Chimaeric Surrogate Light Chains (SLC) Comprising Human VpreB

Abstract

The present invention relates inter alia to improvements in the production of chimaeric antibodies in non-human transgenic vertebrates such as mice and rats bearing one or more chimaeric antibody transgenes. In particular, the invention provides for improved non-human vertebrates and cells in which VpreB has been species-matched with the variable region of the chimaeric antibodies. Also, embodiments also provide for species-matching of the entire surrogate light chain for efficient pairing with chimaeric heavy chains during B-cell development in vivo in a non-human transgenic vertebrate setting.

Description

[0001] This application is a continuation of U.S. application Ser. No. 14/226,698, filed Mar. 26, 2014, which is a continuation of PCT/GB2012/052380, filed Sep. 26, 2012, which claims the benefit of GB 1116495.1, filed Sep. 26, 2011, and GB 1120423.7, filed Nov. 28, 2011, the contents of each which are incorporated by reference herein in their entirety. The attached sequence listing is hereby incorporated by reference.

[0002] The present invention relates inter alia to improvements in the production of chimaeric antibodies in non-human transgenic vertebrates such as mice and rats bearing one or more chimaeric antibody transgenes. In particular, the invention provides for improved non-human vertebrates and cells in which VpreB has been species-matched with the variable region of the chimaeric antibodies. Also, embodiments provide for species-matching of the entire surrogate light chain for efficient pairing with chimaeric heavy chains during B-cell development in vivo in a non-human transgenic vertebrate setting.

[0003] Furthermore, the invention relates to a method of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

BACKGROUND

[0004] Advancements in the construction of transgenic mice bearing human antibody gene loci have led researchers to move away from the provision of entirely human transgenic antibody loci (bearing human variable and constant region gene segments) to chimaeric transgenic antibody loci. The transgenic loci comprise human V(D)J segments operably connected upstream of non-human constant regions. The use of constant regions that are endogenous to the transgenic non-human vertebrate (eg, endogenous mouse or rat constant regions) are desirable to harness the endogenous control of antibody generation and maturation following immunisation of the vertebrate with antigen.

[0005] B-cell development is characterized by the ordered rearrangement of immunoglobulin variable region genes. After the VDJ rearrangement of the H chain gene segments, a precursor (pre)-B-cell is generated. After the functional rearrangement of a light chain gene, the cells develop into surface IgM-bearing mature B-cells. Fully assembled IgH and L chains are transported onto the surface of B-cells, while free H chains are retained in the endoplasmic reticulum (ER) in association with BiP.

[0006] A critical step in B-cell differentiation is the selective expansion of cells with a functional .mu. heavy chain resulting from productive IgH rearrangement. This is achieved by the association of the .mu. heavy chain with the surrogate light chain (SLC) proteins .lamda.5 and VpreB and the signal transducing heterodimer Ig.alpha. and .beta. to form the pre-B-cell receptor (pre-BCR). The expression and formation of a pre-BCR dramatically improves the efficiency of pre-B and B-cell production, by signalling proliferative expansion of pre-B-cells. The major function of the pre-BCR is the selection and expansion of cells that have undergone a productive VDJ rearrangement. The expression of membrane-bound .mu. chains is essential for the clonal expansion, and initiation of L chain gene rearrangement.

[0007] The association of SLC with .mu. chain works as a checkpoint to determine whether the cell has successfully completed VDJ combination and expresses a functional .mu.H chain. More than half of all .mu. chains cannot assemble with the SLC, accounting for most of the changes in the V heavy repertoire during B-cell development.

[0008] VpreB is homologous to variable regions, .lamda.5 is homologous to light chain constant region. The protein encoded by the two genes can form a tightly, but noncovalently bound heterodimer with the general structure of an L chain. .lamda.5 protein can be covalently disulphide-bonded to .mu. H chains in pre-B-cells. Whereas VpreB alone can associate with Ig.mu., .lamda.5 alone cannot. Only when it is non-covalently associated with VpreB does .lamda.5 form a disulphide bridge with the first constant region (CH1) of an Ig.mu. chain to form the pre-BCR.

[0009] The .lamda.5 and VpreB, which together form the SLC, are early markers of B-cell commitment. They are expressed at the pro- and pre-B-cell stages and silenced in immature and mature B-cells. In pre-B-cells, following rearrangement of the heavy chain locus, the surrogate light chain (SLC) acts as a chaperone, mediating transport of the newly synthesised heavy chain mu to the cell surface and together with C mu forms part of the pre-BCR (Immunol Today. 1993 February; 14(2):60-8; The surrogate light chain in B-cell development; Melchers F, Karasuyama H, Haasner D, Bauer S, Kudo A, Sakaguchi N, Jameson B, Rolink A). The pre-BCR mediates signalling, leading to proliferation of pre-B-cells that have a productive heavy chain rearrangement. Mice that lack a functional .lamda.5 show a drastic reduction in the number of B-cells. Using VpreB knock-out mice, it has been shown that VpreB is also required for efficient B-cell development, particularly for the transition to pre-BCR bearing cells (pre-611 stage) (see, Int Immunol. 1999 March; 11(3):453-60; Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice; Martensson A, Argon Y, Melchers F, Dul J L, Martensson IL; and Sabbattini & Dillon 2005 infra). As the degree of proliferation has been proposed to be dependent upon the stability of the association of the mu heavy chain with the components of the SLC, the pre-BCR is thought to influence the choice, and thus the extent and diversity of, the heavy chain variable region repertoire.

[0010] Besides its role in signalling proliferation, the pre-BCR is also thought to mediate down regulation of the RAG genes, thereby preventing the rearrangement of other IgH loci (allelic exclusion) and the occurrence of double-strand breaks in dividing pre-B-cells. The pre-BCR has also been proposed to exert a negative feedback on .lamda.5 and VpreB expression so that, after a phase of clonal expansion, the large pre-BII cells become depleted of pre-BCR, exit the cell cycle and differentiate into resting small pre-BII cells. At this stage, the SLC is repressed while the IgL loci start to be rearranged. The product of a successfully rearranged IgL gene will then pair with the mu heavy chain to form a BCR with antigen-binding capability on the surface of immature B-cells. These cells migrate to the peripheral blood and secondary lymphoid organs, and develop into mature B-cells ready for subsequent encounter with antigen (Sabbattini & Dillon 2005 infra).

SUMMARY OF THE INVENTION

[0011] The inventors were aware of the desirability to use endogenous non-human (eg, mouse or rat) constant regions to harness the endogenous control of antibody generation and maturation following immunisation. They have realised, however, that these considerations do not address earlier B-cell stages in vivo (prior to antigen stimulation) when the animal's B-cell repertoire is maturing. Such maturation includes transition from immature pro-B-cells to pre-B-cells, including the pre-BII stage when B-cells bear surface B-cell receptors (BCR) in which the heavy chain variable region is paired with the VpreB. CH1 regions of heavy chains are paired with .lamda.5 in the pre-BCRs. The inventors realised that the provision of genes encoding chimaeric antibodies is not tailored to the pre-BII stage, and they realised that it is important to address this since this stage features huge clonal expansion required to provide a pool of B-cells for functional heavy chain selection during subsequent B-cell development.

[0012] To this end, the present invention provides for species-matching of human V regions in chimaeric heavy chains with human VpreB. In embodiments, the invention also provides for species-matching of the mu constant region with .lamda.5, so that heavy chains in pre-BCRs are completely species-matched with chimaeric surrogate light chain (SLC).

[0013] Thus, a first configuration of the present invention provides,

[0014] A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene,

[0015] the transgene comprising

[0016] (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

[0017] (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) a chimaeric antibody heavy chain having a human variable region and a constant region;

[0018] the genome further comprising a human VpreB gene capable of expressing a human VpreB.

[0019] The present invention is applicable to the production of 4-chain antibodies in transgenic animals, where the antibodies each contain 2 heavy chains and 2 light chains. Alternatively, the invention can be applied to the production of H2 antibodies (heavy chain antibodies) which are devoid of CH1 and light chains.

[0020] In a second embodiment, the invention provides

[0021] A method of constructing a transgenic non-human vertebrate cell (eg, an ES cell, eg, a mouse or rat ES cell), the method comprising

[0022] (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 (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

[0023] (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;

[0024] and wherein the method further comprises introducing into the cell a human VpreB gene capable of expressing a human VpreB.

[0025] In a third embodiment, the invention provides

[0026] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0027] (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;

[0028] (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

[0029] (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.

[0030] In a fourth embodiment, the invention provides

[0031] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0032] (i) inactivating endogenous heavy chain expression in said vertebrate;

[0033] (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 (optionally an endogenous non-human vertebrate mu constant region); and

[0034] (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;

[0035] Optionally wherein the genome is homozygous for said first transgene.

[0036] An aspect of the invention further provides for the possibility of species-matching in vivo the variable and constant regions of the antibody heavy chain with a chimaeric SLC. This is useful for promoting B-cell development in the transgenic non-human vertebrate (eg, a mouse or rat).

[0037] To this end, the invention provides a fifth embodiment:--

[0038] A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene,

[0039] the transgene comprising

[0040] (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 of a non-human vertebrate (eg, mouse or rat) species so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region; or

[0041] (b) a rearranged VDJ encoding a human variable region operably connected upstream of a constant region gene of a non-human vertebrate (eg, mouse or rat) species so that the transgene encodes (optionally following VDJ combination with the constant region) a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region;

[0042] the genome further comprising one or more genes together encoding a chimaeric surrogate light chain, the surrogate light chain comprising a human VpreB and .lamda.5 of said non-human vertebrate (eg, mouse or rat) species.

[0043] A sixth embodiment of the invention provides

[0044] A method of constructing a transgenic non-human vertebrate cell (eg, an ES cell, eg, a mouse or rat ES cell), the method comprising

[0045] (i) introducing into 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 an endogenous non-human vertebrate constant region gene, 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 a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region; or

[0046] (ii) introducing into 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 an endogenous non-human vertebrate constant region gene, wherein in said cell or a progeny thereof the transgene encodes (optionally following VDJ combination with the constant region) a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region;

[0047] and wherein the method further comprises introducing into the cell a human VpreB gene such that the cell or a progeny thereof is capable of expressing a chimaeric surrogate light chain as well as said chimaeric antibody heavy chain, wherein the surrogate light chain comprises a human VpreB and an endogenous non-human vertebrate .lamda.5.

[0048] A seventh embodiment of the invention further provides

[0049] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0050] (a) providing in a non-human vertebrate embryonic stem (ES) cell an immunoglobulin transgene capable of expressing a chimaeric antibody mu heavy chain, wherein the chimaeric antibody heavy chain comprises a human variable region and an endogenous non-human vertebrate mu constant region; and creating a first non-human vertebrate from said ES cell or a progeny thereof;

[0051] (b) providing in a second ES cell a second transgene capable of expressing a human VpreB so that the human VpreB and an endogenous .lamda.5 form a chimaeric surrogate light chain; and creating a second non-human vertebrate from said ES cell or a progeny thereof; and

[0052] (c) creating by breeding a third non-human vertebrate capable of co-expressing the chimaeric mu antibody heavy chain and chimaeric surrogate light chain wherein a pre-BCR can form to promote B-cell development 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.

[0053] A transgenic mouse according to the vertebrate of this method, or a progeny thereof, is provided.

[0054] An eighth embodiment of the invention provides

[0055] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0056] (i) inactivating endogenous heavy chain expression in said vertebrate;

[0057] (ii) providing in the genome of said vertebrate an immunoglobulin transgene capable of expressing a chimaeric antibody mu heavy chain, wherein the chimaeric antibody heavy chain comprises a human variable region and an endogenous non-human vertebrate mu constant region; and

[0058] (ii) providing in the genome of said vertebrate a second transgene capable of expressing a human VpreB so that the human VpreB and an endogenous .lamda.5 form a chimaeric surrogate light chain; so that the vertebrate is capable of co-expressing the chimaeric mu antibody heavy chain and chimaeric surrogate light chain wherein a pre-BCR can form to promote B-cell development in said vertebrate;

[0059] Optionally wherein the genome is homozygous for said first transgene.

[0060] A transgenic mouse according to the vertebrate of this method, or a progeny thereof, is provided.

BRIEF DESCRIPTION OF THE FIGURES

[0061] FIGS. 1A & B: Schematic representation of the steps involved in replacement of the mouse VpreB1 with human VpreB1. The most 5' gene is Topo3.beta..

[0062] FIG. 2: Upper Schematic: representation of vector for retrieval of the 19 kb mouse DNA sequence containing the human VpreB1 gene; Lower Schematic:representation of the vector containing the retrieved 19 kb mouse DNA sequence containing the human VpreB1 gene. Black rectangular boxes represent homology arms. Black arrows represent the coding sequences of VpreB1 and mouse .lamda.5.

[0063] FIGS. 3A & B: Schematic maps of insert (FIG. 3A) a human BAC (FIG. 3B) containing correctly targeted human VpreB1, .lamda.5 and mouse regulatory elements.

[0064] FIG. 4: Schematic representation of the steps involved in replacement of the mouse .lamda.5 with human .lamda.5.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The invention is useful for promoting the appropriate human IGH repertoire on mature B-cells in the vertebrate (or progeny thereof) or in transgenic vertebrates derived from non-human vertebrate cells. At the transition of pre-BI to pre-BII cells, VH to DH-JH rearrangements are initiated (see, eg, Martensson & Ceredig, Immunology 2000, 101, 435-441; Role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development). Whenever they occur in-frame, a mu chain can be produced and presented on the cell surface only if it associates with the surrogate light chain to form the pre-B-cell receptor (pre-BCR). The pre-BCR on the cell surface is required for clonal expansion of pre-BII cells before light chain rearrangement. Compromised presentation of pre-BCR on the cell surface due to the impairment of association of a mu chain carrying a certain variable region with a surrogate light chain will result in no clonal expansion of such a clone and reduce the representation of this variable region in the mature B cells. Human VpreB1 only shares a low identity with rodent VpreB1/2, suggesting that species specificity for the pre-BCR assembly exists. To this end, the inventors realised the importance for appropriate VpreB/variable region pairing on generation of effective antibody diversity in the context of an antibody heavy chain transgene encoding human variable regions, when this transgene is harboured in a non-human environment such as a transgenic mouse or rat.

[0066] The CH1 constant region pairs with the .lamda.5 protein. Together, the VpreB and .lamda.5 make up the surrogate light chain (SLC) that pairs with mu heavy chains to form the pre-BCR. For H2 antibodies pairing with the CH1 is not possible (since no CH1 is present), but the desirability to pair human VH regions with human VpreB according to the invention still applies.

[0067] Pairing Human Variable Regions of Pre-BCRs with Human VpreB

[0068] The extent of homology between human VpreB with mouse and rat VpreB is relatively low (about 73% amino acid identity). As discussed above, the inventors have realised the advantage of matching human antibody heavy chain variable regions with human VpreB in receptors of pre-B cells for promoting B-cell development in transgenic non-human vertebrates bearing human antibody variable region genes.

[0069] In a first configuration, the invention thus provides:--

[0070] A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene, the transgene comprising

[0071] (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

[0072] (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) a chimaeric antibody heavy chain having a human variable region and a constant region;

[0073] the genome further comprising a human VpreB gene capable of expressing a human VpreB.

[0074] The sequences of VpreB's and .lamda.5's are given below:--

[0075] SEQ ID NO: 1

[0076] Human VpreB1 nucleotide sequence (GenBank Accession No=NG_029387.1)

[0077] SEQ ID NO: 2

[0078] Human VpreB1 amino acid sequence (Gen Bank Accession No=NP_009059.1)

[0079] SEQ ID NO: 3

[0080] Human VpreB3 nucleotide sequence (GenBank Accession No=NC_000022.10)

[0081] SEQ ID NO: 4

[0082] Human VpreB3 amino acid sequence (Gen Bank Accession No=NP_037510)

[0083] SEQ ID NO: 5

[0084] Mouse VpreB1 nucleotide sequence (Gen Bank Accession No=NC_000082.5)

[0085] SEQ ID NO: 6

[0086] Mouse VpreB1 amino acid sequence (GenBank Accession No=NP_058678)

[0087] SEQ ID NO: 7

[0088] Mouse VpreB2 nucleotide sequence (Gen Bank Accession No=NC_000082)

[0089] SEQ ID NO: 8

[0090] Mouse VpreB2 amino acid sequence (GenBank Accession No=NP_058679.1)

[0091] SEQ ID NO: 9

[0092] Rat VpreB1 nucleotide sequence (Gen Bank Accession No=NM_001108845.1)

[0093] SEQ ID NO: 10

[0094] Rat VpreB1 amino acid sequence (GenBank Accession No=NP_001102315.1)

[0095] SEQ ID NO: 11

[0096] Rat VpreB2 nucleotide sequence (Gen Bank Accession No=NC_005110)

[0097] SEQ ID NO: 12

[0098] Rat VpreB2 amino acid sequence (GenBank Accession No=NP_001128260)

[0099] SEQ ID NO: 13

[0100] Human .lamda.5 nucleotide sequence (GenBank Accession No=NG_009791)

[0101] SEQ ID NO: 14

[0102] Human .lamda.5 amino acid sequence (GenBank Accession No=NP_064455)

[0103] SEQ ID NO: 15

[0104] Mouse .lamda.5 nucleotide sequence (Gen Bank Accession No=AC_000038)

[0105] SEQ ID NO: 16

[0106] Mouse .lamda.5 amino acid sequence (Gen Bank Accession No=NP_001177254)

[0107] SEQ ID NO: 17

[0108] Rat .lamda.5 nucleotide sequence (Gen Bank Accession No=NC_005110)

[0109] SEQ ID NO: 18

[0110] Rat .lamda.5 amino acid sequence (Gen Bank Accession No=NP_001177270)

[0111] All sequences listed under these GenBank Accession numbers are incorporated herein by reference as though explicitly written herein and for possible use in the present invention and possible inclusion of any claim below.

[0112] In one embodiment of any configuration of the invention, the human VpreB is human VpreB1. In another embodiment, the human VpreB is human VpreB3.

