Methods and means for producing efficient silencing construct using recombinational cloning
First Claim
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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 byi) 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.
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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.
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Citations
32 Claims
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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:
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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. - 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, 32)
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Specification