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
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Abstract
DNA gene inactivation constructs for homologous recombination in the genome of a mammalian cell are provided.
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Citations
78 Claims
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1-23. -23 (Canceled)
- 24. A DNA gene inactivation construct for homologous recombination in the genome of a mammalian cell, comprising at least 100 bp of a sequence homologous with a gene locus of a subunit of an MHC antigen flanking a sequence encoding a selectable marker gene capable of expression in a mammalian cell, wherein the sequence encoding a selectable marker gene is downstream from a sequence encoding a leader sequence and is fused in frame to a transmembrane coding region of the subunit of the MHC antigen, wherein upon homologous recombination, said gene locus is inactivated, and wherein, as a result of homologous recombination, at least one functional MHC antigen is not expressed.
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29. A DNA construct, comprising DNA encoding in the 5′
- to 3′
direction,a region of homology to a target gene, a foreign promoter/enhancer joined to a first epitope that binds to a ligand for detection, a selectable marker gene, and a second region of homology to said target gene, said target gene encoding a gene product having a second epitope, and said target gene being selected from the group consisting of a gene encoding a subunit of an MHC antigen and a gene encoding a protein that upregulates expression of MHC antigens, wherein, upon homologous recombination of said DNA construct into a genome, a recombinant, secreted fusion protein comprising said first epitope that binds to a ligand for detection and said second epitope is expressed in targeted cells, and wherein, as a result of homologous recombination, at least one functional MHC antigen or protein associated with expression of MHC antigens is not expressed. - View Dependent Claims (30, 33, 34)
- to 3′
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31. A DNA construct comprising DNA encoding a transcriptionally and translationally impaired positive selectable marker gene fused in frame to the transmembrane coding region of an integral membrane protein receptor for a cytokine that upregulates the expression of MHC antigen;
wherein the expression product of said DNA is a fusion protein comprising a functional selectable marker expressed on the cytoplasmic side of said membrane. - View Dependent Claims (32, 35)
- 36. A DNA construct comprising DNA encoding a transcriptionally and tanslationally impaired positive selectable marker gene fused downstream and in frame to the transmembrane coding region of an integral membrane protein that upregulates MHC antigen expression, wherein the expression product of said DNA construct is a fusion protein comprising a functional, selectable marker.
- 38. A DNA gene inactivation construct for homologous recombination in the genome of a mammalian cell, comprising a first sequence homologous with a gene locus present in the genome of the mammalian cell, having a length of at least 150 base pairs, and flanking a sequence encoding a selectable marker gene capable of expression in the mammalian cell, wherein the sequence encoding the selectable marker gene is downstream from a sequence encoding a leader sequence and is fused in frame to a coding sequence encoding an expression product, wherein the leader sequence comprises a second sequence homologous with the gene locus present in the genome of the mammalian cell and having a length of at least 150 base pairs, wherein upon homologous recombination said gene locus is inactivated, and wherein, as a result of homologous recombination, at least one functional expression product encoded by said gene locus is not expressed.
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45. A DNA gene inactivation construct for homologous recombination in the genome of 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, wherein the DNA gene inactivation construct comprises:
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(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, wherein upon insertion of said heterologous DNA sequence 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, said second coding sequence is operably linked to a regulatory sequence allowing the expression of said second product in said mammalian cell. - View Dependent Claims (46, 47, 48, 49, 50, 51)
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52. A DNA construct, comprising DNA encoding in the 5′
- to 3′
direction,a region of homology to a target gene, a foreign promoter/enhancer joined to a first coding sequence that encodes a first gene product, a selectable marker gene, and a second region of homology to said target gene, said target gene comprising a second coding sequence encoding a second gene product, wherein, upon homologous recombination of said DNA construct into a genome, a recombinant, fusion protein comprising said first gene product and part or all of said second gene product is expressed in targeted cells, and wherein, as a result of homologous recombination, at least one functional copy of the target gene is not expressed. - View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)
- to 3′
- 68. A DNA construct comprising a first DNA sequence and a second DNA sequence, wherein said first DNA sequence comprises a first coding sequence that encodes a first gene product that does not confer resistance to a selection agent involved in the selection of transformants, and said second DNA sequence comprises a second coding sequence that encodes a second gene product that confers resistance to a selection agent involved in the selection of transformants, wherein the second DNA sequence is downstream of the first DNA sequence, wherein the expression product of said DNA construct comprises the second product that confers resistance to a selection agent involved in the selection of transformants, in functional form.
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78. A DNA gene inactivation construct for homologous recombination in the genome of a mammalian cell, comprising a first sequence homologous with a gene locus present in the genome of the mammalian cell, having a length of at least 150 base pairs, and flanking a sequence encoding a selectable marker gene capable of expression in the mammalian cell, wherein the sequence encoding the selectable marker gene is downstream from a sequence encoding a leader sequence and is fused in frame to a coding sequence encoding an expression product, wherein the expression product of said DNA construct localizes to the cytoplasm when expressed in a mammalian cell, wherein the leader sequence comprises a second sequence homologous with the gene locus present in the genome of the mammalian cell and having a length of at least 150 base pairs, wherein upon homologous recombination said gene locus is inactivated, and wherein, as a result of homologous recombination, at least one functional expression product encoded by said gene locus is not expressed.
Specification