TABLE-US-00001 TABLE 1 Sequence Identity: VpreB: Human vs. Mouse vs. Rat Human Mouse Mouse Rat Rat VpreB1 VpreB1 VpreB2 VpreB1 VpreB2 Human VpreB1 72.5 72.5 72.6 72.6 Mouse VpreB1 97.2 84.4 83.7 Mouse VpreB2 86.7 85.9 Rat VpreB1 99.3 Rat VpreB2

TABLE-US-00002 TABLE 2 Sequence Identity: Lambda 5: Human vs. Mouse vs. Rat Human .lamda.5 Mouse .lamda.5 Rat .lamda.5 Human .lamda.5 55.8 57.2 Mouse .lamda.5 90.4 Rat .lamda.5

[0113] All nucleotide co-ordinates for the mouse are those corresponding to NCBI m37 for the mouse C57BL/6J strain, e.g. April 2007 ENSEMBL Release 55.37h, e.g. NCBI37 July 2007 (NCBI build 37) (e.g. UCSC version mm9 see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified. Human nucleotides coordinates are those corresponding to GRCh37 (e.g. UCSC version hg 19, http://genome.ucsc.edu/FAQ/FAQreleases.html), February 2009 ENSEMBL Release 55. or are those corresponding to NCBI36, Ensemble release 54 unless otherwise specified. Rat nucleotides are those corresponding to RGSC 3.4 Dec. 2004 ENSEMBL release 55.34w, or Baylor College of Medicine HGSC v3.4 Nov. 2004 (e.g., UCSC rn4, see www.genome.ucsc.edu and http://genome.ucsc.edu/FAQ/FAQreleases.html) unless otherwise specified.

[0114] In one example of the vertebrate or cell, the genome is homozygous for said transgene and endogenous non-human vertebrate antibody heavy chain expression has been inactivated. Alternatively or additionally, endogenous non-human vertebrate antibody light chain expression has been inactivated. Details on possible methods of inactivation are explained in more detail below.

[0115] The present invention is applicable to the production of 4-chain antibodies in transgenic animals, where the antibodies each contain 2 heavy chains and 2 light chains. Alternatively, the invention can be applied to the production of H2 antibodies (heavy chain antibodies) which are devoid of CH1 and light chains (see, eg, Nature. 1993 Jun. 3; 363(6428):446-8; Naturally occurring antibodies devoid of light chains; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, Bendahman N, Hamers R). These antibodies function to specifically bind antigen, such antibodies being found in the blood of Camelidae (eg, llamas, camels, alpacas). Such antibodies with VH pairs can also be synthetically produced to provide therapeutic and prophylactic medicaments (eg, see WO1994004678, WO2004041862, WO2004041863). Transgenic mice also can produce such heavy chain antibodies and the in vivo production of the antibodies allows the mouse's immune system to select for human VH-VH pairings, sometimes selecting for such pairings in which mutations have been introduced in vivo by the mouse to accommodate the pairing (WO2010109165A2). Thus, in an embodiment of the present invention, the heavy chain transgene is devoid of a CH1 gene segment and the genome comprises no functional antibody light chain locus.

[0116] Throughout this text, and with application to any configuration, aspect, embodiment or example of the invention, the term "endogenous" (eg, endogenous constant region gene or endogenous .lamda.5) in relation to a non-human vertebrate or cell indicates that the constant region, .lamda.5 etc is a type of constant region or .lamda.5 that is normally found in the vertebrate or cell (as opposed to an exogenous constant region or .lamda.5 whose sequence is not normally found in such a vertebrate or cell). For example, the endogenous constant region and .lamda.5 can be those encoded by the wild-type genome of the non-human vertebrate/cell. So, in an example wherein the vertebrate cell is a mouse ES cell, the endogenous constant region and .lamda.5 would be mouse constant region and .lamda.5. Going further, the endogenous regions are, in an example, strain-matched to the vertebrate/cell. So, in one embodiment, the vertebrate cell is a mouse 129 ES cell, the endogenous constant region and .lamda.5 would be mouse 129 constant region and .lamda.5. In another embodiment, the vertebrate cell is a mouse JM8 ES cell, the endogenous constant region and .lamda.5 would be mouse JM8 constant region and .lamda.5. In another embodiment, the vertebrate is a Black 6 mouse, the endogenous constant region and .lamda.5 would be mouse Black 6 constant region and .lamda.5.

[0117] In any configuration, aspect, embodiment or example of the invention, the constant region of the heavy chain transgene is a non-human vertebrate constant region (eg, mouse or rat constant region). Optionally, the constant region is endogenous to said non-human vertebrate or cell. Alternatively, the constant region of the heavy chain transgene is human constant region. For example, the constant region is human and devoid of a CH1. This is useful for producing human H2 antibodies (especially when the vertebrate or cell is not capable of expressing light chains).

[0118] In one example of the vertebrate or cell of the invention, the constant region is a Cmu, eg, a mouse or rat Cmu. For example, where the vertebrate is a mouse (or cell is a mouse cell), the Cmu is an endogenous mouse Cmu. The transgene, in an example, comprises a Smu switch 5' of the Cmu and a Cgamma 3' of the Cmu, with a S gamma switch between the Cmu and Cgamma. In an embodiment, the Cmu, Cgamma and switches are endogenous mouse C regions and switches. For example, the C regions and switches are mouse 129 C regions and switches; or the C regions and switches are mouse Black 6 C regions and switches. In another embodiment, the S gamma and C regions are mouse S gamma and C regions, and the Smu is a rat Smu.

[0119] In an example of any configuration, aspect, embodiment or example of the invention, the human VpreB gene has a nucleotide sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 1. The VpreB gene encodes a human VpreB that pairs with the heavy chain human variable region. In an example, the human VpreB gene has a nucleotide sequence that is identical to SEQ ID NO:1.

[0120] In an example of any configuration, aspect, embodiment or example of the invention, the human VpreB gene encodes a human VpreB comprising an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 2. The VpreB gene encodes a human VpreB that pairs with the heavy chain human variable region. In an example, the human VpreB gene encodes a human VpreB comprising an amino acid sequence that is identical to SEQ ID NO:2; or optionally with from one to 15 amino acid changes (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 changes) compared with SEQ ID NO: 2.

[0121] In any configuration, aspect, embodiment or example of the invention, the human VpreB gene encodes a human VpreB that pairs with the heavy chain human variable region, the human VpreB gene having from one to 25 nucleotide changes (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 changes) compared to human VpreB (SEQ ID NO: 1). The VpreB gene can be manipulated in vitro by creation of a targeting vector comprising the gene sequence and homology arms flanking the sequence. This can be introduced into the genome of, for example an ES cell, using the standard technique of homologous recombination as will be apparent to the skilled person. Alternatively, recombinase mediated cassette exchange (RMCE)--another standard technique--can be used to effect precise insertion of the VpreB gene into the genome.

[0122] Homologous recombination or RMCE are generally-useful techniques for the specific, targeted, introduction of sequences into a genome. One or both of these techniques are readily applicable for introducing into a vertebrate or cell genome a human VpreB sequence (wild-type or mutant); V, D, J and C regions; switches; enhancers and other immunoglobulin control elements; and .lamda.5 genes. These and other techniques for target insertion are known to the skilled addressee.

[0123] In an example of any configuration, aspect, embodiment or example of the invention, the genome does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene. In this embodiment, the possibility of non-human VpreB competing with the human VpreB for binding to the human variable regions is eliminated. Thus, pairing of human V regions with human VpreB on pre-B cells is not hampered by such competition, which is advantageous for promoting B-cell development.

[0124] In an example of any configuration, aspect, embodiment or example of the invention, the genome further comprises a .lamda.5 gene (eg, a human, mouse or rat .lamda.5); optionally wherein the .lamda.5 gene is a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region gene is a constant region gene of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene. For example, the constant region and .lamda.5 genes are mouse constant region and .lamda.5 genes, eg, mouse 129 constant region and .lamda.5 genes; or mouse JM8 constant region and .lamda.5 genes). For example, the constant region and .lamda.5 genes are rat constant region and .lamda.5 genes. In an example, constant region gene and .lamda.5 gene are endogenous genes of said vertebrate or cell. Thus, the invention contemplates close matching of not only the V-region/VpreB part of pre-BCRs but also close matching of the Cmu/.lamda.5 part. While not wishing to be bound to any theory, the inventors believe that this is useful for stabilising the pre-BCRs to promote B-cell development in vivo, thus improving the B-cell pool (and potentially diversity) from which to select antigen-specific antibodies following immunisation of vertebrates according to the inventions.

[0125] In another embodiment of any configuration, aspect, embodiment or example of the invention, the .lamda.5 is a chimaeric .lamda.5 comprising a non-human vertebrate .lamda.5 (eg, endogenous mouse or rat .lamda.5) in which amino acid sequences have been replaced by corresponding amino acids from a human .lamda.5. For example, the region of .lamda.5 that interfaces with VpreB to form the SLC is replaced with the corresponding part from a human .lamda.5 to form a chimaeric .lamda.5 with a human part (ie, species-matched with the human VpreB) and a non-human vertebrate (eg, mouse or rat) part that is species matched with the CH1 of the non-human vertebrate Cmu of the heavy chain transgene. In the alternative embodiment where the .lamda.5 is a human .lamda.5, the .lamda.5 is advantageously species-matched to interface with the human .lamda.5.

[0126] In one aspect the non-human vertebrate is able to generate a diversity of at least 1.times.10.sup.6 different functional chimaeric antibody sequence combinations.

[0127] In a second configuration, the method provides

[0128] A method of constructing a transgenic non-human vertebrate cell (eg, an ES cell, eg, a mouse or rat ES cell), the method comprising

[0129] (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 (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

[0130] (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;

[0131] and wherein the method further comprises introducing into the cell (before, after or concomitantly with step (i) or (ii)) a human VpreB gene capable of expressing a human VpreB.

[0132] Targeting of the genome of an ES cell to produce a transgenic cell and subsequently a transgenic vertebrate (eg, a mouse) may be carried out using a protocol as explained by reference to the FIGS. 1-18 and description in WO2011004129, the disclosure of which is incorporated herein by reference. A suitable mouse ES cell is selected from AB2.1 cells (available from Baylor College of Medicine, Texas, USA), AB2.2 cells and JM8 cells.

[0133] In any configuration, aspect, embodiment or example of the invention, the cell is an ES cell is capable of developing into a non-human mammal able to produce a repertoire of antibodies which are chimaeric, said chimaeric antibodies having a non-human mammal constant region and a human variable region. Optionally the genome of the cell is modified to prevent expression of fully host-species specific antibodies.

[0134] In one aspect the cell is an induced pluripotent stem cell (iPS cell).

[0135] In one aspect cells are isolated non-human mammalian cells.

[0136] In one aspect a cell as disclosed herein is preferably a non-human mammalian cell.

[0137] In one aspect the cell is a cell from a mouse strain selected from C57BL/6, M129 such as 129/SV, BALB/c, and any hybrid of C57BL/6, M129 such as 129/SV, or BALB/c. In an example, the mouse is a C57BL/6-129/Sv hybrid.

[0138] Maintaining the performance of the ES cell clones through multiple rounds of manipulation without the need to test the germ line potential of the ES cell line at every step may be important in the method of constructing a transgenic locus. The cell lines currently in use for the KOMP and EUCOMM global knockout projects have been modified twice prior to their use for this project and their germ line transmission rates are unchanged from the parental cells (these lines are publicly available, see www.komp.org and www.eucomm.org). This cell line, called JM8, can generate 100% ES cell-derived mice under published culture conditions (Pettitt, S. J., Liang, Q., Rairdan, X. Y., Moran, J. L., Prosser, H. M., Beier, D. R., Lloyd, K. C., Bradley, A., and Skarnes, W. C. (2009). Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nature Methods.). These cells have demonstrated ability to reproducibly contribute to somatic and germ line tissue of chimaeric animals using standard mouse ES cell culture conditions. This capability can be found with cells cultured on a standard feeder cell line (SNL) and even feeder-free, grown only on gelatine-coated tissue culture plates. One particular sub-line, JM8A3, maintained the ability to populate the germ line of chimeras after several serial rounds of sub-cloning. Extensive genetic manipulation via, for example, homologous recombination--as could be the case in the present method--cannot compromise the pluripotency of the cells. The ability to generate chimeras with such high percentage of ES cell-derived tissue has other advantages. First, high levels of chimaerism correlates with germ line transmission potential and provide a surrogate assay for germ line transmission while only taking 5 to 6 weeks. Second, since these mice are 100% ES cell derived the engineered loci can be directly tested, removing the delay caused by breeding. Testing the integrity of the new Ig loci is possible in the chimera since the host embryo will be derived from animals that are mutant for the RAG-1 gene as described in the next section.

[0139] Another cell line that may be used is an HPRT-negative cell line, such as AB2.1, as disclosed in "Chromosome engineering in mice, Ramirez-Solis R, Liu P and Bradley A, Nature 1995; 378; 6558; 720-4.

[0140] RAG-1 Complementation

[0141] While many clones will generate 100% ES derived mice some will not. Thus, at every step mice can be generated in a RAG-1-deficient background. This provides mice with 100% ES-derived B- and T-cells which can be used directly for immunization and antibody production. Cells having a RAG-2 deficient background, or a combined RAG-1/RAG-2 deficient background may be used, or equivalent mutations in which mice produce only ES cell-derived B cells and/or T cells.

[0142] In order that only the human-mouse IgH (and optionally also light chain) loci are active in these mice, the human-mouse IgH (and optionally light chain loci) can be engineered in a cell line in which one allele of the IgH (and optionally light chain) locus has already been inactivated. Alternatively the inactivation of the host Ig locus/loci can be carried out after insertion.

[0143] Mouse strains that have the RAG-1 gene mutated are immunodeficient as they have no mature B- or T-lymphocytes (U.S. Pat. No. 5,859,307). T- and B-lymphocytes only differentiate if proper V(D)J recombination occurs. Since RAG-1 is an enzyme that is crucial for this recombination, mice lacking RAG-1 are immunodeficient. If host embryos are genetically RAG-1 homozygous mutant, a chimera produced by injecting such an embryo will not be able to produce antibodies if the animal's lymphoid tissues are derived from the host embryo. However, JM8 cells and AB2.1 cells, for example, generally contribute in excess of 80% of the somatic tissues of the chimaeric animal and would therefore usually populate the lymphoid tissue. JM8 cells have wild-type RAG-1 activity and therefore antibodies produced in the chimaeric animal would be encoded by the engineered JM8 ES cell genome only. Therefore, the chimaeric animal can be challenged with an antigen by immunization and subsequently produce antibodies to that antigen. This allows one skilled in the art to test the performance of the engineered human/mouse Ig loci as described in the present invention.

[0144] One skilled in the art could use the chimaeric animal as described to determine the extent of antibody diversity (see e.g. Harlow, E. & Lane, D. 1998, 5.sup.th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.). For example, the existence in the chimaeric animal's serum of certain antibody epitopes could be ascertained by binding to specific anti-idiotype antiserum, for example, in an ELISA assay. One skilled in the art could also sequence the genomes or heavy chain RNA or DNA sequences of B-cell clones derived from the chimaeric animal (naive or immunised) and compare said sequence to wild-type sequence (ie, minus human VpreB) to assess the B-cell repertoire and ascertain the level of hypermutation, such hypermutation being indicative of normal antibody maturation.

[0145] One skilled in the art would also use said chimaeric animal to examine antibody function wherein said antibodies are encoded from the engineered Ig loci (see e.g. Harlow, E. & Lane, D. 1998, 5.sup.th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.). For example, antisera could be tested for binding an antigen, said antigen used to immunize the chimaeric animal. Such a measurement could be made by an ELISA assay. Alternatively, one skilled in the art could test for neutralization of the antigen by addition of the antisera collected from the appropriately immunized chimaeric animal.

[0146] It is well known to those skilled in the art that positive outcomes for any of these tests demonstrate the ability of the engineered Ig loci in the presence of VpreB during B-cell development according to the instant invention, to encode antibodies with human variable regions and non-human vertebrate constant regions, said antibodies capable of functioning in the manner of wild-type antibodies.

[0147] The introduction of human gene segment DNA into the genome can be carried out using vectors carrying the DNA, as more fully explained below. In one aspect such vectors are BACs (bacterial artificial chromosomes). It will be appreciated that other cloning vectors may be used in the invention, and therefore reference to BACs herein may be taken to refer generally to any suitable vector. As described in WO2011004129, in one aspect the inserted DNA is built up in the genome of a cell, such as an ES cell, in a stepwise manner using 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more separate insertions for the heavy chain gene segments. Fragments are suitably inserted at the same or substantially the same cell locus, e.g. ES cell locus, one after another, to form the complete VDJ region.

[0148] In a further aspect the method comprises the insertion of human heavy chain gene segments upstream of the non-human vertebrate constant region by step-wise insertion of multiple fragments by homologous recombination, preferably using an iterative process. Suitably fragments of approximately 100 KB from the human VDJ locus are inserted, suitably to form a VDJ region after the final iteration of the insertion process, as described in WO2011004129.

[0149] In one aspect the insertion process commences at a site where an initiation cassette has been inserted into the genome of a cell, such as an ES cell, providing a unique targeting region. In one aspect the initiation cassette is inserted in the non-human vertebrate heavy chain locus, for use in insertion of human heavy chain DNA. The initiation cassette suitably comprises a vector backbone sequence with which a vector having a human DNA fragment in the same backbone sequence can recombine to insert the human DNA into the cell (e.g. ES) cell genome, and suitably a selection marker, such as a negative selection marker. Suitably the vector backbone sequence is that of a BAC library, to allow BACs to be used in the construction of the ES cells and mammals. The vector backbone sequence may however be any sequence which serves as a target site into which a homologous sequence can insert, for example by homologous recombination and, for example RMCE, and is optionally not DNA encoding any of the VDJ or constant region.

[0150] Suitable BACs are available from the Sanger centre, see "A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction". Adams D J, Quail M A, Cox T, van der Weyden L, Gorick B D, Su Q, Chan W I, Davies R, Bonfield J K, Law F, Humphray S, Plumb B, Liu P, Rogers J, Bradley A. Genomics. 2005 December; 86(6):753-8. Epub 2005 Oct. 27. The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK. BACs containing human DNA are also available from, for example, Invitrogen. A suitable library (source of human heavy chain gene segments) is described in Osoegawa K et al, Genome Research 2001. 11: 483-496 and obtainable from the Roswell Park Cancer Institute (USA)/Invitrogen.

