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Gene targeting methods and vectors

  • US 20040214222A1
  • Filed: 05/19/2004
  • Published: 10/28/2004
  • Est. Priority Date: 06/26/2001
  • Status: Abandoned Application
First Claim
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1. A method for identifying a transformed cell which has undergone site-specific homologous recombination utilizing a PPS vector comprising:

  • a) transforming cells with a PPS vector designed to undergo site-specific homologous recombination wherein the vector comprises;

    a first DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome;

    a second DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to cellular endogenous genomic sequences and therefore incapable of undergoing site-specific homologous recombination;

    a third DNA sequence which is substantially homologous to an endogenous genomic sequence present within the host genome and is different from the first DNA sequence; and

    a fourth DNA sequence which encodes a positive selection characteristic in said cells and is non-homologous to a cellular endogenous genomic sequence and therefore incapable of undergoing site-specific homologous recombination;

    b) propagating cells to select for or enrich for those which have been transformed with said PPS vector by selecting for the presence of the positive selectable marker gene product of said second DNA sequence; and

    c) separating cells which have said second DNA sequence encoding a positive selectable marker from cells which have said fourth DNA sequence encoding a positive selectable marker.

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