Production of antibodies using Cre-mediated site-specific recombination
First Claim
1. A method to produce a cell expressing an antibody from a genomic sequence of said cell comprising a modified immunoglobulin locus, which method uses Cre-mediated site-specific recombination to modify said locus and comprises:
- (a) transfecting an antibody-producing cell with a first homology-targeting vector comprising;
(i) a first lox site, and (ii) a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin locus of said genomic sequence which is to be converted to a modified region, so that said first lox site is inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo;
(b) transfecting said cell with a lox-targeting vector comprising;
(i) a second lox site suitable for Cre-mediated recombination with said first lox site, and (ii) a modifying sequence to convert said region of the immunoglobulin locus to said modified region;
(c) interacting the lox sites with Cre, so that said modifying sequence inserts into said genomic sequence via Cre-mediated site-specific recombination of said lox sites, thereby converting said region of the immunoglobulin locus to said modified region; and
(d) selecting a transfectant in which said region of the immunoglobulin locus is converted to said modified region and which transfectant produces said antibody molecule.
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Abstract
A method to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed. The method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence which is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination. Then the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region. This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites. Also disclosed are a form of the method used to produce a cell expressing a modified antibody molecule using Cre-mediated site-specific recombination, and antibody-producing cells obtainable by the disclosed methods. Class-switching modifications of human antibodies produced in murine hybridoma cells are exemplified.
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Citations
27 Claims
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1. A method to produce a cell expressing an antibody from a genomic sequence of said cell comprising a modified immunoglobulin locus, which method uses Cre-mediated site-specific recombination to modify said locus and comprises:
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(a) transfecting an antibody-producing cell with a first homology-targeting vector comprising;
(i) a first lox site, and (ii) a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin locus of said genomic sequence which is to be converted to a modified region, so that said first lox site is inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo;
(b) transfecting said cell with a lox-targeting vector comprising;
(i) a second lox site suitable for Cre-mediated recombination with said first lox site, and (ii) a modifying sequence to convert said region of the immunoglobulin locus to said modified region;
(c) interacting the lox sites with Cre, so that said modifying sequence inserts into said genomic sequence via Cre-mediated site-specific recombination of said lox sites, thereby converting said region of the immunoglobulin locus to said modified region; and
(d) selecting a transfectant in which said region of the immunoglobulin locus is converted to said modified region and which transfectant produces said antibody molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20)
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19. A method to produce a cell expressing an antibody molecule from a genomic sequence of said cell with a lox site adjacent to or integrated within a region of an immunoglobulin locus encoding said antibody molecule, for use in modifying said region using Cre-mediated site-specific recombination, which method comprises:
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(a) transfecting an antibody-producing cell with a first homology-targeting vector comprising;
(i) a first lox site, and (ii) a targeting sequence homologous to a first DNA sequence adjacent to region of the immunoglobulin locus of said genomic sequence which is to be converted to a modified region, so that said first lox site is inserted into said genomic sequence via site-specific homologous recombination with genomic DNA in vivo; and
(b) selecting a transfectant with said lox site inserted into said genomic DNA adjacent to said region which is to be converted.
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21. A method to modify a region of an immunoglobulin locus in a genomic sequence of a cell expressing an antibody molecule from said immunoglobulin locus wherein said genomic sequence comprises a first lox site adjacent to or integrated within a region of said immunoglobulin locus which is to be modified using Cre-mediated site-specific recombination, which method comprises:
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(a) transfecting said cell with a lox-targeting vector comprising;
(i) a second lox site suitable for Cre-mediated recombination with said first lox site, and (ii) a modifying sequence to convert said region of the immunoglobulin locus to said modified region;
(b) interacting the lox sites with Cre, so that said modifying sequence inserts into said genomic sequence via Cre-mediated site-specific recombination of said lox sites, thereby converting said region of the immunoglobulin locus to said modified region; and
(c) selecting a transfectant in which said region of the immunoglobulin locus is converted to said modified region and which produces said antibody molecule. - View Dependent Claims (22, 23, 25, 27)
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24. An embryonic stem cell of a non-primate mammal comprising a genome comprising:
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at least a functional portion of a human heavy chain immunoglobulin locus or at least a functional portion of a human light chain immunoglobulin locus, wherein a lox site is adjacent to or integrated within a region of said human heavy chain or light chain immunoglobulin locus.
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26. A transgenic non-primate mammal comprising a genome comprising:
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at least a functional portion of a human heavy chain immunoglobulin locus or at least a functional portion of a human light chain immunoglobulin locus, wherein a lox site is adjacent to or integrated within a region of said human heavy chain or light chain immunoglobulin locus.
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Specification