[0151] In any configuration, aspect, embodiment or example of the invention, non-human vertebrates, such as mice, are generated in a RAG-1 or RAG-2-deficient background, or other suitable genetic background which prevents the production of mature host B and T lymphocytes.

[0152] In any configuration, aspect, embodiment or example of the invention, the non-human vertebrate is a rodent, suitably a mouse, and cells of the invention, are rodent cells or ES cells, suitably mouse ES cells.

[0153] The ES cells of the present invention can be used to generate animals using techniques well known in the art, which comprise injection of the ES cell into a blastocyst followed by implantation of chimaeric blastocystys into females to produce offspring which can be bred and selected for homozygous recombinants having the required insertion. In one aspect the invention relates to a chimeric non-human vertebrate comprised of ES cell-derived tissue and host embryo derived tissue, wherein the ES cell is according to the invention. In one aspect the invention relates to genetically-altered subsequent generation non-human vertebrates, which include vertebrates homozygous for the human VDJ region.

[0154] In one any configuration, aspect, example or embodiment, the genome of the cell or vertebrate comprises: one or more human Ig light chain kappa V regions and one or more human Ig light chain kappa J regions upstream of all or part of a kappa constant region.

[0155] In one any configuration, aspect, example or embodiment, the genome of the cell or vertebrate comprises: one or more human Ig light chain lambda V regions and one or more human Ig light chain lambda J regions upstream of all or part of a lambda constant region.

[0156] Suitably the light chain Viand C regions are able to form antibody light chains in vivo capable of forming an antibody with one or more of the chimaeric heavy chains to form an antibody that binds a predetermined antigen.

[0157] In such aspects a human kappa and/or lambda region is inserted into the genome, in combination with insertion of the heavy chain VDJ region or part thereof, upstream of the host heavy chain constant region as disclosed herein.

[0158] Suitably the insertion of the human VJC light chain DNA, or part thereof as disclosed above, is made at the equivalent mouse locus. In one aspect the human light chain kappa VJC DNA, or part thereof, is inserted immediately upstream or downstream of the mouse kappa VJC region. In one aspect, the human light chain lambda VJC region or part thereof is inserted immediately upstream or downstream of the mouse lambda VJC region. In one aspect only the human kappa VJC locus is inserted and not the human lambda VJC locus. In one aspect only the human lambda VJC locus is inserted and not the human kappa VJC locus. Insertions may be made using the techniques disclosed herein, and suitably do not remove the host sequences from the genome. In one aspect the non-human mammal host VJC sequences may be inactivated in some way, by mutation, or inversion, or by insertion of the human variable region DNA, or by any other means. In one aspect the cell or non-human mammal of the invention may comprise an insertion of the complete VJC human region.

[0159] The human kappa variable region DNA might be inserted into the genome in functional arrangement with a lambda constant region, for example inserted upstream of a lambda constant region. Alternatively human lambda region variable DNA might be inserted in functional arrangement with a kappa constant region, for example inserted upstream of a kappa constant region.

[0160] In one any configuration, aspect, example or embodiment, the genome of the cell or vertebrate comprises: one or more non-human vertebrate control sequences such as the enhancer sequence(s) is maintained upstream of the nonhuman vertebrate Mu constant region, suitably in its native position with respect to the distance from the constant region.

[0161] In one any configuration, aspect, example or embodiment, one or more non-human mammal control sequences such as an enhancer sequence(s) are maintained downstream of the nonhuman vertebrate Mu constant region, suitably in its native position with respect to the distance from the constant region.

[0162] In one any configuration, aspect, example or embodiment, a non-human mammal switch sequence, suitably the endogenous switch sequence, is maintained upstream of the non-human vertebrate Mu constant region, suitably in its native position with respect to distance from the constant region.

[0163] In such location the host enhancer or switch sequences are operative in vivo with the host constant region sequence(s).

[0164] In one aspect a switch sequence is neither human, nor native in the non-human mammal, for example in one aspect a non-human mammal switch sequence is not a mouse or human switch sequence. The switch sequence may be, for example, a rodent or primate sequence, or a synthetic sequence. In particular the switch sequence may be a rat sequence where the non-human mammal is a mouse. By way of example, a mouse or human constant mu sequence may be placed under the control of a switch sequence from a rat, or chimp, or other switch sequence, suitably capable of allowing isotype switching to occur in vivo.

[0165] One combination envisaged is a rat switch with mouse enhancer sequences and mouse constant regions in a mouse cell.

[0166] In one aspect the human promoter and/or other control elements that are associated with the different human V, D or J regions are maintained in after insertion of the human VDJ into the mouse genome.

[0167] The functional replacement of human promoter or other control regions by non-human mammal promoter or control regions may be carried out by use of recombineering, or other recombinant DNA technologies, to insert a part of the human Ig region (such as a human V region) into a vector (such as a BAC) containing a non-human Ig region. The recombineering/recombinant technique suitably replaces a portion of the non-human (e.g. mouse) DNA with the human Ig region, and thus places the human Ig region under control of the non-human mammal promoter or other control region. Suitably the human coding region for a human V region replaces a mouse V region coding sequence. Suitably the human coding region for a human D region replaces a mouse D region coding sequence. Suitably the human coding region for a human J region replaces a mouse J region coding sequence. In this way human V, D or J regions may be placed under the control of a non-human mammal promoter, such as a mouse promoter.

[0168] In one any configuration, aspect, example or embodiment, the human DNA inserted into the genome of the non-human vertebrate or cell are placed under control of the host regulatory sequences or other (non-human, non-host) sequences, In one aspect reference to human coding regions includes both human introns and exons, or in another aspect simply exons and no introns, which may be in the form of cDNA.

[0169] It is also possible to use recombineering, or other recombinant DNA technologies, to insert a non-human-mammal (e.g. mouse) promoter or other control region, such as a promoter for a V region or

[0170] VpreB, into a BAC containing a human Ig region. The A recombineering step then places a portion of human DNA under control of the mouse promoter or other control region.

[0171] Generally, insertion of human variable region DNA at or close to the equivalent endogenous locus in the recipient genome is preferred, for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb of the boundary (upstream or downstream) of a host immunoglobulin locus.

[0172] In one embodiment of the method of the second configuration of the invention, said genome is homozygous for said transgene and endogenous non-human vertebrate antibody heavy chain expression is inactivated.

[0173] In one embodiment of the method of the second configuration of the invention, endogenous non-human vertebrate antibody light chain expression is inactivated.

[0174] In one embodiment of the method of the second configuration of the invention, the heavy chain transgene is devoid of a CH1 gene segment and the genome comprises no functional antibody light chain locus.

[0175] In one embodiment of the method of the second configuration of the invention, said constant region is a Cmu. For example, the Cmu is a non-human vertebrate Cmu or an endogenous Cmu of said vertebrate or cell.

[0176] In one embodiment of the method of the second configuration of the invention, said genome does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene.

[0177] In one embodiment of the method of the second configuration of the invention, the genome further comprises a .lamda.5 gene; optionally wherein the .lamda.5 gene is a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region gene is a constant region gene of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene.

[0178] In one embodiment of the method of the second configuration of the invention, said constant region gene and .lamda.5 gene are endogenous genes of said vertebrate or cell.

[0179] In one embodiment of the method of the second configuration of the invention, the method comprises making a progeny of the cell made according to the method, wherein the progeny is homozygous for said heavy chain transgene and endogenous non-human vertebrate antibody expression has been inactivated.

[0180] A third configuration of the invention provides

[0181] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0182] (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;

[0183] (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

[0184] (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.

[0185] In one embodiment of the method of the third configuration of the invention, in (a) the heavy chain transgene is constructed to be devoid of a CH1 gene segment and the genome of the third non-human vertebrate comprises no functional antibody light chain locus.

[0186] In one embodiment of the method of the third configuration of the invention, the genome of the third vertebrate does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene.

[0187] In one embodiment of the method of the third configuration of the invention, the third non-human vertebrate expresses a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region is a constant region of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene; optionally wherein the .lamda.5 and constant region are an endogenous .lamda.5 and constant region of mouse or rat.

[0188] The invention further provides a transgenic mouse according to the third vertebrate, or a progeny thereof.

[0189] In a fourth aspect, the invention provides

[0190] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0191] (i) inactivating endogenous heavy chain expression in said vertebrate;

[0192] (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 (optionally an endogenous non-human vertebrate mu constant region); and

[0193] (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;

[0194] Optionally wherein the genome is homozygous for said first transgene.

[0195] In one embodiment of the method of the fourth configuration of the invention, the heavy chain transgene is devoid of a CH1 gene segment and the genome of the vertebrate comprises no functional antibody light chain locus.

[0196] In one embodiment of the method of the fourth configuration of the invention, the genome of the vertebrate does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene.

[0197] In one embodiment of the method of the fourth configuration of the invention, the non-human vertebrate expresses a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region is a constant region of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene; optionally wherein the .lamda.5 and constant region are an endogenous .lamda.5 and constant region of mouse or rat.

[0198] The invention also provides a transgenic mouse according to the fourth configuration, or a progeny thereof.

[0199] Chimaeric Surrogate Light Chains

[0200] The extent of homology between human .lamda.5 with mouse and rat .lamda.5 is relatively low (about 56% amino acid identity). The inventors realised the advantage of species-matching the .lamda.5 in the SLC in a transgenic non-human vertebrate with the species of mu constant region in the pre-BCRs in such transgenic animals. Thus, the invention further provides aspects that provide for the possibility of species-matching in vivo the variable and constant regions of the antibody heavy chain with both components (VpreB and .lamda.5) of a chimaeric SLC. This is useful for promoting B-cell development in the transgenic non-human vertebrate (eg, a mouse or rat).

[0201] Thus, in an embodiment of the vertebrates, cells and methods of the invention, the genome further comprises a .lamda.5 gene; optionally wherein the .lamda.5 gene is a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region gene is a constant region gene of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene. For example, the vertebrate is a mouse or rat (or cell is a mouse or rat cell) and the .lamda.5 is a .lamda.5 that is endogenous to the mouse or rat (or mouse or rat cell). In this example, the constant region of the heavy chain transgene is an constant region (eg, Cmu) endogenous to the mouse or rat. In this way, the .lamda.5 is matched for pairing to the constant region at the cell surface in the pre-BII stage, and the human VpreB is matched for pairing with the human variable region encoded by the transgene.

[0202] To this end, the invention provides in a fifth configuration

[0203] A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene,

[0204] the transgene comprising

[0205] (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 of a non-human vertebrate (eg, mouse or rat) species so that the transgene is capable of undergoing VDJ recombination in vivo to produce an antibody gene comprising a rearranged VDJC encoding a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region; or

[0206] (b) a rearranged VDJ encoding a human variable region operably connected upstream of a constant region gene of a non-human vertebrate (eg, mouse or rat) species so that the transgene encodes (optionally following VDJ combination with the constant region) a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region;

[0207] the genome further comprising one or more genes together encoding a chimaeric surrogate light chain, the surrogate light chain comprising a human VpreB and .lamda.5 of said non-human vertebrate (eg, mouse or rat) species.

[0208] In one embodiment of the method of the fifth configuration of the invention, said genome is homozygous for said transgene and endogenous non-human vertebrate antibody heavy chain expression has been inactivated.

[0209] In one embodiment of the method of the fifth configuration of the invention, said constant region gene is a Cmu gene.

[0210] In one embodiment of the method of the fifth configuration of the invention, said constant region gene and .lamda.5 gene are endogenous genes of said vertebrate or cell.

[0211] In one embodiment of the method of the fifth configuration of the invention, said genome does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene.

[0212] In a sixth configuration, the invention further provides

[0213] A method of constructing a transgenic non-human vertebrate cell (eg, an ES cell, eg, a mouse or rat ES cell), the method comprising

[0214] (i) introducing into 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 an endogenous non-human vertebrate constant region gene, 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 a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region; or

[0215] (ii) introducing into 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 an endogenous non-human vertebrate constant region gene, wherein in said cell or a progeny thereof the transgene encodes (optionally following VDJ combination with the constant region) a chimaeric antibody heavy chain having a human variable region and a non-human vertebrate constant region;

[0216] and wherein the method further comprises introducing into the cell a human VpreB gene such that the cell or a progeny thereof is capable of expressing a chimaeric surrogate light chain as well as said chimaeric antibody heavy chain, wherein the surrogate light chain comprises a human VpreB and an endogenous non-human vertebrate .lamda.5.

[0217] In one embodiment of the method of the sixth configuration of the invention, said constant region gene is a Cmu gene.

[0218] In one embodiment of the method of the sixth configuration of the invention, each endogenous non-human vertebrate VpreB gene is inactivated or deleted from said genome.

[0219] In one embodiment, the method of the sixth configuration of the invention comprises making a progeny of the cell made according to the method, wherein the progeny is homozygous for said heavy chain transgene and endogenous non-human vertebrate antibody heavy chain expression has been inactivated.

[0220] In a seventh configuration, the invention provides

[0221] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0222] (a) providing in a non-human vertebrate embryonic stem (ES) cell an immunoglobulin transgene capable of expressing a chimaeric antibody mu heavy chain, wherein the chimaeric antibody heavy chain comprises a human variable region and an endogenous non-human vertebrate mu constant region; and creating a first non-human vertebrate from said ES cell or a progeny thereof;

[0223] (b) providing in a second ES cell a second transgene capable of expressing a human VpreB so that the human VpreB and an endogenous .lamda.5 form a chimaeric surrogate light chain; and creating a second non-human vertebrate from said ES cell or a progeny thereof; and

[0224] (c) creating by breeding a third non-human vertebrate capable of co-expressing the chimaeric mu antibody heavy chain and chimaeric surrogate light chain wherein a pre-BCR can form to promote B-cell development 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.

[0225] The invention provides a transgenic mouse according to the vertebrate of this method, or a progeny thereof.

[0226] In an eighth configuration, the invention provides

[0227] A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising

[0228] (i) inactivating endogenous heavy chain expression in said vertebrate;

[0229] (ii) providing in the genome of said vertebrate an immunoglobulin transgene capable of expressing a chimaeric antibody mu heavy chain, wherein the chimaeric antibody heavy chain comprises a human variable region and an endogenous non-human vertebrate mu constant region;

[0230] and

[0231] (ii) providing in the genome of said vertebrate a second transgene capable of expressing a human VpreB so that the human VpreB and an endogenous .lamda.5 form a chimaeric surrogate light chain; so that the vertebrate is capable of co-expressing the chimaeric mu antibody heavy chain and chimaeric surrogate light chain wherein a pre-BCR can form to promote B-cell development in said vertebrate;

[0232] Optionally wherein the genome is homozygous for said first transgene.

[0233] A transgenic mouse according to the vertebrate of this method, or a progeny thereof, is provided.

[0234] In any configuration, aspect, embodiment or example of the invention, the inserted human genes may be derived from the same individual or different individuals, or be synthetic or represent human consensus sequences.

[0235] Techniques for constructing non-human vertebrates and vertebrate cells whose genomes comprise a transgene containing human V, J and D regions are well known in the art. For example, reference is made to WO2011004192, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364, WO2009/076464 and U.S. Pat. No. 6,586,251, the disclosures of which are incorporated herein by reference in their entirety.

[0236] In one embodiment in any configuration of the invention, the vertebrate is a non-human mammal and the vertebrate cell is a non-human mammalian cell. In one embodiment in any configuration of the invention, the vertebrate is a mouse, rat, rabbit, Camelid (eg, a llama, alpaca or camel) or shark; or the vertebrate cell is a mouse, rat, rabbit, Camelid (eg, a llama, alpaca or camel) or shark cell.

[0237] In one aspect the human heavy chain gene segments are inserted into the genome so that they are placed under control of the host regulatory sequences (eg, enhancers, promoters and/or switches) or other (non-human, non-host) sequences. In one aspect reference to human coding regions includes both human introns and exons, or in another aspect simply exons and no introns, which may be in the form of cDNA.

[0238] Alternatively it is possible to use recombineering, or other recombinant DNA technologies, to insert a non human-vertebrate (e.g. mouse) promoter or other control region, such as a promoter for a V region, into a BAC containing a human Ig region. The recombineering step then places a portion of human DNA under control of the mouse promoter or other control region.

[0239] The invention also relates to a cell line which is grown from or otherwise derived from cells as described herein, including an immortalised cell line. The cell line may comprise inserted human V, D or J genes as described herein, either in germline configuration or after rearrangement following in vivo maturation. The cell may be immortalised by fusion to a tumour cell to provide an antibody producing cell and cell line, or be made by direct cellular immortalisation.

[0240] In one aspect the non-human vertebrate of any configuration of the invention is able to generate a diversity of at least 1.times.10.sup.6 different functional chimaeric immunoglobulin sequence combinations.

[0241] Optionally in any configuration of the invention the constant region is endogenous to the vertebrate and optionally comprises an endogenous switch. In one embodiment, the constant region comprises a Cgamma (CY) region and/or a Smu (S.mu.) switch. Switch sequences are known in the art, for example, see Nikaido et al, Nature 292: 845-848 (1981) and also WO2011004192, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364, WO2009/076464 and U.S. Pat. No. 6,586,251, eg, SEQ ID NOs: 9-24 disclosed in

[0242] U.S. Pat. No. 7,501,552. Optionally the constant region comprises an endogenous S gamma switch and/or an endogenous Smu switch. One or more endogenous switch regions can be provided, in one embodiment, by constructing a transgenic immunoglobulin locus in the vertebrate or cell genome in which at least one human V region, at least one human J region, and optionally at least one human D region, or a rearranged VDJ or VJ region, are inserted into the genome in operable connection with a constant region that is endogenous to the vertebrate or cell. For example, the human V(D)J regions or rearranged VDJ or VJ can be inserted in a cis orientation onto the same chromosome as the endogenous constant region. A trans orientation is also possible, in which the human V(D)J regions or rearranged VDJ or VJ are inserted into one chromosome of a pair (eg, the chromosome 6 pair in a mouse or the chromosome 4 in a rat) and the endogenous constant region is on the other chromosome of the pair, such that trans-switching takes place in which the human V(D)J regions or rearranged VDJ or VJ are spliced inoperable linkage to the endogenous constant region. In this way, the vertebrate can express antibodies having a chain that comprises a variable region encoded all or in part by human V(D)J or a rearranged VDJ or VJ, together with a constant region (eg, a Cgamma or Cmu) that is endogenous to the vertebrate.

