Methods and means for producing efficient silencing construct using recombinational cloning

• US 8,877,435 B2
• Filed: 09/21/2010
• Issued: 11/04/2014
• Est. Priority Date: 01/26/2001
• Status: Active Grant

• Current Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Original Assignee: Commonwealth Scientific & Industrial Research Organisation (Commonwealth Of Australia As Represented By Department Of Industry, Innovation, Climate Change, Science, Research And Tertiary Education)
• Inventors: Helliwell, Christopher A., Wesley, Susan V., Waterhouse, Peter M.
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US12/886,905
• Publication Number:
  US 20130017976A1
• Time in Patent Office: 1,505 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/320.1, 435/455, 435/462, 536/23.1, 536/24.3, 536/24.5
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8249 involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
• CPC Class Codes: C12N 15/825 involving pigment biosynthesis
• CPC Class Codes: C12N 15/827 Flower development or morphology, e.g. flowering promoting factor [FPF]

Abstract

Methods and means are provided for producing chimeric nucleic acid constructs capable of producing dsRNA for silencing target nucleic acid sequences of interest using recombinational cloning.

Claims

1. A method for isolating a nucleic acid molecule involved in determining a particular phenotype in a eukaryotic non-human organism or cell, the method comprising:

a) preparing a library of chimeric DNA constructs capable of expressing a dsRNA in cells of said eukaryotic non-human organism or cell by

i) combining in vitro;

1) a vector comprising the following operably linked DNA fragments;

a) an origin of replication allowing replication in a recipient cell, preferably in bacteria;

particularly in Escherichia coli;

b) a selectable marker region capable of being expressed in said recipient cell; and

c) a chimeric DNA construct comprising in sequence;

 

i) a promoter or promoter region capable of being recognized by RNA polymerases of a non-human eukaryotic cell;

 

ii) a first recombination site, a second recombination site, a third recombination site and a fourth recombination site;

 

iii) a 3′

transcription terminating and polyadenylation region functional in said non-human eukaryotic cell;

wherein said first recombination site and said fourth recombination site are capable of reacting with a same recombination site, preferably are identical, and said second recombination site and said third recombination site, are capable of reacting with a same recombination site, preferably are identical; and

wherein said first recombination site and said second recombination site do not recombine with each other or with a same recombination site or said third recombination site and said fourth recombination site do not recombine with each other or with a same recombination site;

2) an insert DNA comprising a DNA segment of interest flanked by a fifth recombination site which is capable of recombining with said first or fourth recombination site on said vector; and

a sixth recombination site which is capable of recombining with said second or third recombination site on said vector;

3) at least one site specific recombination protein capable of recombining said first or fourth and said fifth recombination site and said second or third and said sixth recombination site;

ii) allowing recombination to occur so as to produce a reaction mixture comprising product DNA molecules, said product DNA molecules comprising in sequence;

1) said promoter or promoter region capable of being recognized by RNA polymerases of said non-human eukaryotic cell;

2) a recombination site which is the recombination product of said first and said fifth recombination site;

3) said DNA fragment of interest;

4) a recombination site which is the recombination product of said second and said sixth recombination site;

5) a recombination site which is the recombination product of said third and said sixth recombination site;

6) said DNA fragment of interest in opposite orientation;

7) a recombination site which is the recombination product of said fourth and said fifth recombination site; and

8) said 3′

transcription terminating and polyadenylation region functional in said non-human eukaryotic cell;

iii) selecting said product DNA molecules;

b) introducing individual representatives of said library of chimeric DNA constructs in cells of said eukaryotic non-human organism; and

c) isolating a non-human eukaryotic organism or cell exhibiting said particular phenotype; and

d) isolating said nucleic acid molecule.

2. The method according to claim 1, wherein said non-human eukaryotic organism is a plant.

3. The method according to claim 1, wherein said first and second recombination sites flank a second selectable marker gene and said third and fourth recombination sites flank a third selectable marker gene.

4. The method according to claim 1, wherein said chimeric DNA construct comprises a region flanked by intron processing signals, functional in said non-human eukaryotic cell, located between said second recombination site and said third recombination site.

