In vivo assembly of transcription units

Patent metadata

Field	Value
Publication number	US-9611484-B2
Application number	US-201314109835-A
Country	US
Kind code	B2
Filing date	Dec 17, 2013
Priority date	Feb 13, 2004
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

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 assembly of a recombinant transcription unit, said method comprising:
introducing into a chromosome in a plant cell a first T-DNA and a distinct second T-DNA, wherein said first and second T-DNAs are not in operable linkage, and wherein said first and second T-DNAs each comprise a part of a recombinant transcription unit;
wherein said first T-DNA comprises, between left and right T-DNA borders, a promoter operably linked to a first transcribable DNA;
wherein said second T-DNA comprises, between left and right T-DNA borders, a second transcribable DNA, wherein said second T-DNA does not include an operable promoter;
wherein said first and second T-DNAs are randomly inserted into said chromosome in right border to right border configuration to form a transcription unit comprising in 5′ to 3′ order said promoter, said first transcribable DNA, and said second transcribable DNA, and
wherein said first transcribable DNA and said second transcribable DNA are in the same orientation in said transcription unit; and

selecting a transgenic cell in which said transcription unit is produced.

2. The method of claim 1 , wherein said first and second T-DNAs are introduced from a common plasmid.

3. The method of claim 1 , wherein said first and second T-DNAs are introduced from separate plasmids.

4. The method of claim 1 , wherein at least one of said first and second T-DNAs further comprises a selectable marker transcription unit.

5. The method of claim 1 , further comprising introducing a third T-DNA comprising a selectable marker transcription unit.

6. The method of claim 5 , wherein said third T-DNA is introduced from a second plasmid.

7. The method of claim 1 , wherein said first transcribable DNA is: (a) in an anti-sense orientation with respect to transcription initiated by said promoter, or (b) in a sense orientation with respect to transcription initiated by said promoter.

8. The method of claim 1 , wherein said first transcribable DNA is adjacent to said right T-DNA border of said first T-DNA, and wherein said second transcribable DNA is located adjacent to said right T-DNA border of said second T-DNA.

9. The method of claim 1 , 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.

10. 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 transcription unit, 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 plant and is operably linked to a first transcribable DNA; and
(b) a second T-DNA comprising, between left and right T-DNA borders, a second transcribable DNA, wherein said second T-DNA does not include an operable promoter;
wherein, when said first and second T-DNAs are co-integrated in tandem and joined at said T-DNA borders in right border to right border configuration, a transcription unit is formed comprising, in order, said promoter operably linked to said first transcribable DNA and to said second transcribable DNA.

11. The plasmid vector of claim 10 , wherein at least one of said T-DNAs further comprises a marker gene transcription unit.

12. The plasmid vector of claim 10 , 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.

13. The plasmid vector of claim 10 , in which said first transcribable DNA is: (a) in an anti-sense orientation with respect to transcription initiated by said promoter, or (b) in a sense orientation with respect to transcription initiated by said promoter.

14. A method of in vivo assembling of a recombinant transcription unit from a first T-DNA and a second T-DNA which are co-integrated in tandem into the genome of a plant by Agrobacterium tumefaciens -mediated transformation, said method comprising:
introducing into a cell of said plant a binary plasmid vector comprising said first and second T-DNAs;
wherein said first T-DNA comprises, between left and right T-DNA borders, a promoter operably linked to a first transcribable DNA,
wherein said second T-DNA comprises, between left and right T-DNA borders, a second transcribable DNA, wherein said second T-DNA does not include an operable promoter;
wherein said first and second T-DNAs are randomly inserted into a chromosome of said cell in right border to right border configuration to form a transcription unit comprising in 5′ to 3′ order said promoter, said first transcribable DNA, and said second transcribable DNA, and wherein said first transcribable DNA and said second transcribable DNA are in the same orientation in said transcription unit; and
selecting a transgenic cell in which said transcription unit is produced.

15. The method of claim 14 , wherein at least one of the T-DNAs further 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

• C12N15/8247

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

• C12N15/8218

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

• C12N15/8203

  Virus mediated transformation · CPC title

• C12N15/8213

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