Cells and non-human organisms containing predetermined genomic modifications and positive-negative selection methods and vectors for making same
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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
116 Claims
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1-90. -90. (canceled)
- 91. An isolated mouse embryonic stem cell having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.
- 98. An isolated mouse embryonic stem cell having a genome comprising a human gene in place of a corresponding mouse gene, wherein said human gene is inserted into said genome by a homologous recombination event between said genome and a targeting vector, without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.
- 101. A transgenic mouse having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genomic DNA is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.
- 108. A transgenic mouse having a genome comprising a human gene replacing a corresponding mouse gene, wherein said human gene is inserted into said genome by a homologous recombination event between said genome and a targeting vector, without a duplication of genomic DNA sequence by said even and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event
- 110. An isolated cell having a genome comprising a positive selection marker DNA sequence inserted into a non-selectable gene by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.
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116. An isolated mouse embryonic stem cell having a genome comprising a positive selection marker DNA sequence inserted into a gene other than hprt by a homologous recombination event between said genome and a targeting vector, thereby inactivating said gene, wherein said gene is inactivated without a duplication of genomic DNA sequence by said event and wherein said genome is free of an additional copy of the positive selection marker DNA sequence integrated by a random recombination event.
Specification