second T-DNA comprising in series a T-DNA border and at least a part of said DNA from at least one gene targeted for suppression;
wherein, when said first and second T-DNAs are co-integrated in tandem and joined at said T-DNA borders, a transcription unit is formed comprising said promoter operably linked to DNA from at least one gene targeted for suppression; and wherein RNA transcribed by said transcription unit can form double-stranded RNA.

14 . A plasmid vector of claim 13 wherein at least one of said T-DNAs further comprises a marker gene transcription unit.

15 . A plasmid vector of claim 13 wherein said T-DNAs are co-integrated in tandem and joined at T-DNA borders which are
(a) both right T-DNA borders,
(b) both left T-DNA borders, or
(c) one left T-DNA border and one right T-DNA border.

16 . A plasmid vector of claim 13 wherein in each of said T-DNAs there is common DNA of identical sequence adjacent to one of said T-DNA borders, wherein said common DNA is oriented in each T-DNA to enable annealing of T-strands produced from the two T-DNAs, whereby said borders are eliminated from the assembled transcription unit.

17 . A plasmid vector of claim 13 in which DNA from at least one gene targeted for suppression in said first T-DNA is in an anti-sense orientation with respect to DNA transcribed by said promoter.

18 . A method of in vivo assembling of a recombinant transcription unit for gene suppression from two T-DNAs which are co-integrated in tandem into the genome of an eukaryotic organism by Agrobacterium tumefaciens -mediated transformation using a binary plasmid vector comprising said T-DNAs, said method comprising introducing into a cell of said organism a said binary plasmid vector comprising said two T-DNAs and a tumor-inducing plasmid;
wherein said first T-DNA comprises, in series, a promoter, transcribable DNA from at least one gene targeted for suppression in an anti-sense orientation and a T-DNA border and said second T-DNA comprises, in series, a T-DNA border and at least a part of said transcribable DNA in a sense orientation with respect to said border; wherein, when inserted in tandem into a chromosome in said organism, said first and second T-DNAs can form an assembled transcription unit comprising said promoter operably linked to transcribable DNA from the first T-DNA linked to T-DNA border linked to transcribable DNA from the second T-DNA; and wherein RNA transcribed from said assembled transcription unit is capable of forming double-stranded RNA; and selecting a transgenic cell in which said double-stranded RNA is produced.

19 . A method of claim 18 wherein said organism is a plant.

20 . A method of claim 18 wherein at least one of the T-DNAs comprises a selectable marker transcription unit.

Assignees

• Monsanto Technology Llc

Inventors

• Fillatti Joanne
• Gilbertson Larry
• Voelker Toni

Classifications

• C12N15/8201 Primary

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N15/8218

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

• C12N15/8247

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

• C12N15/8203

  Virus mediated transformation · CPC title