Positive-negative selection methods and vectors

Claim 1

A positive-negative selection (PNS) vector for modifying a target DNA sequence contained in the genomes of murine embryonic stem cells, said PNS vector comprising: a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence, a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells, a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence, and a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, thereby allowing killing of 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.

Claim 20

A method for enriching for a transformed murine embryonic stem cell containing a modification in a target DNA sequence in the genome of said cell comprising: (a) transfecting cells capable of mediating homologous recombination with a positive-negative selection vector comprising: a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence, a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells, a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence, and a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, thereby allowing killing of 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 the target DNA sequence by homologous recombination of said first and second homologous vector sequences with the first and second regions of said target sequence; (b) selecting for transformed cells in which said positive-negative selection vector has integrated into said target DNA sequence by homologous recombination by sequentially or simultaneously selecting against transformed cells containing said negative selection marker and selecting for cells containing said positive selection marker; and (c) analyzing the DNA of transformed cells surviving the selecting step to identify a cell containing the modification.

Claim 21

A method for enriching for a transformed murine embryonic stem cell containing a modification in a target DNA sequence in the genome of said cell comprising: (a) transfecting cells capable of mediating homologous recombination with a positive-negative selection vector comprising: a first homologous vector DNA sequence capable of homologous recombination with a first region of said target DNA sequence, a positive selection marker DNA sequence capable of conferring a positive selection characteristic in said cells, a second homologous vector DNA sequence capable of homologous recombination with a second region of said target DNA sequence, and a negative selection marker DNA sequence, capable of conferring a negative selection characteristic in said cells, thereby allowing killing of 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 the target DNA sequence by homologous recombination of said first and second homologous vector sequences with the first and second regions of said target sequence; (b) selecting for transformed cells in which said positive-negative selection vector has integrated into said target DNA sequence by homologous recombination by sequentially or simultaneously selecting against transformed cells containing said negative selection marker and selecting for cells containing said positive selection marker; and (c) analyzing the DNA of transformed cells surviving the selecting step to identify a cell containing the modification.