5. The method according to claim 4, wherein said region flanked by intron processing signals is an intron sequence functional in said non-human eukaryotic cell.

6. The method according to claim 4, further comprising a fourth selectable marker gene, located between said second and third recombination site.

7. The method according to claim 1, wherein said selectable marker genes are selected from the group consisting of an antibiotic resistance gene, a tRNA gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an antisense oligonucleotide;

a restriction endonuclease;

a restriction endonuclease cleavage site, an enzyme cleavage site, a protein binding site, and a sequence complementary PCR primer.

8. The method according to claim 1, wherein said promoter is a plant expressible promoter.

9. The method according to claim 1, wherein said chimeric DNA construct is flanked by left and right border T-DNA sequences.

10. The method according to claim 1, wherein said vector further comprises a selectable marker gene capable of being expressed in plant cells located between said left and said right T-DNA border sequences.

11. The method according to claim 1, wherein said vector further comprises an origin of replication capable of functioning in Agrobacterium sp.

12. The method according to claim 1, wherein said first second and third and fourth recombination sites are selected from the group consisting of attB, attP, attL, attR and loxP sites.

13. The method according to claim 1, wherein said first and fourth recombination site is attR1 comprising the nucleotide sequence of SEQ ID No 4 and said second and third recombination site is attR2 comprising the nucleotide sequence of SEQ ID No 5.

14. The method according to claim 1, wherein said first and fourth recombination site is attP1 comprising the nucleotide sequence of SEQ ID No 10 and said second and third recombination site is attP2 comprising the nucleotide sequence of SEQ ID No 11.

15. The method according to claim 1, wherein said first and fourth recombination site is attR1 comprising the nucleotide sequence of SEQ ID No 4 and said second and third recombination site is attR3 comprising the nucleotide sequence of SEQ ID No 6.

16. The method according to claim 1, wherein said first and fourth recombination site is attR2 comprising the nucleotide sequence of SEQ ID No 5 and said second and third recombination site is attR3 comprising the nucleotide sequence of SEQ ID No 6.

17. The method according to claim 1, wherein said first and fourth recombination site is attL1 comprising the nucleotide sequence of SEQ ID No 7 and said second and third recombination site is attL2 comprising the nucleotide sequence of SEQ ID No 8.

18. The method according to claim 1, wherein said first and fourth recombination site is attL1 comprising the nucleotide sequence of SEQ ID No 7 and said second and third recombination site is attL3 comprising the nucleotide sequence of SEQ ID No 9.

19. The method according to claim 1, wherein said first and fourth recombination site is attL2 comprising the nucleotide sequence of SEQ ID No 8 and said second and third recombination site is attL3 comprising the nucleotide sequence of SEQ ID No 9.

20. The method according to claim 1, wherein said first and fourth recombination site is attB1 comprising the nucleotide sequence of SEQ ID No 1 and said second and third recombination site is attB2 comprising the nucleotide sequence of SEQ ID No 2.

21. The method according to claim 1, wherein said first and fourth recombination site is attB1 comprising the nucleotide sequence of SEQ ID No 1 and said second and third recombination site is attB3 comprising the nucleotide sequence of SEQ ID No 3.

22. The method according to claim 1, wherein said first and fourth recombination site is attB2 comprising the nucleotide sequence of SEQ ID No 1 and said second and third recombination site is attB3 comprising the nucleotide sequence of SEQ ID No 3.

23. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No 13.

24. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No 23.

25. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No 24.

26. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No 25.

27. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No 26.

28. The method according to claim 1, wherein said insert DNA is a linear DNA molecule.

29. The method according to claim 1, wherein said insert DNA is a circular DNA molecule.

30. The method according to claim 1, wherein said at least one recombination protein is selected from (i) Int (λ

integrase) and IHF (integration host factor) and (ii) Int, Xis (λ

excisionase), and IHF.

31. The method of claim 1, wherein said non-human eukaryotic organism is selected from a plant, a fungus, a nematode.

32. The method of claim 1, wherein said non-human eukaryotic organism is selected from an animal.