[0243] Human variable regions are suitably inserted upstream of non-human vertebrate constant region, the latter comprising all of the DNA required to encode the full constant region or a sufficient portion of the constant region to allow the formation of an effective chimaeric antibody capable of specifically recognising an antigen.

[0244] In one aspect the chimaeric antibodies or antibody chains have a part of a host constant region sufficient to provide one or more effector functions seen in antibodies occurring naturally in a host vertebrate, for example that they are able interact with Fc receptors, and/or bind to complement.

[0245] Reference to a chimaeric antibody or antibody chain having a non-human vertebrate constant region herein therefore is not limited to the complete constant region but also includes chimaeric antibodies or chains which have all of the host constant region, or a part thereof sufficient to provide one or more effector functions. This also applies to non-human vertebrate mammals and cells and methods of the invention in which human variable region DNA may be inserted into the host genome such that it forms a chimaeric antibody chain with all or part of a host constant region. In one aspect the whole of a host non-human vertebrate constant region is operably linked to human variable region DNA.

[0246] The host non-human vertebrate constant region herein is optionally the endogenous host wild-type constant region located at the wild type locus, as appropriate for the heavy or light chain. For example, the human heavy chain DNA is suitably inserted on mouse chromosome 12, suitably adjacent the mouse heavy chain constant region, where the vertebrate is a mouse.

[0247] In one optional aspect where the vertebrate is a mouse, the insertion of the human DNA, such as the human VDJ region is targeted to the region between the J4 exon and the C.mu. locus in the mouse genome IgH locus, and in one aspect is inserted between coordinates 114,667,090 and 114,665,190, suitably at coordinate 114,667,091, after 114,667,090. In one aspect the insertion of the human DNA, such as the human light chain kappa VJ is targeted into mouse chromosome 6 between coordinates 70,673,899 and 70,675,515, suitably at position 70,674,734, or an equivalent position in the lambda mouse locus on chromosome 16.

[0248] In one aspect the host non-human vertebrate constant region for forming the chimaeric antibody may be at a different (non endogenous) chromosomal locus. In this case the inserted human DNA, such as the human variable VDJ or VJ region(s) may then be inserted into the non-human genome at a site which is distinct from that of the naturally occurring heavy or light constant region. The native constant region may be inserted into the genome, or duplicated within the genome, at a different chromosomal locus to the native position, such that it is in a functional arrangement with the human variable region such that chimaeric antibodies of the invention can still be produced.

[0249] In one aspect the human DNA is inserted at the endogenous host wild-type constant region located at the wild type locus between the host constant region and the host VDJ region.

[0250] Reference to location of the variable region upstream of the non-human vertebrate constant region means that there is a suitable relative location of the two antibody portions, variable and constant, to allow the variable and constant regions to form a chimaeric antibody or antibody chain in vivo in the vertebrate. Thus, the inserted human DNA and host constant region are in operable connection with one another for antibody or antibody chain production.

[0251] In one aspect the inserted human DNA is capable of being expressed with different host constant regions through isotype switching. In one aspect isotype switching does not require or involve trans switching. Insertion of the human variable region DNA on the same chromosome as the relevant host constant region means that there is no need for trans-switching to produce isotype switching.

[0252] In the present invention, optionally host non-human vertebrate constant regions are maintained and it is preferred that at least one non-human vertebrate enhancer or other control sequence, such as a switch region, is maintained in functional arrangement with the non-human vertebrate constant region, such that the effect of the enhancer or other control sequence, as seen in the host vertebrate, is exerted in whole or in part in the transgenic animal. This approach is designed to allow the full diversity of the human locus to be sampled, to allow the same high expression levels that would be achieved by non-human vertebrate control sequences such as enhancers, and is such that signalling in the B-cell, for example isotype switching using switch recombination sites, would still use non-human vertebrate sequences.

[0253] A non-human vertebrate having such a genome would produce chimaeric antibodies with human variable and non-human vertebrate constant regions, but these are readily humanized, for example in a cloning step. Moreover the in vivo efficacy of these chimaeric antibodies could be assessed in these same animals.

[0254] In one aspect the inserted human IgH VDJ region comprises, in germline configuration, all of the V, D and J regions and intervening sequences from a human. Optionally, non-functional V and/or D and/or J gene segments are omitted. For example, VH which are inverted or are pseudogenes may be omitted.

[0255] In one aspect 800-1000 kb of the human IgH VDJ region is inserted into the non-human vertebrate IgH locus, and in one aspect a 940, 950 or 960 kb fragment is inserted. Suitably this includes bases 105,400,051 to 106,368,585 from human chromosome 14.

[0256] In one aspect the inserted IgH human fragment consists of bases 105,400,051 to 106,368,585 from chromosome 14. In one aspect the inserted human heavy chain DNA, such as DNA consisting of bases 105,400,051 to 106,368,585 from chromosome 14, is inserted into mouse chromosome 12 between the end of the mouse J4 region and the Eli region, suitably between co-ordinates 114,667,090 and 114,665,190, or at co-ordinate 114,667,091, after 114,667,090. In one aspect the insertion is between co-ordinates 114,667,089 and 114,667,090 (co-ordinates refer to NCBI m37, for the mouse C57BL/6J strain), or at equivalent position in another non-human vertebrate genome.

[0257] A cell or non-human vertebrate of the invention, in one embodiment, comprises an insertion of human heavy chain variable region DNA between co-ordinates 114, 666, 183 and 114, 666, 725, such as between 114 666 283 and 114 666 625, optionally between co-ordinates 114,666,335 and 114,666,536, optionally between 114,666,385 and 114,666,486, or between 114,666,425 and 114,666,446, or between 114,666,435 and 114,666,436 of mouse chromosome 12 with reference to NCBIM37 for the mouse genome, relating to mouse strain C57BL/6J or an equivalent position of mouse chromosome 12 from a different mouse strain or an equivalent position in the genome of another non-human vertebrate, e.g., a rat. The insertion between co-ordinates 114,666,435 and 114,666,436 relating to mouse strain C57BL/6J is equivalent to an insertion between co-ordinates 1207826 and 1207827 on chromosome 12 with reference to the 129/SvJ genomic sequence of the geneBank access number NT114985.2. An insertion may be made at equivalent position in another genome, such as another mouse genome. In an example of this embodiment, the cell or mammal of the invention comprises a human IgH VDJ region which comprises or consists of nucleotides 106,328,851-107,268,544, such as nucleotides 106,328,901-107,268,494, such as nucleotides 106,328,941-107,268,454, such as nucleotides 106,328,951-107,268,444 of human Chromosome 14, with reference to the GRCH37/hg19 sequence database, or insertion of equivalent nucleotides relating to chromosome 14 from a different human sequence or database. The human insertion may be made between the regions indicated above.

[0258] In one aspect there is inserted into the genome the human kappa VJ region which comprises, in germline configuration, all of the V and J regions and intervening sequences from a human. Optionally, non-functional V and/or J gene segments are omitted.

[0259] Suitably this includes bases 88,940,356 to 89,857,000 from human chromosome 2, suitably approximately 917 kb. In a further aspect the light chain VJ insert may comprise only the proximal clusters of V segments and J segments. Such an insert would be of approximately 473 kb.

[0260] In one aspect the human light chain kappa DNA, such as the human Ig.kappa. fragment of bases 88,940,356 to 89,857,000 from human chromosome 2, is suitably inserted into mouse chromosome 6 between coordinates 70,673,899 and 70,675,515, suitably at position 70,674,734.

[0261] In one aspect the human lambda V (or VJ) region is inserted into the genome, which comprises, in germline configuration, all of the V (and optionally J) regions and intervening sequences from a human (which will thus include the human VpreB gene and a promoter). Suitably this includes analogous bases to those selected for the kappa fragment, from human chromosome 2. Optionally, non-functional V and/or J gene segments are omitted.

[0262] All specific human fragments described above may vary in length, and may for example be longer or shorter than defined as above, such as 500 bases, 1 KB, 2K, 3K, 4K, 5 KB, 10 KB, 20 KB, 30 KB, 40 KB or 50 KB or more, which suitably comprise all or part of the human V(D)J region, whilst preferably retaining the requirement for the final insert to comprise human genetic material encoding the complete heavy chain region and light chain region, as appropriate, as described above.

[0263] In one aspect the 3' end of the last inserted human sequence, generally the last human J sequence, is inserted less than 2 kb, preferably less than 1 KB from the human/non-human vertebrate (eg, human/mouse or human/rat) join region.

[0264] Optionally, the genome is homozygous at at least the IgH, or a second, or all three immunoglobulin loci (IgH, Ig.lamda. and Ig.kappa..

[0265] In another aspect the genome may be heterozygous at one or more of the loci, such as heterozygous for DNA encoding a chimaeric antibody chain and native (host cell) antibody chain. In one aspect the genome may be heterozygous for DNA capable of encoding 2 different antibody heavy chains encoded by immunoglobulin transgenes of the invention, for example, comprising 2 different chimaeric heavy chains and a human lambda light chain.

[0266] In one aspect the invention relates to a non-human vertebrate or cell, and methods for producing said vertebrate or cell, as described herein, wherein the inserted human DNA, such as the human IgH VDJ region and/or light chain V, J regions are found on only one allele and not both alleles in the mammal or cell. In this aspect a mammal or cell has the potential to express both an endogenous host antibody heavy or light chain and a chimaeric heavy or light chain.

[0267] In one embodiment in any configuration of the invention, the genome has been modified to prevent or reduce the expression of fully-endogenous antibody. Examples of suitable techniques for doing this can be found in WO2011004192, U.S. Pat. No. 7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364, WO2009/076464, EP1399559 and U.S. Pat. No. 6,586,251, the disclosures of which are incorporated herein by reference. In one embodiment, the non-human vertebrate VDJ region of the endogenous heavy chain immunoglobulin locus, and optionally VJ region of the endogenous light chain immunoglobulin loci (lambda and/or kappa loci), have been inactivated. For example, all or part of the non-human vertebrate VDJ region is inactivated by inversion in the endogenous heavy chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus. For example, all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous kappa chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus. For example, all or part of the non-human vertebrate VJ region is inactivated by inversion in the endogenous lambda chain immunoglobulin locus of the mammal, optionally with the inverted region being moved upstream or downstream of the endogenous Ig locus. In one embodiment the endogenous heavy chain locus is inactivated in this way as is one or both of the endogenous kappa and lambda loci.

[0268] Additionally or alternatively, the vertebrate has been generated in a genetic background which prevents the production of mature host B and T lymphocytes, optionally a RAG-1-deficient and/or RAG-2 deficient background. See U.S. Pat. No. 5,859,301 for techniques of generating RAG-1 deficient animals.

[0269] In one embodiment in any configuration of the invention, the human V, J and optional D regions are provided by all or part of the human IgH locus; optionally wherein said all or part of the IgH locus includes substantially the full human repertoire of IgH V, D and J regions and intervening sequences. A suitable part of the human IgH locus is disclosed in WO2011004192. In one embodiment, the human IgH part includes (or optionally consists of) bases 105,400,051 to 106,368,585 from human chromosome 14 (coordinates from NCB136). Additionally or alternatively, optionally wherein the vertebrate is a mouse or the cell is a mouse cell, the human V, J and optional D regions are inserted into mouse chromosome 12 at a position corresponding to a position between coordinates 114,667,091 and 114,665,190, optionally at coordinate 114,667,091 (coordinates from NCBIM37, relating to mouse strain C57BL/6J).

[0270] In one embodiment of any configuration of the vertebrate or vertebrate cell of the invention when the vertebrate is a mouse, (i) the constant region comprises a mouse or rat S.mu. switch and optionally a mouse C.mu. region. For example the constant region is provided by the constant region endogenous to the mouse, eg, by inserting human V(D)J region sequences into operable linkage with the endogenous constant region of a mouse genome or mouse cell genome.

[0271] In one embodiment of any configuration of the vertebrate or vertebrate cell of the invention when the vertebrate is a rat, (i) the constant region comprises a mouse or rat S.mu. switch and optionally a rat C.mu. region. For example the constant region is provided by the constant region endogenous to the rat, eg, by inserting human V(D)J region sequences into operable linkage with the endogenous constant region of a rat genome or rat cell genome.

[0272] In one embodiment of any configuration of the vertebrate or vertebrate cell of the invention the genome comprises an antibody light chain transgene which comprises all or part of the human Ig.lamda. locus including at least one human JA region and at least one human CA region, optionally C.sub..lamda.6 and/or C.sub..lamda.7. Optionally, the transgene comprises a plurality of human JA regions, optionally two or more of J.sub..lamda.1, J.sub..lamda.2, J.sub..lamda.6 and J.sub..lamda.7, optionally all of J.sub..lamda.1, J.sub..lamda.2, and J.sub..lamda.7. The human lambda immunoglobulin locus comprises a unique gene architecture composed of serial J-C clusters. In order to take advantage of this feature, the invention in optional aspects employs one or more such human J-C clusters inoperable linkage with the constant region in the transgene, eg, where the constant region is endogenous to the non-human vertebrate or non-human vertebrate cell. Thus, optionally the transgene comprises at least one human J.sub..lamda.-C.sub..lamda. cluster, optionally at least J.sub..lamda.7-C.sub..lamda.7. The construction of such transgenes is facilitated by being able to use all or part of the human lambda locus such that the transgene comprises one or more J-C clusters in germline configuration, advantageously also including intervening sequences between clusters and/or between adjacent J and C regions in the human locus. This preserves any regulatory elements within the intervening sequences which may be involved in VJ and/or JC recombination and which may be recognised by AID (activation-induced deaminase) or AID homologues.

[0273] Where endogenous regulatory elements are involved in CSR (class-switch recombination) in the non-human vertebrate, these can be preserved by including in the transgene a constant region that is endogenous to the non-human vertebrate. In the invention, one can match this by using an AID or AID homologue that is endogenous to the vertebrate or a functional mutant thereof. Such design elements are advantageous for maximising the enzymatic spectrum for SHM (somatic hypermutation) and/or CSR and thus for maximising the potential for antibody diversity.

[0274] Optionally, the lambda transgene comprises a human EX enhancer. Optionally, the kappa transgene comprises a human E.kappa. enhancer. Optionally, the heavy chain transgene comprises a heavy chain human enhancer.

[0275] In one embodiment of any configuration of the invention the constant region is endogenous to the non-human vertebrate or derived from such a constant region. For example, the vertebrate is a mouse or the cell is a mouse cell and the constant region is endogenous to the mouse. For example, the vertebrate is a rat or the cell is a rat cell and the constant region is endogenous to the rat.

[0276] In one embodiment of any configuration of the invention the heavy chain transgene comprises a plurality human IgH V regions, a plurality of human D regions and a plurality of human J regions, optionally substantially the full human repertoire of IgH V, D and J regions.

[0277] In one embodiment of any configuration of the invention, the vertebrate or cell comprises a heavy chain further transgene, the further transgene comprising at least one human IgH V region, at least one human D region and at least one human J region, optionally substantially the full human repertoire of IgH V, D and J regions.

[0278] In one embodiment of any configuration of the invention,

[0279] (i) the heavy chain transgene comprises at least one human IgH V region, at least one human J region, and optionally at least one human D region; and

[0280] (ii) the vertebrate or cell comprises a kappa transgene, the kappa transgene comprising at least one human Ig.kappa. V region and at least one human J region.

[0281] In one embodiment of any configuration of the invention,

[0282] (i) the heavy chain transgene comprises at least one human IgH V region, at least one human J region, and optionally at least one human D region; and

[0283] (ii) the vertebrate or cell comprises a lambda transgene, the lambda transgene comprising at least one human Ig.lamda. V region and at least one human J region.

[0284] In one embodiment of any configuration of the invention,

[0285] (i) the heavy chain transgene comprises substantially the full human repertoire of IgH V, D and J regions; and

[0286] (ii) the vertebrate or cell comprises substantially the full human repertoire of Ig.kappa. V and J regions and/or substantially the full human repertoire of Ig.lamda. V and J regions.

[0287] An aspect provides a B-cell, hybridoma or a stem cell, optionally an embryonic stem cell or haematopoietic stem cell, according to any configuration of the invention. In one embodiment, the cell is a BALB/c, JM8 or AB2.1 or AB2.2 embryonic stem cell (see discussion of suitable cells, and in particular JM8 and AB2.1 cells, in WO2011004192, which disclosure is incorporated herein by reference). In one aspect the ES cell is derived from the mouse BALB/c, C57BL/6N, C57BL/6J, 12955 or 129Sv strain.

[0288] An aspect provides a method of isolating an antibody or nucleotide sequence encoding said antibody, the method comprising

[0289] (a) immunising (see e.g. Harlow, E. & Lane, D. 1998, 5.sup.th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259) a vertebrate according to any configuration or aspect of the invention with an antigen such that the vertebrate produces antibodies; and

[0290] (b) isolating from the vertebrate an antibody that specifically binds to said antigen and/or a nucleotide sequence encoding at least the heavy and/or the light chain variable regions of said antibody;

[0291] optionally wherein the variable regions of said antibody are subsequently joined to a human constant region. Such joining can be effected by techniques readily available in the art, such as using conventional recombinant DNA and RNA technology as will be apparent to the skilled person. See e.g. Sambrook, J and Russell, D. (2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).

[0292] Suitably an immunogenic amount of the antigen is delivered. The invention also relates to a method for detecting a target antigen comprising detecting an antibody produced as above with a secondary detection agent which recognises a portion of that antibody.

[0293] Isolation of the antibody in step (b) can be carried out using conventional antibody selection techniques, eg, panning for antibodies against antigen that has been immobilised on a solid support, optionally with iterative rounds at increasing stringency, as will be readily apparent to the skilled person.

[0294] As a further optional step, after step (b) the amino acid sequence of the heavy and/or the light chain variable regions of the antibody are mutated to improve affinity for binding to said antigen.

[0295] Mutation can be generated by conventional techniques as will be readily apparent to the skilled person, eg, by error-prone PCR. Affinity can be determined by conventional techniques as will be readily apparent to the skilled person, eg, by surface plasmon resonance, eg, using Biacore.TM..

[0296] Additionally or alternatively, as a further optional step, after step (b) the amino acid sequence of the heavy and/or the light chain variable regions of the antibody are mutated to improve one or more biophysical characteristics of the antibody, eg, one or more of melting temperature, solution state (monomer or dimer), stability and expression (eg, in CHO or E coli).

