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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
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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