Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made
First Claim
1. An in vitro process for providing a recombinant DNA sequence, wherein the process comprises:
- (A) providing a mammalian cell having a recipient DNA sequence, wherein said recipient DNA sequence comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;
(B) transfecting said mammalian cell in vitro with a transfection DNA comprising;
(1) a third nucleotide sequence homologous to said first nucleotide sequence;
(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and
(3) a DNA sequence heterologous with respect to said recipient DNA sequence, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a first coding sequence that encodes a first product that does not confer resistance to a selection agent involved in the selection of transformants, and said second insertion DNA sequence comprises a second coding sequence that encodes a second product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said second product in said mammalian cell;
(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient DNA sequence by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing the recombinant DNA sequence, and in addition, the second insertion DNA sequence encoding the selective agent; and
(D) recovering said recombinant DNA sequence from said mammalian cell.
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Abstract
The present invention relates to a process for targeted replacement of at least a part of an endogenous gene by at least a part of a foreign gene or targeted insertion of at least a part of a foreign gene at a targeted site in an endogenous gene in a cell by homologous recombination. This process includes (A) providing a vector which contains (1) at least a part of the foreign gene which is heterologous with respect to the endogenous gene, (2) a first flanking DNA sequence homologous to a first genomic sequence situated on one side of the part of the endogenous gene to be replaced or the targeted site; and (3) a second flanking DNA sequence homologous to a second genomic sequence situated on the other side of the part of the endogenous gene to be replaced or the targeted site, the foreign gene being located between the first and second flanking DNA sequences and is complementary to the part of the endogenous gene to be replaced; (B) transfecting a cell with the vector; (C) and selecting a transfected cell that contains the foreign gene at the targeted site or where the part of the endogenous gene is to be replaced. This invention also pertains to the vector, and the cell transfected by the vector.
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Citations
44 Claims
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1. An in vitro process for providing a recombinant DNA sequence, wherein the process comprises:
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(A) providing a mammalian cell having a recipient DNA sequence, wherein said recipient DNA sequence comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;
(B) transfecting said mammalian cell in vitro with a transfection DNA comprising;
(1) a third nucleotide sequence homologous to said first nucleotide sequence;
(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and
(3) a DNA sequence heterologous with respect to said recipient DNA sequence, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a first coding sequence that encodes a first product that does not confer resistance to a selection agent involved in the selection of transformants, and said second insertion DNA sequence comprises a second coding sequence that encodes a second product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said second product in said mammalian cell;
(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient DNA sequence by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing the recombinant DNA sequence, and in addition, the second insertion DNA sequence encoding the selective agent; and
(D) recovering said recombinant DNA sequence from said mammalian cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. An in vitro process for providing a recombinant DNA sequence, wherein the process comprises:
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(A) providing a mammalian cell having a recipient DNA sequence, wherein said recipient DNA sequence comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;
(B) transfecting said mammalian cell in vitro with a transfection DNA comprising;
(1) a third nucleotide sequence homologous to said first nucleotide sequence;
(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and
(3) a DNA sequence heterologous with respect to said recipient DNA sequence, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a first coding sequence that encodes a first product that does not confer resistance to a selection agent involved in the selection of transformants, and a regulatory sequence for regulating the expression of said first product, and said second insertion DNA sequence comprises a second coding sequence that encodes a second product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said second product in said mammalian cell;
(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient DNA sequence by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing the recombinant DNA sequence, and in addition, the second insertion DNA sequence encoding the selective agent; and
(D) recovering said recombinant DNA sequence from said mammalian cell. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. An in vitro process for providing a recombinant DNA sequence, wherein the process comprises:
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(A) providing a mammalian cell having a recipient DNA sequence, wherein said recipient DNA sequence comprises complementing DNA comprising a first nucleotide sequence and a second nucleotide sequence downstream of said first nucleotide sequence;
(B) transfecting said mammalian cell in vitro with a transfection DNA comprising;
(1) a third nucleotide sequence homologous to said first nucleotide sequence;
(2) a fourth nucleotide sequence homologous to said second nucleotide sequence; and
(3) a DNA sequence heterologous with respect to said recipient DNA sequence, wherein said heterologous DNA sequence is between said third and said fourth nucleotide sequences and said heterologous DNA sequence comprises a first insertion DNA sequence and a second insertion DNA sequence, wherein said first insertion DNA sequence comprises a regulatory sequence, and said second insertion DNA sequence comprises a coding sequence that encodes a product that confers resistance to a selection agent involved in the selection of transformants, and a promoter allowing the expression of said product in said mammalian cell;
(C) selecting a transfected cell in which said heterologous DNA sequence has been inserted between said first and said second nucleotide sequences in said recipient DNA sequence by homologous recombination with said third and said fourth nucleotide sequences to thereby provide a mammalian cell containing the recombinant DNA sequence, and in addition, the second insertion DNA sequence encoding the selective agent; and
(D) recovering said recombinant DNA sequence from said mammalian cell. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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Specification