[0297] An aspect provides an antibody produced by the method of the invention, optionally for use in medicine, eg, for treating and/or preventing a medical condition or disease in a patient, eg, a human.

[0298] An aspect provides a nucleotide sequence encoding the antibody of the invention, optionally wherein the nucleotide sequence is part of a vector. Suitable vectors will be readily apparent to the skilled person, eg, a conventional antibody expression vector comprising the nucleotide sequence together in operable linkage with one or more expression control elements.

[0299] An aspect provides a pharmaceutical composition comprising the antibody of the invention and a diluent, excipient or carrier, optionally wherein the composition is contained in an IV container (eg, and IV bag) or a container connected to an IV syringe.

[0300] An aspect provides the use of the antibody of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition in a patient, eg a human.

[0301] In a further aspect the invention relates to a method for producing an antibody specific to a desired antigen the method comprising immunizing a transgenic non-human vertebrate as above with a predetermined antigen and recovering a chimaeric antibody (see e.g. Harlow, E. & Lane, D. 1998, 5.sup.th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259). Suitably an immunogenic amount of the antigen is delivered. The invention also relates to a method for detecting a target antigen comprising detecting an antibody produced as above with a secondary detection agent which recognises a portion of that antibody.

[0302] In a further aspect the invention relates to a method for producing a fully humanised antibody comprising immunizing a transgenic non-human vertebrate as above with a predetermined antigen, recovering a chimaeric antibody or cells expressing the antibody, and then replacing the non-human vertebrate constant region with a human constant region. This can be done by standard cloning techniques at the DNA level to replace the non-human vertebrate constant region with an appropriate human constant region DNA sequence--see e.g. Sambrook, J and Russell, D. (2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).

[0303] In a further aspect the invention relates to humanised antibodies and antibody chains produced according to the present invention, both in chimaeric and fully humanised form, and use of said antibodies in medicine. The invention also relates to a pharmaceutical composition comprising such an antibody and a pharmaceutically acceptable carrier or other excipient.

[0304] Antibody chains containing human sequences, such as chimaeric human-non-human antibody chains, are considered humanised herein by virtue of the presence of the human protein coding regions region. Fully humanised antibodies may be produced starting from DNA encoding a chimaeric antibody chain of the invention using standard techniques.

[0305] Methods for the generation of both monoclonal and polyclonal antibodies are well known in the art, and the present invention relates to both polyclonal and monoclonal antibodies of chimaeric or fully humanised antibodies produced in response to antigen challenge in non-human vertebrates of the present invention.

[0306] In a yet further aspect, chimaeric antibodies or antibody chains generated in the present invention may be manipulated, suitably at the DNA level, to generate molecules with antibody-like properties or structure, such as a human variable region from a heavy chain absent a constant region, for example a domain antibody; or a human variable region with any constant region from either heavy or light chain from the same or different species; or a human variable region with a non-naturally occurring constant region; or human variable region together with any other fusion partner. The invention relates to all such chimaeric antibody derivatives derived from chimaeric antibodies identified according to the present invention.

[0307] In an example, the genome of the cell or non-human vertebrate of the invention encodes an antibody comprising an antibody chain having a human heavy chain variable region upstream of a mouse light chain constant region in combination with one of:

[0308] a fully human antibody light chain;

[0309] a non-human vertebrate (e.g., mouse or rat) antibody light chain;

[0310] a chimaeric non-human vertebrate (e.g., mouse or rat)--human antibody chain; or an antibody chain having a human heavy chain variable region upstream of a non-human vertebrate (e.g., mouse or rat) light chain constant region;

[0311] In a further aspect, the invention relates to use of non-human vertebrates of the present invention in the analysis of the likely effects of drugs and vaccines in the context of a quasi-human antibody repertoire.

[0312] The invention also relates to a method for identification or validation of a drug or vaccine, the method comprising delivering the vaccine or drug to a vertebrate of the invention and monitoring one or more of: the immune response, the safety profile; the effect on disease.

[0313] The invention also relates to a kit comprising an antibody or antibody derivative as disclosed herein and either instructions for use of such antibody or a suitable laboratory reagent, such as a buffer, antibody detection reagent.

[0314] The invention also relates to a method for making an antibody, or part thereof, the method comprising providing:

[0315] (i) a nucleic acid encoding an antibody, or a part thereof, obtained according to the present invention; or

[0316] (ii) sequence information from which a nucleic acid encoding an antibody obtained according to the present invention, or part thereof, can be expressed to allow an antibody to be produced.

[0317] In an embodiment, the invention provides

[0318] The non-human vertebrate, mouse, rat, cell or method of any preceding configuration, wherein the genome comprises

[0319] (a) said antibody heavy chain transgene; and

[0320] (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;

[0321] 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.

[0322] In an embodiment, the the kappa and lambda chain transgenes comprise constant regions of said non-human vertebrate species capable of pairing with the constant region of the heavy chain.

[0323] In an embodiment, the heavy chain transgene comprises a substantially complete human functional VH, D and JH repertoire.

[0324] In an embodiment, the kappa chain transgene comprises a substantially complete human functional V.kappa. and J.kappa. repertoire; and the lambda chain transgene comprises a substantially complete human functional VA and JA repertoire.

[0325] In a ninth embodiment, the invention provides

[0326] A transgenic mouse or rat, or a transgenic mouse or rat cell (eg, an ES cell), whose genome comprises

[0327] (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;

[0328] (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene; [0329] wherein all of the V, D and J in said transgenes are human V, D and J; [0330] wherein the kappa chain transgene comprises a substantially complete human functional V.kappa. and J.kappa. repertoire; and the lambda chain transgene comprises a substantially complete human functional VA and JA repertoire; [0331] wherein the kappa and/or lambda chain transgenes comprise endogenous constant regions capable of pairing with the constant region of the heavy chain;

[0332] (c) a human VpreB gene capable of expressing a human VpreB; [0333] optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter; and

[0334] (d) an endogenous .lamda.5 gene capable of expressing an endogenous .lamda.5; [0335] wherein endogenous antibody heavy and light chain expression has been inactivated; and [0336] optionally wherein said genome is homozygous for said heavy and light chain transgenes.

[0337] In a tenth embodiment, the invention provides

[0338] A transgenic mouse or rat, or a transgenic mouse or rat cell (eg, an ES cell), whose genome comprises

[0339] (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; [0340] wherein all of the V, D and J in said transgene are human V, D and J; [0341] wherein the heavy chain transgene is devoid of a CH1 gene segment and the genome of the vertebrate comprises no functional antibody light chain locus; [0342] wherein endogenous antibody heavy chain expression has been inactivated; and [0343] optionally wherein said genome is homozygous for said heavy chain transgene;

[0344] (c) a human VpreB gene capable of expressing a human VpreB; [0345] optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter; and

[0346] (d) an endogenous .lamda.5 gene capable of expressing an endogenous .lamda.5.

[0347] Insertion of Human VpreB Gene

[0348] As described above, precise insertion of exogenous DNA (such as human VpreB or antibody gene segment DNA) can be effected by homolgous recombination, RMCE or other techniques that will be apparent to the skilled person. Vector manipulation can be effected using recombineering as is well known.

[0349] The human VpreB gene is found in human cells within the human antibody VA gene segment cluster (in the nucleotide region between human Ig.lamda. V gene segments IV-53 and 5-52 (the nucleotide region being positions 22599200 to 22599926 on human chromosome 22)). Thus, in one embodiment, a nucleotide sequence found between human Ig.lamda. V gene segments IV-53 and 5-52, eg, positions 22599200 to 22599926 on human chromosome 22, is inserted into the genome of the cell or vertebrate, wherein the nucleotide sequence comprises a human VpreB gene and optionally the associated human promoter. For example, the nucleotide sequence is inserted in the endogenous lambda antibody locus upstream of the endogenous lambda constant region. In one embodiment, a region of the human VA gene segment cluster comprising at least one (or substantially all) human VA gene segments and the human VpreB and promoter is inserted upstream of the endogenous (eg, mouse or rat) CA. In one example, the insertion is between the most 3' endogenous JA and the endogenous CA. In another embodiment, the insertion is in the endogenous VA cluster, optionally replacing the VA in whole or in part. In one embodiment, all of the human lambda VA gene segment cluster (including introns) is inserted into the genome so that it replaces the enogenous VA gene segment cluster. In one embodiment, the cell or vertebrate is a mouse or rat, so "endogenous" refers to the regions found in the genome of a mouse or rat.

[0350] Expression of the .lamda.5 and VpreB genes is B-cell lineage restricted and is also subject to stage-specific regulation during B-cell development. Binding sites for the transcription factors EBF, E47, Pax-5 and Ikaros are present in the the .lamda.5 and VpreB promoters. The mouse VpreB1 and .lamda.5 genes are both located in the .lamda.5--VpreB1 locus and 4 kb away from each other. A locus control region (LCR) located within the .lamda.5--VpreB1 locus provides the regulatory elements for these genes are. Hence, the region around the two genes are important for the transcription of the VpreB1 and .lamda.5 genes. Furthermore, these two genes are subject to tight stage-specific control during B-cell development. For activation at the pro-B cell stage, early B cell factor (EBF) initiates the activation of the .lamda.5--VpreB1 locus. It has been observed that the EBF with some other factors (E2As) bind the promoters of the two genes. The transcription of these two genes has to be silenced during the transition from the pre-B to the immature B cell stage, in order for light chain rearrangement and binding with heavy chain. Ikaros gene acts as a repressor by competing with EBF for binding to the same sites in the promoters of the VpreB1 and .lamda.5 genes. In summary, the .lamda.5--VpreB1 locus seems to be important for the temporally-controlled activation and inactivation of the surrogate light chain during B-cell development. See:-- [0351] Mol Cell Biol. 1999 January; 19(1):671-9; Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the lambda5-VpreB1 locus control region; Sabbattini P, Georgiou A, Sinclair C, Dillon N. [0352] Seminars in Immunology, 2005 April; 17(2):121-7; The lambda5-VpreB1 locus--a model system for studying gene regulation during early B cell development; Sabbattini P, Dillon N.

[0353] Thus, in one embodiment the invention provides a non-human vertebrate, mouse, rat, cell or method according to any configuration, aspect, embodiment or example, wherein the expression of the human VpreB gene is under endogenous control. For example, the vertebrate is a mouse or rat or the cell is a mouse or rat cell (eg, an ES cell) and the human VpreB is under the control of the gene expression control elements of the mouse or rat (cell). This can be achieved, in one example, by replacing an endogenous VpreB (eg, VpreB1) gene with a human VpreB gene at the location where the former usually resides in the genome. Precise gene replacement in genomes can be carried out by homolgous recombination or RMCE as herein described.

[0354] Mouse VpreB1 and VpreB2 are found at the following position in a mouse genome:--

[0355] Mouse VpreB1: chromosome 16: 16868494 to 16869348

[0356] Mouse VpreB2: chromosome 16: 17980658 to Ser. No. 17/981,173

[0357] Thus, the invention provides a non-human vertebrate, mouse, rat, cell or method according to any configuration, aspect, embodiment or example, wherein the human VpreB gene is operably linked to an endogenous promoter, optionally an endogenous VpreB promoter (eg, a VpreB1 or VpreB2 promoter). Thus, where the vertebrate (or cell) is a mouse or rat (cell), the human VpreB is operably linked to a VpreB1 or VpreB2 promoter of said mouse or rat (cell). Operable linkage enables the promoter to control expression of the human gene. It is desirable to use endogenous gene expression control (eg, using an endogenous promoter) for human VpreB expression since this will harness the endogenous (eg, mouse or rat) temporal expression control for turning on and off the human VpreB gene (and coordinating this with .lamda.5 expression) during the various stages of B-cell development, particularly up to the point where light chain pairing with heavy chains occurs in immature B-cells. Thereafter, the usual switching off of SLC-component gene expression can be controlled properly by the endogenous control mechanisms when the pre-BCR is no longer participating in B-cell maturation.

[0358] Thus, the invention provides a non-human vertebrate, mouse, rat, cell or method according to any configuration, aspect, embodiment or example, wherein the human VpreB gene is operably linked to an endogenous VpreB-.lamda.5 locus control region (LCR). Thus, where the vertebrate (or cell) is a mouse or rat (cell), the human VpreB is operably linked to a VpreB-.lamda.5 LCR of said mouse or rat (cell). LCRs are dominant activating sequences that are able to activate gene expression at any location in the genome. Optionally, the LCR is present at the wild-type position in the genome of the vertebrate or cell. In another example, the LCR is not at the wild-type position in the genome of the vertebrate or cell. In this case, therefore, the human VpreB is positioned away from the endogenous VpreB gene in the genome, which may be advantageous for controlling the human VpreB expression independently of the endogenous .lamda.5.

[0359] In an embodiment, the invention provides a non-human vertebrate, mouse, rat, cell or method according to any configuration, aspect, embodiment or example, wherein the human VpreB gene is operably linked to an endogenous VpreB locus control region (LCR). Thus, where the vertebrate (or cell) is a mouse or rat (cell), the human VpreB is operably linked to a VpreB LCR of said mouse or rat (cell). Optionally, the LCR is present at the wild-type position in the genome of the vertebrate or cell. In another example, the LCR is not at the wild-type position in the genome of the vertebrate or cell. In this case, therefore, the human VpreB is positioned away from the endogenous VpreB gene in the genome, which may be advantageous for controlling the human VpreB expression independently of the endogenous .lamda.5.

[0360] Optionally one or more DNase I hypersensitive sites (HS) is operably linked in the genome to the human VpreB gene. For example, the HS forms part of the LCR or is 3' of the LCR or 3' of the LCR-human VpreB (see, Sabbattini et al 1999, supra).

[0361] Optionally in any configuration, aspect, embodiment or example of the inventions, the human VpreB gene is present in multiple copies in the genome, eg, the genome comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the human VpreB gene. For example, 2, 3, 4 or 5 copies are operably linked to the control element (eg, promoter and/or LCR) mentioned above.

[0362] Assessing the Development of the B-Cell Repertoire

[0363] Some assays can be used to investigate if the invention leads to promotion of B-cell development and repertoire. Although described in the context of transgenic mice below, these comments apply equally to other transgenic non-human vertebrates of the invention.

[0364] 1. In chimaeric mouse, after introducing the human DNA fragment covering several human Vs, all Ds and Js between mouse Js and mouse Cs, the usage of the inserted human Vs can be tested by sequencing the mRNA products of the IgH locus in chimaeric mouse. In this case, human Vs will compete with endogenous mouse Vs for formation of the heavy chain. The usage of human Vs vs mouse Vs will be compared in mice with and without introduction of human VpreB gene into the chimaeric mouse with/without the endogenous mouse VpreB gene. More detail is provided in the non-limiting example below.

[0365] 2. The use of human VpreB or mouse VpreB protein for pre-BCR assembly can be analysed in pre-B cell populations from mice with or without the human VpreB. Surrogate light chain can only be detected in the transitional pre-BI and large pre-BII stages in bone marrow. To enrich the cell populations in bone marrow for investigating pre-BCR, the deregulation of surrogate light chain can be blocked by inactivation of SLP65 gene (Nature Immunol. 2003 4, 38-43; The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Flemming, A., Brummer, T., Reth, M. & Jumaa, H.) or both IRF4 (interferon-regulatory factor 4) and IRF8 genes (Genes Dev. 2003 17, 1703-1708; IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development. Lu, R., Medina, K. L., Lancki, D. W. & Singh, H). It has been reported that pre-BII cells from mice that are deficient in these factors continue to express surrogate-light-chain genes and therefore retain pre-BCR expression. The unlimited supply of surrogate light chains lead to a hyperplastic pre-Ill-cell phenotype in vivo and provide large number of pre-B cells for us to analyze.

[0366] 3. In instances (eg, as described in part 1 above), where mouse Vs are retained following insertion of human gene segments, the mouse endogenous heavy chain VDJ is inactivated, and this may be done while leaving mouse Vs intact. Thus, for the formation of chimaeric heavy chain genes, the heavy chain V regions should only be transcribed from human heavy VDJs. To assess the relative use of human v mouse Vs for heavy chain gene formation, the B-cell repertoire and diversity will be compared between mice having human VpreB gene v mice lacking the human VpreB (eg, mice with mouse VpreB only). Pairing of human Vs with human VpreB will be preferable to pairing of mouse Vs with human VpreB, and thus antibody heavy chains comprising human variable regions will be selected during B-cell development.

[0367] As cells transit from the pre-BI to pre-BII stage, the pre-BCR induces a signal for proliferative expansion. Pre-BCR controls the number of developing pre-B cells by controlling proliferation, which represents clonal expansion. Pre-BII cells enter two to seven rounds of cell division when Ig.mu. chain assembles into pre-BCRs. This ultimately results in a more diverse antibody repertoire, as later in development, each cell will recombine and express a unique L chain. In this process, the association of surrogate light chain with .mu. chain monitors the cells to determine whether it has successfully completed VDJH recombination and expresses a functional .mu.H chain which will form the pre-PCR for downstream signalling. In this case, the strength of the pairing between the .mu.H and SL chains may determine the amplitude of the transmitted signal, and thereby, the extent of proliferation (J. Immunol. 2006, 177 pp. 2242-2249; Pre-B cell receptor assesses the quality of IgH chains and tunes the Pre-B cell repertoire by delivering differential signals. Y. Kawano, S. Yoshikawa, Y. Minegishi and H. Karasuyama). Therefore, in the mouse with chimaeric heavy chain (human VDJs and mouse Cs)--by introducing the human VpreB gene to provide complete species-matching with the chimaeric heavy chain, It is expected that the repertoire and so the diversity of chimaeric antibodies will be improved by using human VpreB instead of mouse VpreB for association with the heavy chains. The improvement for B cell repertoire and diversity can be showed by increased usage of human heavy VDJs and/or a shift in VH gene family usage in mouse expressing human VpreB (see the examples below for possible techniques).

