Human VpreB & Chimaeric Surrogate Light Chains in Transgenic Non-Human Vertebrates

A non-human vertebrate or cell, wherein said vertebrate is optionally a rat or a mouse, and wherein said vertebrate cell is optionally a rat cell or a mouse cell, whose genome comprises an antibody heavy chain transgene for producing heavy chains that are devoid of a CHI, transgene comprising

• (a) one or more human VH gene segments, one or more human D gene segments and one or more human JH gene segments operably connected upstream of a constant region gene so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and a constant region;
• or
• (b) a rearranged VDJ encoding a human variable region operably connected upstream of a constant region gene so that the transgene encodes (optionally following VDJ combination with the constant region) an antibody heavy chain having a human variable region and a constant region;
• the genome further comprising a human VpreB gene capable of expressing a human VpreB;
• wherein the constant region is devoid of a functional CHI gene;
• and wherein the vertebrate or cell is capable of expressing a human VpreB for pairing with the heavy chains devoid of CHI.

A method of constructing a transgenic non-human vertebrate cell, wherein when said cell is an ES cell, said ES cell is a mouse or rat ES cell, the method comprising

• (i) introducing into the genome of a non-human vertebrate cell, or an ancestor thereof, one or more human VH gene segments, one or more human D gene segments and one or more human JH gene segments so that said gene segments are operably connected upstream of a constant region gene, wherein said constant region gene is optionally an endogenous non-human vertebrate constant region gene, to form a heavy chain transgene, wherein in said cell or a progeny thereof the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and a constant region;
• or
• (ii) introducing into the genome of a non-human vertebrate cell (or an ancestor thereof) a rearranged VDJ encoding a human variable region so that said VDJ is operably connected upstream of a constant region gene (optionally an endogenous non-human vertebrate constant region gene) to form a heavy chain transgene, wherein in said cell or a progeny thereof the transgene encodes (optionally following VDJ combination with the constant region) an antibody heavy chain having a human variable region and a constant region;
• and wherein the method further comprises introducing into the cell a human VpreB gene in the absence of a λ5 sequence, wherein the cell (or a progeny cell or vertebrate) is capable of expressing human VpreB for pairing with the human variable region of the heavy chain.

A method of promoting B-cell development in a non-human vertebrate, wherein said vertebrate is optionally a mouse or rat species, the method comprising

• (a) providing in a non-human vertebrate embryonic stem (ES) cell genome an immunoglobulin transgene capable of expressing an antibody mu heavy chain, wherein the antibody heavy chain comprises a human variable region and a mu constant region (optionally an endogenous non-human vertebrate mu constant region);
• and creating a first non-human vertebrate from said ES cell or a progeny thereof;
• (b) providing in a second ES cell genome a second transgene comprising a human VpreB gene capable of expressing a human VpreB;
• and creating a second non-human vertebrate from said ES cell or a progeny thereof;
• and
• (c) creating by breeding a third non-human vertebrate capable of co-expressing the mu antibody heavy chain and human VpreB wherein a pre-B-cell receptor can form to promote B-cell development of cells bearing a mu heavy chain in said third vertebrate;
• the third vertebrate being made by crossing said first and second vertebrates or progeny thereof by breeding to create said third vertebrate, the third vertebrate comprising the first and second transgenes,
• and wherein endogenous heavy chain expression has been inactivated in said third vertebrate.

A method of promoting B-cell development in a non-human vertebrate, wherein said vertebrate is optionally a mouse or rat), the method comprising

• (i) inactivating endogenous heavy chain expression in said vertebrate;
• (ii) providing in the genome of said vertebrate an immunoglobulin transgene capable of expressing an antibody mu heavy chain, wherein the antibody heavy chain comprises a human variable region and a non-human vertebrate mu constant region, wherein said non-human vertebrate mu constant region is optionally an endogenous non-human vertebrate mu constant region;
• and
• (ii) providing in the genome of said vertebrate a second transgene capable of expressing a human VpreB wherein a pre-B-cell receptor can form to promote B-cell development of cells bearing a mu heavy chain in said vertebrate;
• optionally wherein the genome is homozygous for said first transgene.

A transgenic mouse or rat, or a transgenic mouse or rat cell, wherein said cell is optionally an ES cell, whose genome comprises

• (a) an antibody heavy chain transgene, the transgene comprising a substantially complete human functional VH, D and J H repertoire operably connected upstream of an endogenous (mouse or rat) mu constant region gene so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and an endogenous mu constant region;
• (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene;
• wherein all of the V, D and J in said transgenes are human V, D and J;
• wherein the kappa chain transgene comprises a substantially complete human functional VK and JK repertoire;
• and the lambda chain transgene comprises a substantially complete human functional νλ and Jλ repertoire;
• wherein the kappa and/or lambda chain transgenes comprise endogenous constant regions capable of pairing with the constant region of the heavy chain;
• (c) a human VpreB gene capable of expressing a human VpreB;
• optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter or its associated human promoter;
• and
• (d) an endogenous λ5 gene capable of expressing an endogenous λ5;
• wherein endogenous antibody heavy and light chain expression has been inactivated;
• and
• optionally wherein said genome is homozygous for said heavy and light chain transgenes.

A transgenic mouse or rat, or a transgenic mouse or rat cell, wherein said cell is optionally an ES cell, whose genome comprises

• (a) an antibody heavy chain transgene, the transgene comprising a substantially complete human functional VH, D and JH repertoire operably connected upstream of an endogenous (mouse or rat) mu constant region gene so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding an antibody heavy chain having a human variable region and an endogenous mu constant region;
• wherein all of the V, D and J in said transgene are human V, D and J;
• wherein the heavy chain transgene is devoid of a CH1 gene segment, optionally a gamma CH1, and the genome of the vertebrate comprises no functional antibody light chain locus;
• wherein endogenous antibody heavy chain expression has been inactivated;
• and
• optionally wherein said genome is homozygous for said heavy chain transgene;
• (b) a human VpreB gene capable of expressing a human VpreB;
• optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter or its associated human promoter;
• and
• (c) optionally an endogenous λ5 gene capable of expressing an endogenous λ5.