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Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same

  • US 6,204,061 B1
  • Filed: 01/09/1997
  • Issued: 03/20/2001
  • Est. Priority Date: 08/22/1989
  • Status: Expired due to Fees
First Claim
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1. A method of culturing a cell, comprising culturing a cell having a genome comprising a modification of a target DNA sequence, wherein the modification was introduced into the genome of the cell or an ancestor thereof by(a) transforming a population of cells with a PNS vector;

  • (b) identifying a cell having said genome comprising the modification of the target DNA sequence by selecting for cells containing a positive selection characteristic and against cells containing a negative selection characteristic;

    (c) propagating the cell, wherein said PNS vector comprises;

    (1) a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence;

    (2) a positive selection marker DNA sequence capable of conferring the positive selection characteristic in said cells;

    (3) a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence; and

    (4) a negative selection marker DNA sequence, capable of conferring the negative selection characteristic in said cells, but substantially incapable of homologous recombination with said target DNA sequence;

    wherein the spatial order of said sequences in said PNS vector is;

    said first homologous vector DNA sequence, said positive selection marker DNA sequence, said second homologous vector DNA sequence and said negative selection marker DNA sequence as shown in FIG. 1;

    wherein the 5′

    -3′

    orientation of said first homologous vector sequence relative to said second homologous vector sequence is the same as the 5′

    -3′

    orientation of said first region relative to said second region of said target sequence;

    wherein the vector is capable of modifying said target DNA sequence by homologous recombination of said first homologous vector DNA sequence with said first region of said target sequence and of said second homologous vector DNA sequence with said second region of said target sequence.

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