[0368] The invention provides the following aspects:-- [0369] 1. A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene, [0370] the transgene comprising [0371] (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 [0372] (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; [0373] the genome further comprising [0374] (i) a human VpreB gene capable of expressing a human VpreB, and [0375] (ii) a non-human vertebrate (eg, mouse or rat) .lamda.5 gene; [0376] wherein the vertebrate or cell is capable of expressing a chimaeric surrogate light chain comprising human VpreB and non-human vertebrate .lamda.5 for pairing with the heavy chain. [0377] 2. The vertebrate or cell of aspect 1, wherein the constant region is a non-human vertebrate (eg, mouse or rat) constant region so that the chimaeric surrogate light chain is species- or strain-matched with the heavy chain. [0378] 3. A mouse or mouse cell according to aspect 1 or 2, wherein the constant region and the .lamda.5 gene are mouse constant region and mouse .lamda.5 gene, optionally of the same mouse strain. [0379] 4. The vertebrate or cell of aspect 2 or 3, wherein the strain is a 129-derived, mouse black 6-derived or JM8-derived strain (eg, a C57BL/6--129/Sv hybrid strain). [0380] 5. The vertebrate or cell of aspect 2 or 3, wherein the constant region is a mouse 129 or mouse

[0381] Black 6 constant region and the .lamda.5 gene is a mouse 129 or mouse Black 6 .lamda.5 gene. [0382] 6. The vertebrate or cell of any preceding aspect, wherein said constant region gene and .lamda.5 gene are endogenous genes of said vertebrate or cell. [0383] 7. The vertebrate or cell of any preceding aspect, wherein said genome is homozygous for said transgene, human VpreB gene and non-human vertebrate .lamda.5 gene. [0384] 8. The vertebrate or cell of any preceding aspect, wherein endogenous non-human vertebrate antibody heavy chain expression has been inactivated. [0385] 9. The vertebrate or cell of any preceding aspect, wherein endogenous non-human vertebrate antibody light chain expression has been inactivated. [0386] 10. The vertebrate or cell of any preceding aspect, wherein the heavy chain transgene is devoid of a

[0387] CH1 gene segment and the genome comprises no functional antibody light chain locus. [0388] 11. The vertebrate or cell of any preceding aspect, wherein the heavy chain transgene is devoid of a gamma CH1 gene segment and the genome comprises no functional antibody light chain locus. [0389] 12. The vertebrate or cell of any preceding aspect, wherein the heavy chain transgene is devoid of a mu CH1 gene segment and the genome comprises no functional antibody light chain locus. [0390] 13. The vertebrate or cell of any preceding aspect, wherein said constant region is a mu constant region, optionally endogenous mu constant region. [0391] 14. The vertebrate or cell of any preceding aspect, wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1. [0392] 15. The vertebrate or cell of any preceding aspect, wherein said genome does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene. [0393] 16. The cell of any preceding aspect, wherein the cell is an ES cell, an iPS cell, a hybridoma, an immortalised cell or a B-cell (eg, an immortalised B-cell). [0394] 17. The cell of aspect 16, wherein the cell is derived from C57BL/6, M129 (eg, 129/SV), BALB/c or a hybrid of C57BL/6, M129 or BALB/c (eg, a C57BL/6--129/Sv hybrid). [0395] 18. The vertebrate or cell of any preceding aspect, wherein the genome comprises an insertion of a human lambda V (or VJ) region comprising all of the V (and optionally also J) regions and intervening sequences, wherein the lambda V (or VJ) region comprises a human VpreB gene. [0396] 19. The vertebrate or cell of aspect 18, wherein non-functional V and/or J gene segments are omitted. [0397] 20. The vertebrate or cell of aspect 18 or 19, wherein the lambda V (or VJ) region comprises a human VpreB gene and its associated human promoter. [0398] 21. The vertebrate or cell of any preceding aspect, wherein the genome comprises an insertion of DNA corresponding to positions 22599200 to 22599926 on human chromosome 22. [0399] 22. The vertebrate or cell of any one of aspects 18 to 21, wherein the insertion is an insertion into an antibody light chain locus; optionally an insertion into endogenous lambda locus upstream of the endogenous lambda constant region or an insertion into endogenous kappa locus upstream of the endogenous kappa constant region. [0400] 23. The vertebrate or cell of any one of aspects 18 to 22, wherein the insertion replaces the endogenous VA in whole or in part. [0401] 24. The vertebrate or cell of any preceding aspect, wherein the human VpreB gene is not within the endogenous (non-human vertebrate or cell) VpreB-.lamda.5 locus. [0402] 25. The vertebrate or cell of any preceding aspect, wherein the expression of the human VpreB gene is under endogenous (non-human vertebrate or cell) control. [0403] 26. The vertebrate or cell of any preceding aspect, wherein the human VpreB gene is operably linked to one or more DNase I hypersensitive sites. [0404] 27. The vertebrate or cell of any preceding aspect, wherein the human VpreB gene is present in the genome in 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies. [0405] 28. The vertebrate or cell of any preceding aspect, which is a rodent, mouse or rat; or a rodent, mouse or rat cell. [0406] 29. The vertebrate or cell of any preceding aspect, wherein all .lamda.5 sequences in the genome are non-human vertebrate .lamda.5 sequences. [0407] 30. The vertebrate or cell of any preceding aspect, wherein the genome is devoid of a human .lamda.5 nucleotide sequence. [0408] 31. A non-human vertebrate (eg, a mouse or rat) or cell (eg, a mouse cell or rat cell) whose genome comprises an antibody heavy chain transgene for producing heavy chains that are devoid of a CH1, [0409] the transgene comprising [0410] (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 [0411] (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; [0412] the genome further comprising a human VpreB gene capable of expressing a human VpreB; [0413] wherein the constant region is devoid of a functional CH1 gene; and [0414] wherein the vertebrate or cell is capable of expressing a human VpreB for pairing with the heavy chains devoid of CH1. [0415] 32. The vertebrate or cell of aspect 31, wherein the human VpreB gene does not comprise a .lamda.5 sequence or constant region sequence. [0416] 33. The vertebrate or cell of aspect 31 or 32, wherein the constant region is a human constant region. [0417] 34. The vertebrate or cell of aspect 31 or 32, wherein the constant region is a non-human vertebrate (eg, mouse or rat) constant region; optionally of a strain as recited in aspect 4. [0418] 35. The vertebrate or cell of any one of aspects 31 to 34, wherein said genome is homozygous for said transgene and human VpreB gene. [0419] 36. The vertebrate or cell of any one of aspects 31 to 35, wherein endogenous non-human vertebrate antibody heavy chain expression has been inactivated. [0420] 37. The vertebrate or cell of any one of aspects 31 to 36, wherein endogenous non-human vertebrate antibody light chain expression has been inactivated. [0421] 38. The vertebrate or cell of any one of aspects 31 to 37, wherein the genome comprises no functional antibody light chain locus. [0422] 39. The vertebrate or cell of any one of aspects 31 to 38, wherein the heavy chain transgene is devoid of a gamma CH1 gene segment and the genome comprises no functional antibody light chain locus. [0423] 40. The vertebrate or cell of any one of aspects 31 to 39, wherein the heavy chain transgene is devoid of a mu CH1 gene segment and the genome comprises no functional antibody light chain locus. [0424] 41. The vertebrate or cell of any aspects 31 to 40, wherein said constant region is a mu constant region (optionally endogenous mu constant region) or a gamma constant region (optionally endogenous gamma constant region). [0425] 42. The vertebrate or cell of any one of aspects 31 to 41, wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO: 1. [0426] 43. The vertebrate or cell of any one of aspects 31 to 42, wherein said genome does not comprise a non-human species VpreB gene. [0427] 44. The cell of any one of aspects 31 to 43, wherein the cell is an ES cell, an iPS cell, a hybridoma, an immortalised cell or a B-cell (eg, an immortalised B-cell). [0428] 45. The cell of aspect 44, wherein the cell is derived from C57BL/6, M129 (eg, 129/SV), BALB/c or a hybrid of C57BL/6, M129 or BALB/c (eg, a C57BL/6--129/Sv hybrid). [0429] 46. The vertebrate or cell of any one of aspects 31 to 45, wherein the genome comprises an insertion of a human lambda V (or VJ) region comprising all of the V (and optionally also J) regions and intervening sequences, wherein the lambda V (or VJ) region comprises a human VpreB gene. [0430] 47. The vertebrate or cell of aspect 46, wherein non-functional V and/or J gene segments are omitted. [0431] 48. The vertebrate or cell of aspect 46 or 47, wherein the lambda V (or VJ) region comprises a human VpreB gene and its associated human promoter. [0432] 49. The vertebrate or cell of any one of aspects 31 to 48, wherein the genome comprises an insertion of DNA corresponding to positions 22599200 to 22599926 on human chromosome 22. [0433] 50. The vertebrate or cell of any one of aspects 46 to 49, wherein the insertion is an insertion into an antibody light chain locus; optionally an endogenous lambda locus upstream of the endogenous lambda constant region. [0434] 51. The vertebrate or cell of any one of aspects 46 to 50, wherein the insertion replaces the endogenous VA in whole or in part. [0435] 52. The vertebrate or cell of any one of aspects 31 to 51, wherein the human VpreB gene is not within the endogenous (non-human vertebrate or cell) VpreB-.lamda.5 locus. [0436] 53. The vertebrate or cell of any one of aspects 31 to 52, wherein the expression of the human VpreB gene is under endogenous (non-human vertebrate or cell) control. [0437] 54. The vertebrate or cell of any one of aspects 31 to 53, wherein the human VpreB gene is operably linked to one or more DNase I hypersensitive sites. [0438] 55. The vertebrate or cell of any one of aspects 31 to 54, wherein the human VpreB gene is present in the genome in 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies. [0439] 56. The vertebrate or cell of any one of aspects 31 to 55, which is a rodent, mouse or rat; or a rodent, mouse or rat cell. [0440] 57. The vertebrate or cell of any one of aspects 31 to 56, wherein the genome is devoid of a human .lamda.5 gene. [0441] 58. The vertebrate or cell of any one of aspects 31 to 57, wherein the genome is devoid of a .lamda.5 gene. [0442] 59. A method of constructing a transgenic non-human vertebrate cell (eg, an ES cell, eg, a mouse or rat ES cell), the method comprising [0443] (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 (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 [0444] (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; [0445] and wherein the method further comprises introducing into the cell a human VpreB gene in the absence of a .lamda.5 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. [0446] 60. The method of aspect 59, wherein the genome of the product cell of the method is as recited in any one of aspects 1 to 58. [0447] 61. The method of aspect 59 or 60, further comprising making a progeny (progeny cell or vertebrate) of the product cell made according to aspect 59 or 60, wherein the progeny is homozygous for said heavy chain transgene and endogenous non-human vertebrate antibody expression has been inactivated. [0448] 62. The method of aspect 61, wherein the progeny cell is an ES cell, an iPS cell, a hybridoma, an immortalised cell or a B-cell (eg, an immortalised B-cell); optionally a cell derived from a strain or hybrid as recited in aspect 17. [0449] 63. The method of aspect 61, wherein the progeny vertebrate is a rodent, mouse or rat. [0450] 64. A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising [0451] (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; [0452] (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 [0453] (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. [0454] 65. The method of aspect 64, wherein in (a) the heavy chain transgene is constructed to be devoid of a CH1 gene segment and the genome of the third non-human vertebrate comprises no functional antibody light chain locus. [0455] 66. The method of aspect 64 or 65, wherein the genome of the third non-human vertebrate is as recited in any one of aspects 1 to 58. [0456] 67. The method of any one of aspects 64 to 65, wherein the genome of the third vertebrate does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene. [0457] 68. The method of any one of aspects 64 to 67, wherein the third non-human vertebrate expresses a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region is a constant region of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene; optionally wherein the .lamda.5 and constant region are an endogenous .lamda.5 and constant region of mouse or rat. [0458] 69. A transgenic mouse or rat according to the third vertebrate of any one of aspects 64 to 68, or a progeny thereof. [0459] 70. A method of promoting B-cell development in a non-human vertebrate (eg, a mouse or rat), the method comprising [0460] (i) inactivating endogenous heavy chain expression in said vertebrate; [0461] (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 (optionally an endogenous non-human vertebrate mu constant region); and [0462] (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;

[0463] Optionally wherein the genome is homozygous for said first transgene. [0464] 71. The method of aspect 70, wherein in (a) the heavy chain transgene is constructed to be devoid of a CH1 gene segment and the genome of the third non-human vertebrate comprises no functional antibody light chain locus. [0465] 72. The method of aspect 70 or 71, wherein the genome of the vertebrate is as recited in any one of aspects 1 to 58. [0466] 73. The method of any one of aspects 70 to 72, wherein the genome of the vertebrate does not comprise a non-human vertebrate (eg, mouse or rat) species VpreB1 and/or VpreB2 gene. [0467] 74. The method of any one of aspects 70 to 73, wherein the third vertebrate expresses a .lamda.5 of a non-human vertebrate (eg, mouse or rat) species and said constant region is a constant region of the same non-human vertebrate (eg, mouse or rat) species as the .lamda.5 gene; optionally wherein the .lamda.5 and constant region are an endogenous .lamda.5 and constant region of mouse or rat. [0468] 75. A transgenic mouse or rat obtained or obtainable by the method of any one of aspects 70 to 74, or a progeny thereof. [0469] 76. A non-human vertebrate, mouse, rat, cell or method according to any preceding aspect, wherein the expression of the human VpreB gene is under edogenous control. [0470] 77. The non-human vertebrate, mouse, rat, cell or method according to aspect 76, wherein the human VpreB gene is operably linked to an endogenous promoter, optionally an endogenous VpreB promoter (eg, a VpreB1 promoter). [0471] 78. The non-human vertebrate, mouse, rat, cell or method of any preceding aspect, wherein the genome comprises [0472] (a) said antibody heavy chain transgene; and [0473] (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene; [0474] wherein all of the V, D and J in said transgenes are human V, D and J; [0475] wherein endogenous antibody heavy and light chain expression has been inactivated; and [0476] optionally wherein said genome is homozygous for said heavy and light chain transgenes. [0477] 79. The non-human vertebrate, mouse, rat, cell or method of aspect 78, wherein the kappa and lambda chain transgenes comprise constant regions of said non-human vertebrate species capable of pairing with the constant region of the heavy chain. [0478] 80. The non-human vertebrate, mouse, rat, cell or method of aspect 78 or 79, wherein the heavy chain transgene comprises a substantially complete human functional VH, D and JH repertoire. [0479] 81. The non-human vertebrate, mouse, rat, cell or method of aspect 78, 79 or 80, wherein the kappa chain transgene comprises a substantially complete human functional V.kappa. and J.kappa. repertoire; and the lambda chain transgene comprises a substantially complete human functional VA and JA repertoire. [0480] 82. A transgenic mouse or rat, or a transgenic mouse or rat cell (eg, an ES cell), whose genome comprises [0481] (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; [0482] (b) an antibody kappa light chain transgene and/or an antibody lambda chain transgene; [0483] wherein all of the V, D and J in said transgenes are human V, D and J; [0484] wherein the kappa chain transgene comprises a substantially complete human functional V.kappa. and J.kappa. repertoire; and the lambda chain transgene comprises a substantially complete human functional VA and JA repertoire; [0485] wherein the kappa and/or lambda chain transgenes comprise endogenous constant regions capable of pairing with the constant region of the heavy chain; [0486] (c) a human VpreB gene capable of expressing a human VpreB; [0487] optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter or its associated human promoter; and [0488] (d) an endogenous .lamda.5 gene capable of expressing an endogenous .lamda.5; [0489] wherein endogenous antibody heavy and light chain expression has been inactivated; and [0490] optionally wherein said genome is homozygous for said heavy and light chain transgenes. [0491] 83. A transgenic mouse or rat, or a transgenic mouse or rat cell (eg, an ES cell), whose genome comprises [0492] (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; [0493] wherein all of the V, D and J in said transgene are human V, D and J; [0494] wherein the heavy chain transgene is devoid of a CH1 gene segment (eg, a gamma CH1) and the genome of the vertebrate comprises no functional antibody light chain locus; [0495] wherein endogenous antibody heavy chain expression has been inactivated; and [0496] optionally wherein said genome is homozygous for said heavy chain transgene; [0497] (c) a human VpreB gene capable of expressing a human VpreB; [0498] optionally wherein the human VpreB gene is operably linked to an endogenous VpreB promoter or its associated human promoter; and [0499] (d) optionally an endogenous .lamda.5 gene capable of expressing an endogenous .lamda.5. [0500] As demonstrated in Example below, the present invention is useful for influencing the repertoire of human heavy chain variable domains and variable region DNA and RNA sequences that can be produced in a non-human vertebrate. Thus, the invention provides:-- [0501] 84. A mouse according to any one of aspects 1 to 58 or 75 to 83, which expresses a repertoire of Ig heavy chain variable regions that significantly differs (eg, as indicated by a probability of less than 0.05 in a chi-squared test) from the heavy chain variable region repertoire of a control vertebrate in the proportion (eg, percentage) of use of heavy chain variable gene segments, wherein the control and said non-human vertebrate genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. [0502] In an example of any aspect, the mouse and control strains are 129 or 129-derived (eg, 129 crossed with C57BL/6). [0503] In an example of any aspect, each repertoire is a repertoire of heavy chain variable region RNA. Optionally, the variable regions are provided as part of IgH heavy chains or corresponding RNA. [0504] In an example of any aspect, each transgene comprises at least 11 human gene segments, all or substantially all human D gene segments and all or substantially all human JH gene segments. For example, each transgene comprises human D1-1, 2-2, 3-9, 3-10, 4-11, 5-12, 6-13, 1-14, 2-15, 3-16, 4-17, 5-18, 6-19, 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 1-26 and 7-27. For example, each transgene comprises human J1, J2, J3, J4, J5 and J6. For example, each transgene comprises (optionally in 5' to 3' order) VH3-13, 3-11, 3-9, 1-8, 3-7, 2-5, 7-4-1, 4-4, 1-3, 1-2 and 6-1. [0505] 85. A mouse according to any one of aspects 1 to 58 or 75 to 84, which expresses a repertoire of Ig heavy chain variable regions that significantly differs (eg, as indicated by a probability of less than 0.05 in a binomial distribution test) from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion (eg, percentage) of use of heavy chain JH gene segments, wherein the control and said non-human vertebrate genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. [0506] 86. A mouse according to any one of aspects 1 to 58 or 75 to 85, which expresses a repertoire of Ig heavy chain variable regions that significantly differs (eg, as indicated by a probability of less than 0.05 in a binomial distribution test) from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion (eg, percentage) of use of one, more or all heavy chain variable gene segments selected from VH6-1, VH1-3, VH3-7, VH1-8, VH3-9, VH3-11, JH1 and JH6, wherein the control and said non-human vertebrate genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB. [0507] 87. The mouse of any one of aspects 84 to 86, wherein use of one, more or all of VH6-1, VH1-3, VH3-9 and JH1 is higher in the repertoire of said mouse than in the repertoire of the control. [0508] 88. The mouse of any one of aspects 84 to 87, wherein use of one, more or all of VH3-7, VH1-8, VH3-11 and JH6 is lower in the repertoire of said mouse than in the repertoire of the control. [0509] In an example of any of aspects 84 onwards, the IgH heavy chain variable region repertoire is naive, ie, has not been preselected against a predetermined target antigen. [0510] In an example of any of aspects 84 onwards, the scope is not limited to a mouse, but the scope of the aspect relates to a non-human vertebrate (eg, a mouse or a rat). Aspects 84 onwards can in the alternative, therefore, be read with this scope and for possible inclusion in claims herein.

