Targeted replacement of an immunoglobulin gene without endogenous and selectable residual sequences in mice
First Claim
1. A method of producing a transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment comprising, selecting a transgenic mouse comprising a targeted, functional replacement of the mouse immunoglobulin constant region gene segment with said human immunoglobulin constant region gene segment, wherein said mouse is the progeny of a surrogate mother that contained a mouse blastocyst comprising a mouse embryonic stem cell, wherein said mouse embryonic stem cell was transfected with a recombination vector comprising a human immunoglobulin constant region gene segment, DNA sequences homologous to the mouse immunoglobulin constant region gene segment, a selectable marker gene and recombinase recognition sites flanking said selectable marker gene, wherein said selectable marker gene is removed by a non-mammalian recombinase.
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Abstract
Transgenic mice that produce high levels of humanized antibodies are described. Targeted gene replacement exchanges constant regions of the mouse immunoglobulin heavy and light chain genes with human genes, either through conventional gene targeting, or by use of the bacteriophage-derived Cre-loxP recombination system. The transgenic animals undergo antibody affinity maturation, and a class switch from the native immunoglobulin to the humanized form.
67 Citations
31 Claims
- 1. A method of producing a transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment comprising, selecting a transgenic mouse comprising a targeted, functional replacement of the mouse immunoglobulin constant region gene segment with said human immunoglobulin constant region gene segment, wherein said mouse is the progeny of a surrogate mother that contained a mouse blastocyst comprising a mouse embryonic stem cell, wherein said mouse embryonic stem cell was transfected with a recombination vector comprising a human immunoglobulin constant region gene segment, DNA sequences homologous to the mouse immunoglobulin constant region gene segment, a selectable marker gene and recombinase recognition sites flanking said selectable marker gene, wherein said selectable marker gene is removed by a non-mammalian recombinase.
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10. A method of producing a transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment comprising the steps of:
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i) transferring a mouse blastocyst comprising a mouse embryonic stem cell into a surrogate mother, wherein said embryonic stem cell is transfected with a recombination vector comprising a human immunoglobulin constant region gene segment, DNA sequences homologous to the mouse immunoglobulin constant region gene segment, a selectable marker gene and recombinase recognition sites flanking said selectable marker gene, wherein said selectable marker gene is removed by a non-mammalian recombinase;
ii) selecting surrogate mother progeny comprising a targeted, functional replacement of the mouse immunoglobulin constant region gene segment with said human immunoglobulin constant region gene segment. - View Dependent Claims (11, 12)
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- 13. A transgenic mouse, or progeny thereof, having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein said transgenic mouse produces antibodies comprising a human constant region joined to a mouse variable region in response to an antigen.
- 22. A method for producing humanized antibodies in response to an antigen, comprising the step of selecting antibodies comprising a human constant region gene segment from a transgenic mouse immunized with said antigen, said mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein said transgenic mouse produces antibodies comprising a human constant region joined to a mouse variable region in response to an antigen.
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30. A method for producing humanized antibodies in response to an antigen, comprising the steps of:
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i) immunizing a transgenic mouse with said antigen, said mouse containing a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein said transgenic mouse produces antibodies comprising a human constant region joined to a mouse variable region in response to said antigen; and
ii) selecting antibodies comprising a human constant region gene segment.
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31. A method of producing a transgenic mouse comprising a human immunoglobulin light chain gene segment and a human immunoglobulin heavy chain gene segment comprising mating a first transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein in said first transgenic mouse, said human immunoglobulin constant region gene segment is from the Cγ
- 1 gene, with a second transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein in said second transgenic mouse said human immunoglobulin constant region gene segment is from the Ck gene and selecting progeny that comprises a gene segment from the Cγ
1 gene and a gene segment from the Ck gene.
- 1 gene, with a second transgenic mouse having a genome comprising a targeted, functional replacement of a mouse immunoglobulin constant region gene segment with a human immunoglobulin constant region gene segment, wherein in said second transgenic mouse said human immunoglobulin constant region gene segment is from the Ck gene and selecting progeny that comprises a gene segment from the Cγ
Specification