Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
First Claim
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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Accused Products
Abstract
Positive-negative selector (PNS) vectors are provided for modifying a target DNA sequence contained in the genome of a target cell capable of homologous recombination. The vector comprises a first DNA sequence which contains at least one sequence portion which is substantially homologous to a portion of a first region of a target DNA sequence. The vector also includes a second DNA sequence containing at least one sequence portion which is substantially homologous to another portion of a second region of a target DNA sequence. A third DNA sequence is positioned between the first and second DNA sequences and encodes a positive selection marker which when expressed is functional in the target cell in which the vector is used. A fourth DNA sequence encoding a negative selection marker, also functional in the target cell, is positioned 5′ to the first or 3′ to the second DNA sequence and is substantially incapable of homologous recombination with the target DNA sequence. The invention also includes transformed cells containing at least one predetermined modification of a target DNA sequence contained in the genome of the cell. In addition, the invention includes organisms such as non-human transgenic animals and plants which contain cells having predetermined modifications of a target DNA sequence in the genome of the organism.
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Citations
7 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A population of cells comprising:
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(a) first cells having a genome comprising a target sequence modified by insertion of a positive selection marker DNA sequence, wherein said target sequence is the same in each of said first cells and said insertion is effected by homologous recombination of the target sequence with a PNS vector; and
(b) second cells having genomes modified by insertion of a PNS vector fragment comprising a positive selection marker DNA sequences and a negative selection marker DNA sequence, wherein said PNS vector fragment is randomly distributed in the genomes of said second cells;
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 a 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;
(4) a negative selection marker DNA sequence, capable of conferring a 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 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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Specification