[0511] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0512] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps

[0513] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0514] Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

[0515] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0516] The present invention is described in more detail in the following non limiting prophetic exemplification (Examples 1-5). An additional example, Example 6, not prophetic, but was actually performed and yielded data whose results demonstrate the present invention.

[0517] EXAMPLES

Example 1: Introduction of Human DNA Gene Segments into ES Cells

[0518] By way of illustration, the following example is provided on the introduction of human antibody gene segment DNA into Cell culture of C57BL/6N-derived cell lines, such as the JM8 male ES cells. This will follow standard techniques. The JM8 ES cells have been shown to be competent in extensively contributing to somatic tissues and to the germline, and are being used for large mouse mutagenesis programs at the Sanger Institute such as EUCOMM and KOMP (Pettitt, S. J., Liang, Q., Rairdan, X. Y., Moran, J. L., Prosser, H. M., Beier, D. R., Lloyd, K. C., Bradley, A., and Skarnes, W. C. (2009). Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nature Methods.). JM8 ES cells (1.0.times.10.sup.7) will be electroporated (500 .mu.F, 230V; BioRad) with 10 .mu.g I-Scel linearized human BAC DNA. The transfectants will be selected with either Puromycin (3 .mu.g/ml) or G418 (150 .mu.g/ml). The selection will begin either 24 hours (with G418) or 48 hours (with Puromycin) post electroporation and proceed for 5 days. 10 .mu.g linearized human BAC DNA can yield up to 500 Puromycin or G418 resistant ES cell colonies. The antibiotic resistant ES cell colonies will be picked into 96-well cell culture plates for genotyping to identify the targeted clones.

[0519] Once targeted mouse ES cell clones are identified, they will be analyzed by array Comparative Genomic Hybridization (CGH) for total genome integrity (Chung, Y. J., Jonkers, J., Kitson, H., Fiegler, H., Humphray, S., Scott, C., Hunt, S., Yu, Y., Nishijima, I., Velds, A., et al. (2004). A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization. Genome research 14, 188-196. and Liang, Q., Conte, N., Skarnes, W. C., and Bradley, A. (2008). Extensive genomic copy number variation in embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America 105, 17453-17456.). ES cells that have abnormal genomes do not contribute to the germline of the chimaeric mice efficiently. BAC integrity will be examined by PCR-amplifying each known functional V gene in the BAC. For example, in one approach the first human BAC chosen for the IgH locus has 6 functional VH gene segments and all human D and JH gene segments. To confirm the integrity of this BAC for the presence of these IgH genes, pairs of PCR primers will be designed and used to PCR-amplify genomic DNA from the targeted ES cells. The human wild-type size and sequence of these fragments will ensure that the inserted BAC has not been rearranged. More detailed CGH will also confirm the integrity of the inserted BACs. For example, one skilled in the art could use an oligo aCGH platform, which is developed by Agilent Technologies, Inc. This platform not only enables one to study genome-wide DNA copy number variation at high resolution (Barrett, M. T., Scheffer, A., Ben-Dor, A., Sampas, N., Lipson, D., Kincaid, R., Tsang, P., Curry, B., Baird, K., Meltzer, P. S., et al. (2004). Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proceedings of the National Academy of Sciences of the United States of America 101, 17765-17770.), but permit examination of a specific genome region using custom designed arrays. Comparing the traditional aCGH techniques which rely on cDNA probes or whole BAC probes, the 60-mer oligonucleotides probes can ensure specific hybridization and high sensitivity and precision that is needed in order to detect the engineered chromosome alterations that we have made. For example, oligos designed to hybridize at regular intervals along the entire length of the inserted BAC would detect even quite short deletions, insertions or other rearrangements. Also, this platform provides the greatest flexibility for customized microarray designs. The targeted ES cell genomic DNA and normal human individual genomic DNA will be labelled separately with dyes and hybridized to the array. Arrays slides will be scanned using an Aglient Technologies DNA microarray scanner. Reciprocal fluorescence intensities of dye Cy5 and dye Cy3 on each array image and the log 2 ratio values will be extracted by using Bluefuse software (Bluegnome). Spots with inconsistent fluorescence patterns ("confidence"<0.29 or "quality"=0) will be excluded before normalizing all log 2 ratio values. Within an experiment, Log 2 ratio between -0.29 and +0.29 for the signal from any oligo probe are regarded as no copy number change. The log 2 ratio threshold for "Duplication" is usually >0.29999, and for deletion is <0.29999.

Example 2: Introducing the Human VpreB Gene into the Mouse Lambda Locus by Inserting the Human Lambda V Gene Segment Cluster

[0520] The human VpreB gene is inserted into a mouse genome by introducing human lambda gene segment DNA, wherein the nucleotide sequence of the DNA comprises a human VpreB gene and optionally the associated human promoter. Human VpreB1 and VpreB3 genes are both located in chromosome 22. The human VpreB1 gene is between human Ig.lamda. V gene segments IV-53 and 5-52 (Chromosome 22: 22599200 to 22599926). Therefore, the human lambda gene segment which covers the human VpreB1 gene and contains multiple Vs flanking this gene is inserted in the endogenous lambda antibody locus upstream of the endogenous lambda constant region between the most 3' endogenous JA and the endogenous CA following the approach in Example 1 and as further explained in WO2011004129.

Example 3: Introducing the Human VpreB Gene within the Mouse VpreB-.lamda.5 Locus and Targeting into the Mouse Heavy or Lambda Locus

[0521] The following example illustrates the insertion of the human VpreB1 gene within the mouse VpreB-.lamda.5 locus to bring the human gene under the endogenous LCR influence. The mouse VpreB1 and .lamda.5 genes are closely associated, both within a 19-kb fragment called the .lamda.5-VpreB1 locus. Apart from these two genes, this locus contains a locus control region (LCR) required for correct levels of expression and tissue-specificity of .lamda.5 and VpreB1. Locus control regions (LCRs) are sequences that mediate reorganisation of chromatin and activation of transcription by sequence-specific transcription factors. The characteristic of an LCR is the ability to drive gene expression in transgenic mice at any site of integration at levels that are substantially equivalent to those of the gene in its natural location. It has been shown that the LCR in the .lamda.5-VpreB1 locus is able to promote efficient and stage-specific expression of both genes in transgenic mice at all sites of integration tested.

[0522] To make sure that the expression of the inserted human VpreB1 gene is properly controlled in mouse, the mouse .lamda.5-VpreB1 locus is retrieved and cloned into a vector (pBlueScript II SK(+)). The mouse VpreB1 nucleotide sequence is replaced by the human VpreB1 sequence by cloning. The construct containing the fragment of mouse .lamda.5-human VpreB1 locus is then cloned into a BAC containing a human heavy chain locus fragment. Then it is targeted into the genome of a mouse ES cell following the introduction of the human BAC into the mouse heavy chain locus upstream of the endogenous heavy chain mu constant region. In this case, the human VpreB function can be directly tested in the F1 mouse generated from the ES cells. Manipulation of constructs and BACs can be effected by standard recombineering.

[0523] Insertion of the mouse .lamda.5-human VpreB1 can be carried out using homologous recombination or RMCE. Homologous combination can employ homology arms corresponding to the endogenous sequences flanking the mouse .lamda.5-mouse VpreB1 in the mouse ES genome, so that insertion of the mouse .lamda.5-human VpreB1 replaces (and thus deletes) the endogenous mouse .lamda.5-mouse VpreB1 and places the inserted construct under the control of the endogenous LCR.

[0524] RMCE can be used to insert the mouse .lamda.5-human VpreB1 construct (optionally with the endogenous mouse .lamda.5-mouse VpreB1 LCR) anywhere in the genome. RMCE can also be used to precisely delete the endogenous mouse .lamda.5-mouse VpreB1 so that the inserted mouse .lamda.5-human VpreB1 construct is the only source of a VpreB1 gene.

[0525] In the alternative, the method can be modified to insert the mouse lambda 5-human VpreB1 construct into the endogenous lambda5-VpreB locus on the mouse lambda locus, for example to bring the human VpreB1 gene under endogenous control (eg, control of the lambda5-VpreB LCR). To do this, the cloned mouse lambda 5-human VpreB1 construct (produced by standard recombineering) is inserted into a homologous recombination vector using in vitro recombineering to add homology arms flanking the construct. Using homologous recombination in the presence of a marker carried by the vector, the construct is inserted precisely to replace the endogenous mouse lambda 5-mouse VpreB1 in the genome of a mouse ES cell. Alternatively, instead of homologous recombination, standard RMCE can be used to effect precise insertion into the genome.

[0526] A suitable protocol is provided in Example 5.

Example 4: Assessing the B-Cell Development and Repertoire in Chimaeric Mouse Containing Human VpreB Gene

[0527] The following example is provided on assessing the advantage of having the human VpreB gene in the chimaeric mouse.

[0528] The mouse embryonic stem cells (eg, AB2.1 cells; Baylor College of Medicine) containing the targeted human VpreB1 gene are grown on a feeder layer of SNL7 fibroblasts (Baylor College of Medicine) in embryonic stem cell medium containing 15% serum. Then the cells are microinjected into blastocysts which are transferred to the uteri of pseudopregnant F.sub.1 female mice. To test for germline transmission, male chimeras are bred to C57BL/6-Tyr.sup.c-Brd albino female mice (Baylor College of Medicine). After obtaining mice with both chimaeric heavy chain gene (human V region and mouse C region) and human VpreB1 gene, the following assays are performed to show the advantage of human VpreB v mouse VpreB.

[0529] 1. Pre-B Cell Populations Containing Pre-BCR from Human VpreB v Mouse VpreB

[0530] In the pre-B cells, .mu. heavy chain associated with surrogate light chain will deposit on the cell surface to signal the cell survival and proliferation. The pre-B cell populations with the pre-BCR from human VpreB protein v mouse VpreB protein are analyzed by flow cytometry.

[0531] Cells from bone marrow are surface stained with fluorescence-labelled mAb and then analysied with MACSQuant (Miltenyi Biotech) on B220.sup.+ cells. Pre-B cell population can be detected using pre-BCR-specific mAB and/or mouse .lamda.5.sup.+ cells. The cells with pre-BCR of human VpreB v mouse VpreB are distinguished by using monoclonal antibody specifically to human or mouse VpreB.

[0532] 2. Usage of Human Vs in the Mouse with Chimaeric Antibody

[0533] In the mouse with the insertion of human V.sub.HD.sub.HJ.sub.Hs between endogenous mouse V.sub.HD.sub.HJ.sub.Hs and C.sub.Hs, the usage of human Vs v endogenous mouse Vs are compared by sequencing the heavy chain products in naive mice.

[0534] Approach: [0535] a. Total RNA extraction (use TRIzol.RTM. Reagent) from mouse spleen [0536] b. By a standard 5' RACE (Rapid amplification of 5' complementary DNA ends) method, using a mouse C.mu.-specific primer, PCR fragments from mouse synthesized heavy chain mRNAs can be amplified. [0537] c. The PCR products are cloned into a vector and then sequenced. [0538] d. The sequences map to heavy V D J sequences which contain either mouse V or human V. Then we compare the usage of human Vs v mouse Vs. [0539] e. The usage of human Vs v mouse Vs is analysed in the mice with/without a human VpreB1 gene. We expect by introducing human VpreB1 gene, the human V usage will increase in the mouse with chimaeric IgH gene.

[0540] 3. Improvement of Repertoire and Diversity of Chimaeric Antibody

[0541] In this embodiment, in mice with the chimaeric heavy locus, endogenous mouse V genes are inactivated by deletion or inversion of the mouse V locus. Therefore, all the heavy chain products are transcribed from human Vs.

[0542] Using the same methods in Example 3, the heavy chain products from naive and/or immunized mice are sequenced and map to human V.sub.HD.sub.HJ.sub.HS with different CDR sequences.

[0543] The repertoire of heavy V D J selected by surrogate light chain from human VpreB and mouse VpreB can be compared from several aspects. First is the improvement of usage of human heavy V D Js and hypervariabilities for mice having human VpreB. Second is the shift of usage in terms of human heavy V gene families caused by human VpreB v mouse VpreB.

Example 5: Surrogate Light Chain Targeting Protocol

[0544] Mouse .lamda.5 and VpreB1 sequences will be substituted with human .lamda.5 and VPREB1 sequences. The regulation and expression will be controlled by mouse endogenous regulatory elements (FIG. 1; references 1 & 2) that are used during B cell development. In a mouse genome this VpreB1 is found on chromosome 16 at coordinates 16,868,494-16,869,348. In a human genome VPREB1 is found on chromosome 22 at coordinates 22,599,087-22,599,927.

REFERENCES

[0545] 1. Minaee S, Farmer D, Georgiou A, Sabbattini P, Webster Z, Chow C M, Dillon N, Mapping and functional analysis of regulatory sequences in the mouse lambda5-VpreB1 domain, Mol Immunol. 2005 July; 42(11):1283-92. [0546] 2. Sabbattini P, Georgiou A, Sinclair C, Dillon N., Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the lambda5-VpreB1 locus control region, Mol Cell Biol. 1999 January; 19(1):671-9.

[0547] Insertion of Human VpreB1 into Mouse BAC

[0548] A bacterial artificial chromosome (BAC) containing mouse .lamda.5 and VpreB1 genes will be used for recombineering in bacteria (E. coli) to replace mouse genes with human genes. A suitable source of BACs is mouse BAC library collection, RP23-220N17 (BacPac Resource Center; http://bacpac.chori.org). The following steps will be followed to generate a targeting construct that will be used further for targeting of DNA into mouse AB2.1 ES cells and generation of a transgenic mouse harbouring the human sequences.

[0549] The first step is to delete the mouse VpreB1 gene on a BAC containing this mouse gene, by using a positive/negative selection cassette flanked by 5' and 3' 50 bp homology arms (ie, stretches of mouse genomic sequence that flank the endogenous VpreB1 gene in the mouse genome--see sequences A and B below and denoted as BAC Arms A and B respectively in FIG. 1B). The cassette contains ampicillin and streptomycin genes between the homology arms. This cassette is able to select clones that have been correctly targeted, FIGS. 1A and 1B. The selection cassette will be inserted and targeted using homologous recombination to replace the endogenous mouse VpreB1 gene. Positive clones will be checked using positive selection, ampicillin, followed by PCR based confirmation (denoted by the arrows on the second schematic in FIG. 1A and by means of PRIMER1 to PRIMER4 in FIG. 1B). As shown, the PCR based confirmation will be performed using specific primers situated within the inserted DNA sequence whereas, other primers will be outside the targeted region. A positive amplification product can only be achieved if the newly inserted DNA is targeted correctly to the desired locus. Using 50 bp homology arms (sequences A and B below) that are added to flank the human VPREB1 gene, a vector will be constructed and used to target and replace the positive/negative selection cassette specifically using homologous recombination (bottom schematics in FIGS. 1A and B). Correctly targeted clones containing human VPREB1 (targeted mouse BACs) will be screened using a negative selection, streptomycin and PCR based method. A source of human DNA for the human VPREB1 gene is RP11-373H24 BAC (Invitrogen; Roswell Park Cancer Institute).

TABLE-US-00003 Sequence A: 5' Arm acctggccaaactgagcatgacctttgacctagccagctc ttaaacttgttctgagatcacaaaccagccagaccaaatt Sequence B: 3' Arm tcctcccagaatgcttccctgggtcaaacccagagccaca aaggcttccattagaccattctggtaagtgacagagtcac

[0550] Retrieval of 19 kb DNA from Mouse BAC

[0551] Next, the 19 kb of DNA (see Sabbattini references cited supra) containing human VPREB1, mouse .lamda.5 and the mouse regulatory elements will be removed from the BAC using homologous recombination in E coli. The region will be retrieved from the previously targeted mouse BAC into a smaller vector containing 5' and 3' mouse homology arms (FIG. 2; Mouse Arms 1 and 2). The arms are constructed using mouse BAC as a DNA template to maintain high nucleotide homology. Amplified arms are digested with specific restriction enzymes and cloned into targeting vector (the resulting targeting vector is shown in the top schematic in FIG. 2). The targeting vector is linearised between two homology arms and electroporated into E. coli which contains the previously targeted human VPREB1 gene.

