Methods and means for producing efficient silencing construct using recombinational cloning
First Claim
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1. A vector comprising the following operably linked DNA fragments:
- an origin of replication (1) allowing replication in a recipient cell;
a selectable marker region (2) capable of being expressed in said recipient cell; and
a chimeric DNA construct comprising in sequence;
a promoter or promoter region (3) capable of being recognized by a prokaryotic RNA polymerase;
a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7);
a 3′
transcription terminating region (8) functional in said recipient cell;
whereinsaid first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site, and said second recombination site (5 ) and said third recombination site (6), are capable of reacting with a same recombination site;
and whereinsaid first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site;
orsaid third recombination site (6 ) and said fourth recombination site (7) do not recombine with each other or with a same recombination site.
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Abstract
Methods and vectors and kits are provided for producing chimeric nucleic acid constructs capable of producing dsRNA for silencing target nucleic acid sequences of interest using recombinational cloning.
24 Citations
31 Claims
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1. A vector comprising the following operably linked DNA fragments:
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an origin of replication (1) allowing replication in a recipient cell; a selectable marker region (2) capable of being expressed in said recipient cell; and a chimeric DNA construct comprising in sequence; a promoter or promoter region (3) capable of being recognized by a prokaryotic RNA polymerase; a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7); a 3′
transcription terminating region (8) functional in said recipient cell;wherein said first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site, and said second recombination site (5 ) and said third recombination site (6), are capable of reacting with a same recombination site; and wherein said first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site;
orsaid third recombination site (6 ) and said fourth recombination site (7) do not recombine with each other or with a same recombination site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
allowing recombination to occur so as to produce a reaction mixture comprising product DNA molecules, said product DNA molecules comprising in sequence; said promoter or promoter region (3) capable of being recognized by RNA polymerases of said prokaryotic cell; a recombination site (15) which is the recombination product of said first (4) and said fifth (13) recombination site; said DNA fragment of interest (12); a recombination site (16) which is the recombination product of said second (4) and said sixth (14) recombination site; a recombination site (17) which is the recombination product of said third (5) and said sixth (14) recombination site; said DNA fragment of interest (12) in opposite orientation; a recombination site (18) which is the recombination product of said fourth (7) and said fifth (13) recombination site; and said 3′
transcription terminating region (8) functional in said recipient cell; andselecting said product DNA molecules.
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11. The method according to claim 10, wherein said insert DNA is a linear DNA molecule.
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12. The method according to claim 10, wherein said insert DNA is a circular DNA molecule.
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13. The method according to claim 10, 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.
- integrase) and IHF (integration host factor) and (ii) Int, Xis (λ
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14. The method according to claim 10, wherein multiple insert DNAs comprising different DNA fragments of interest are processed simultaneously.
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15. A vector according to claim 1, wherein the origin of replication allows replication in bacteria.
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16. A vector according to claim 15, wherein the bacteria is Escherichia coli.
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17. A vector according to claim 1, wherein said first recombination site (4) and said fourth recombination site (7) are identical.
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18. A vector according to claim 1, wherein said second recombination site (5) and said third recombination site (6) are identical.
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19. A prokaryotic host comprising the vector according to claim 1.
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20. Feed for an animal comprising the prokaryotic host of claim 19.
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21. The feed for an animal of claim 20, wherein said animal is a nematode.
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22. The vector of claim 1, wherein said first (4) second (5) and third (6) and fourth (7) recombination site are selected from the group consisting of attB, attP, attL, attR and loxP sites.
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23. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attR1 comprising the nucleotide sequence of SEQ ID No 4 and said second (5) and third (6) recombination site is attR3 comprising the nucleotide sequence of SEQ ID No 6.
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24. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attR2 comprising the nucleotide sequence of SEQ ID No 5 and said second (5) and third (6) recombination site is attR3 comprising the nucleotide sequence of SEQ ID No 6.
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25. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attL1 comprising the nucleotide sequence of SEQ ID No 7 and said second (5) and third (6) recombination site is attL2 comprising the nucleotide sequence of SEQ ID No 8.
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26. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attL1 comprising the nucleotide sequence of SEQ ID No 7 and said second (5) and third (6) recombination site is attL3 comprising the nucleotide sequence of SEQ ID No 9.
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27. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attL2 comprising the nucleotide sequence of SEQ ID No 8 and said second (5) and third (6) recombination site is attL3 comprising the nucleotide sequence of SEQ ID No 9.
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28. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attB1 comprising the nucleotide sequence of SEQ ID No 1 and said second (5) and third (6) recombination site is attB2 comprising the nucleotide sequence of SEQ ID No 2.
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29. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attB1 comprising the nucleotide sequence of SEQ ID No 1 and said second (5) and third (6) recombination site is attB3 comprising the nucleotide sequence of SEQ ID No 3.
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30. The vector of claim 1, wherein said first (4) and fourth (7) recombination site is attB2 comprising the nucleotide sequence of SEQ ID No 1 and said second (5) and third (6) recombination site is attB3 comprising the nucleotide sequence of SEQ ID No 3.
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31. The method of claim 10, wherein said first (4), second (5), third (6), fourth (7), fifth (13) and sixth (14) recombination site are selected from the group consisting of attB, attP, attL, attR and loxP sites.
Specification