TABLE-US-00004 Mouse Arm 1 Sequence: AAAAAAAAAAAAGCCCAGCTAGCTTAGTTGGTAGAGCATGAGACTCTT AATCTCAGAGTCATGGGTTCAGGCCTCATGTTTGGCACCATCTATAGT GTGCAGTTATAAATCAAACAGTTCACGATGTGGCTGGCTAGGCACTGG CAACTGCAGTCTCACCTGCTCCCATGGTTCCCAGTTCCCACAGCTAGT TTGCTGCCAGGCTGTTCACACTTCCCAGGTCATCTACCCACTGTGGCA AGCCTGCAGAAAGCCTGCTATTGCTAGCTCAGATTCCCTTAGCCCTAT AAAATGATAACACCACAGACTTTTACTATACCATCCAGATTTATAACA GTAATTCTCCAACCC Mouse Arm 2 Sequence: CAGGAGTACACCAAAATGTCTAGCCAAAATTTTTATATATGATCACTT AAATAAGACTCCTTAACATAAACCTACATGATATACCAAGTCTTTTCT GCCAAGGCTCTGACACTATAGTTTGTCCTATTCTGGAGTTAGGTAAGC AAAGGGCTATTTAGGTGTGGATTGCAAAGAGAGAATAGCAAGACAACC TGCCCATTCTTTGCCACACCTCACTAATCAGTGTCCCTTGGAAAGCAC TGTAAATATGGAGGTTTCTTTTTGTATTATGTAGTAGTGGATTTAACT TGAGGAGCCCCAAAAGGGGTCAGCAAAGCATGGGAAATCTAAGAATTT AACACTTCAGTGACTTTTAATCACCTACAGATCCAGGAAAATAAGCCT GTCTCTT

[0552] The resulting vector contains the 19 kb sequence containing mouse .lamda.5 and human VPREB1 genes (second schematic in FIG. 2). This vector will allow for amplification of the retrieved 19 kb sequence and use for further insertion into a human BAC of choice (eeg, a BAC containing human antibody gene segments, such as a BAC obtainable from the RPCI-11 library available from Invitrogen). The insertion of the 19 kb DNA fragment will be targeted into a BAC containing human IgH V gene segments (FIGS. 3A and 3B; which can be read in conjunction with the disclosure of WO2011004192, the disclosure of which is incorporated herein by reference), eg, to target the 19 kb sequence directly 5' of the most distal 5' end of the human IgH locus to avoid any possible interference with normal IgH rearrangement subsequently in a mouse or B-cell progeny. To this end, using a series of BACs, human Ig gene segments (VDJ) are inserted in a series of steps into the genome of a mouse ES cell (eg, an AB2.1 cell) using sequential recombinase mediated cassette exchange (sRMCE) or homologous recombination as is known in the art. The 19 kb sequence can be inserted directly 5' of the 5' VH in the last BAC to be used, thus providing the 19 kb sequence immediately 5' of the first human VH in a transgenic IgH locus after insertion of the 19 kb sequence and human VH sequences into the mouse ES cell genome. Thus, a human VPREB1/mouse .lamda.5 gene insert is provided for expression of chimaeric surrogate light chain (SLC) in a subsequent progeny mouse or B-cell.

[0553] Humanisation of .lamda.5

[0554] Similar procedures of targeting and genetic engineering steps will be used to modify the second gene of SLC, namely the .lamda.5 (FIG. 4), in order to replace mouse .lamda.5 with human .lamda.5.

[0555] Correctly targeted ES will be used to generate a transgenic mouse. Analyses will be performed using animals which harbour (i) the human VPREB1 and mouse .lamda.5 (humanVB1/mouse .lamda.5) or (ii) human VPREB1 and human .lamda.5 (human VB1/.lamda.5). Differences in populations of expressed antibodies between a control wild type (WT) mouse and (i) or (ii) will be recorded using Fluorescence-Activated Cell Sorting (FACS). The end point data will be generated using mice spleen to analyse the contribution of human VPREB1 only or in conjunction with human .lamda.5 in expansion of B cell population harbouring human sequences and the generation of more diverse antibody repertoires.

Example 6: Engineering of Non-Human Vertebrate Genome--Chimaeric Surrogate Light Chains Significantly Change Human Variable Region Repertoires In Vivo

[0556] Construction of Transgenic IgH Loci

[0557] Insertion of human heavy gene segments from a 1st IGH BAC (RP11-1065N8) into the IGH locus of mouse AB2.1 ES cells (Baylor College of Medicine) was performed to create a heavy chain allele denoted the 51 allele. The inserted human sequence corresponds to the sequence of human chromosome 14 from position 106494908 to position 106328951 and comprises functional heavy gene segments V.sub.H2-5, V.sub.H7-4-1, V.sub.H4-4, V.sub.H1-3, V.sub.H1-2, V.sub.H6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, J.sub.H1, J.sub.H2, J.sub.H3, J.sub.H4, J.sub.H5 and J.sub.H6 (in 5' to 3' order), wherein the JH6 was chosen to be the human JH6*02 variant. The insertion was made between positions 114666435 and 114666436 on mouse chromosome 12, which is upstream of the mouse C.mu. region. The mouse V.sub.H, D and J.sub.H gene segments were retained in the locus, immediately upstream of (5' of) the inserted human heavy chain DNA.

[0558] A second allele, S2 was constructed in which more human functional V.sub.H gene segments were inserted upstream (5') of the 5'-most V.sub.H inserted in the 51 allele by the sequential insertion of human DNA from a second BAC (BAC2). The inserted human sequence from BAC2 corresponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments V.sub.H3-13, V.sub.H3-11, V.sub.H3-9, V.sub.H1-8, V.sub.H3-7. The mouse V.sub.H, D and J.sub.H gene segments were retained in the locus, immediately upstream of (5' of) the inserted human heavy chain DNA.

[0559] Mice bearing the S2 insertion into an endogenous heavy chain locus were generated from the ES cells using standard procedures. The other endogenous heavy chain locus was inactivated in the mice by insertion of an inactivating sequence comprising neo.sup.n into the mouse J.sub.H-C.mu. intron (to produce the "HA" allele). The mice retained mouse VpreB and .lamda.5, but no human VpreB gene (control, S2 mouse) or a human VpreB gene was included in the genome (S2/hVpreB mouse, ie, chimaeric surrogate light chain mouse according to the invention comprising a human VpreB1 gene with mouse VpreB1 promoter-driven expression). The human VpreB was inserted from a BAC using recombinase mediated cassette exchange (RMCE) upon construction of the S2/hVpreB mouse according to the invention. The latter mouse was a chimaeric mouse having around 30% Agouti coat colour, which is indicative of the potential fraction of B cells derived from the injected ES cells bearing the human VpreB gene. Thus, in the chimaeric mouse of the invention most B-cells do not have the human VpreB. This provides for a more challenging test versus control, as explained below. An alternative would be to breed a mouse of the invention where all cells harbour a human VpreB gene, and this is expected to show the advantage of the invention in producing a different repertoire as demonstrated below for the chimaeric test mouse.

[0560] Specifically, the following human gene segments were included (in 5' to 3' order):-- [0561] human VH gene segments VH3-13, 3-11, 3-9, 1-8, 3-7, 2-5, 7-4-1, 4-4, 1-3, 1-2 and 6-1; [0562] human D gene segments D1-1, 2-2, 3-9, 3-10, 4-11, 5-12, 6-13, 1-14, 2-15, 3-16, 4-17, 5-18, 6-19, 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 1-26 and 7-27; and. [0563] Human JH gene segments J1, J2, J3, J4, J5 and J6.

[0564] RNA was extracted from the spleens of both the control and S2/hVpreB mouse of the invention. RT-PCR was performed as described in the VDJ sequencing methodology below. The mouse C.mu. transcripts were captured and sequenced, and only human JH transcripts were used for usage analysis to ensure use of transcripts only derived from the transgenic IgH loci bearing human variable region gene segments.

[0565] VDJ-Sequencing Methodology

[0566] The following methodology was performed to sequence human IgH variable regions from RNA samples obtained from the mouse of the invention comprising a transgenic IgH locus and a human VpreB gene. Comparison of the control with a chimaeric mouse of the invention is more challenging than comparison with a mouse of the invention that is 100% transgenic (ie, where all cells bear the transgenic IgH locus and human VpreB gene). In the chimaeric mouse, only a proportion of the cells comprise the transgenic IgH locus and human VpreB gene, and in those cells the IgH is present in one copy (heterozygous). Thus, the comparison in this example surprisingly was still able to show a difference in repertoires produced in the control and invention mice despite the chimaeric nature of the latter.

[0567] Sequences were filtered so that the resulting data set related to human variable regions only (and not variable regions having mouse sequence).

[0568] V gene usage frequency was assessed by next generation sequencing of VDJ recombined transcripts using the Illumina Miseq platform (see, eg, Nature. 2008 Nov. 6; 456(7218):53-9; "Accurate whole human genome sequencing using reversible terminator chemistry"; Bentley D R et al). Libraries of VDJ sequences were generated with standard adapter sequences at either end that were compatible with Illumina sequencers. The Illumina adapter sequences are termed P5 and P7 permit the binding of DNA fragments to the Illumina flowcell and are the initiation sites of the sequencing reactions. The VDJ libraries were generated using the following methodology: 5' RACE was performed on 5 .mu.g of total RNA to generate cDNA with a known sequence at the 5' end using the ExactSTART Eukaryotic mRNA 5'- & 3' RACE kit (Epicentre) using the manufacturer's protocol with the following modifications: The 5' RACE acceptor oligo was replaced with an RNA oligo (P7 RNA oligo: AGACGUGUGCUCUUCCGAUCU) specific to a 21 bp portion of the Illumina P7 adapter. The first-strand cDNA was synthesised using a reverse primer (IgM RT primer: GAAGACATTTGGGAAGGACTG) specific to the first exon of the IgM constant region. Second strand synthesis, and enrichment of VDJ sequences was achieved by performing 15 cycles of PCR using a primer specific to the ligated RNA oligo (P7 PCR 1: [GTGACTGGAGTTC]AGACGTGTGCTCTTCCGATCT) that also included additional Illumina P7 sequence at the 5' end (shown by square brackets). Paired with a primer specific to the IgM constant region/J segment splice junction (IgM P5 PCR 1: [ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN]GGGAAGGACTGACTCTCTGA) that also included additional Illumina P5 sequence, and two bases of random nucleotides, at the 5' end (shown by square brackets).

[0569] The PCR reaction was purified using gel electrophoresis and the major PCR product that corresponded to VDJ transcripts (.about.600 bp) was excised and purified using a Qiagen gel extraction kit according to the manufacturer's protocol. A second round of 15-cycle PCR was performed to add the remaining portions of the Illumina P5 & P7 adapter sequence and to incorporate a barcode, so multiple samples could be pooled and sequenced together in a single run. The forward primer was specific to the P5 sequence with the additional P5 flowcell binding sequence shown within square brackets (P5 PCR 2: [AATGATACGGCGACCACCGAGATCT]ACACTCTTTCCCTACACGACGCTCTT). The reverse primer was specific to the P7 sequence and incorporated a 6 bp barcode and the P7 flowcell binding sequence shown within square brackets (P7 PCR 2 index X: [CAAGCAGAAGACGGCATACGAGAT******]GTGACTGGAGTTCAGACGTGT). The 6 bp barcodes were standard Illumina 6 bp indexes, 4 different indexes were used in this case and the sequences were: CGTGAT, GCCTAA, GATCTG, & TCAAGT. The final PCR products were purified using AMPure XP beads (Beckman Coulter) and were pooled together and sequenced on the consisted of a VDJ transcribed gene with a complete P5 and P7 Illumina adapter on either end. This construct was suitable for next generation sequencing on the Illumina Mi-Seq system using the TruSeq DNA sequencing program2.times.150 bp paired end sequencing program.

[0570] Bioinformatics & Statistical Analysis

[0571] The sequences from the Illumina MiSeq were obtained as a set of 4 BAM files from the machine, each file corresponding to a different barcode. Barcodes 1 and 3 were from the surrogate light chain samples, while barcodes 7 and 8 were from the control samples. These BAM files were converted into fastq using bam2fastq using the command: bam2fastq <file>.bam -o<file>#.fastq which splits the paired reads into two different fastq files.

[0572] These fastq files were then converted into fasta using fastq_to_fasta -I<file>.fastq -o<file>.fasta -Q33. This yielded a set of 8 fasta files.

[0573] The fasta files were then analysed with Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/) to find J and V segments from mice and humans. The paired reads were used to identify the J and V used, with the read from the constant region end being used to find the J and the read from the 5' end of the RNA being used to identify the V gene used. As the transcription occurred before the start of the V segment, 5' UTR sequence from Ensemble (http://www.ensembl.org) was used to search against to find the V genes used.

[0574] An InforSense workflow was used to then combine the results from the paired reads by matching their unique identifier to the results from the two Blast searches. These combined results were then filtered for sequences which used a Human J gene and a Human V gene. Because the sample with the chimaeric surrogate light chain was a wild type/transgenic chimaera, there were far fewer samples with a Human J than in the control, which is expected.

[0575] To avoid any errors due to PCR duplication events sequences with identical CDR3 regions, as determined by the IMGT numbering scheme, were treated as one sequence. This reduced the number of sequences in the control sample from 2,632,503 to 168,551 and in the surrogate light chain sample from 2,543,248 to 590,462.

[0576] The final counts after filtering for Human J, Human V and treating each CDR3 string as single sequence gave 10,781 sequences for the control, and 267 for the surrogate light chain sample. The low percentage of sequences with an identified Human V is due to the 150 bp reads from the Illumina MiSeq not extending very far into the variable part of the V genes. The results are shown in Table 3. In the table, the mouse of the invention is labelled "Chimaeric SLC" and significant changes in gene segment usage are underlined.

TABLE-US-00005 TABLE 3 Chimaeric Chimaeric Control Total SLC Total Control % SLC % V6-1 587 38 5.44% 14.23% V1-2 155 3 1.44% 1.12% V1-3 1167 43 10.82% 16.10% V4-4 984 33 9.13% 12.36% V2-5 409 7 3.79% 2.62% V3-7 4095 66 37.98% 24.72% V1-8 1315 21 12.20% 7.87% V3-9 893 40 8.28% 14.98% V3-11 443 3 4.11% 1.12% V3-13 733 13 6.80% 4.87% sum 10781 267 IGHJ1*01 131 13 1.21% 4.87% IGHJ2*01 260 13 2.41% 4.87% IGHJ3*02 1011 34 9.37% 12.73% IGHJ4*02 5467 153 50.69% 57.30% IGHJ5*02 867 12 8.04% 4.49% IGHJ6*02 3050 42 28.28% 15.73% sum 10786 267

[0577] The usage between the two samples was compared using a standard Pearson's chi-squared test (see eg, NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/, April 2012). This gave the probabilities that the distribution of the surrogate light chain sample and the control sample being the same was 5.8E-15 for the V genes and 8.2E-12 for the J genes. These are below the standard 5% (ie, 0.05) probability cut off and thus are significant.

[0578] The individual V and J genes which showed the most significant differences between the two are summarised in the Table 4 below. The probability shown is the probability of obtaining by chance that number or more in the case of an increase or that number or less in the cast of a decrease, using a binomial distribution using the probabilities from the ratios obtained from the control sample. The individual V and J genes in the table have a probability score below the standard 5% (ie, 0.05) probability cut off and thus are significant.

TABLE-US-00006 TABLE 4 Expected Chimaeric from Gene Usage SLC Count Control Probability IGHV6-1 Increased 38 14.5 2.38E-08 IGHJ1 Increased 13 3.2 6.31E-06 IGHV3-9 Increased 40 22.1 9.80E-05 IGHV1-3 Increased 43 28.9 3.21E-03 IGHJ6 Decreased 42 75.5 1.09E-06 IGHV3-7 Decreased 66 101.4 2.94E-06 IGHV3-11 Decreased 3 11.0 4.42E-03 IGHV1-8 Decreased 21 32.574 1.52E-02

TABLE-US-00007 SEQUENCE LISTING SEQ ID NO: 1 Human VpreB1 nucleotide sequence (GenBank Accession No = NG_029387.1) gagtcagagctctgcatgtctgcaccatgtcctgggctcctgtcctgctcatgctgtttgtctactgcacaggt- gaggga acccccagatcccaaagactcctgccccttccttcatcctgccctgcccccacggcccacatgcatctgtgtca- ccaggt tgtggtcctcagccggtgctgcatcagccgccggccatgtcctcggcccttggaaccacaatccgcctcacctg- caccct gaggaacgaccatgacatcggtgtgtacagcgtctactggtaccagcagaggccgggccaccctcccaggttcc- tgctga gatatttctcacaatcagacaagagccagggcccccaggtcccccctcgcttctctggatccaaagatgtggcc- aggaac agggggtatttgagcatctctgagctgcagcctgaggacgaggctatgtattactgtgctatgggggcccgcag- ctcgga gaaggaggagagggagagggagtgggaggaagaaatggaacccactgcagccaggacacgtgtcccttgaactg- aagaca gcagaggcacgcatccccttggagagactgtcatggaagagggtggagccgccgcccgaagcgccgaggaggct- gagcca ctcagcatctcctggtcctgcagtgttgctgtaaatccccattggagactgcattagggaattaaagctgcttg- tcactt tttgctg SEQ ID NO: 2 Human VpreB amino acid sequence (GenBank Accession No = NP_009059.1) mswapvllmlfvyctgcgpqpvlhqppamssalgttirltctlrndhdigvysvywyqqrpghpprfllryfsq- sdksqg pqvpprfsgskdvarnrgylsiselqpedeamyycamgarssekeerereweeemeptaartrvp SEQ ID NO: 3 Human VpreB3 nucleotide sequence (GenBank Accession No = NC_000022.10) cttcccagccctgtgccccaaagcacctggagcatatagccttgcagaacttctacttgcctgcctccctgcct- ctggcc atggcctgccggtgcctcagcttccttctgatggggaccttcctgtcaggtgaatctttcctgggcctcagtca- cctggg tgtggggtggggaacagtactggccccagaaggcccctgcaaggaggcaaatcgatggggacagtagggcaggt- cctggg aggggtattttttttttttttgagacggagttttgctcttgttgcccaggctggagtgcaacggtgcaatctcg- gctcac tgcacctctgcctcccaggttcaagcgattcttctgcctcagcgtcctgagtagctgggattacaggcatgcgc- caccac gcctggctaattttgtatttttagtagagacaaggtttcttcatgttggtcaggctggtctcaaactcctgacc- tcaggt gatccgcccacctcagcttcccaaagtgctaggattacaggcttgagccactgcacctggctgggaggcgtatt- caggta gaggggtgacctggcttcagacagctctgcccttgacctggggcaaatcacttccctgtgtgggccttggtttt- ctcatt cgtgaatgaccagatcactagagccctggactctgactttgggtctccttcttagtttttcacaggggaagcta- ttgtgg gttggtccctaccccacagggcctgaggcaatctaacctccctgagagggtccctgcagccagttgcctgaggc- tgagtt gatgtgtgggacagcccaggaggttcctgggggtggttagtctgtattcagggtttggaagagctgaagtgaag- tgggcg gggaagtgggggagaggggtgcagttctgcagagaaacgtgggtgggtagcagagggacctagaggctgctagt- ccaacc ttctgagctctgggcctttaactgaacacagatccttgaggatatggcactaatggagatttgggggctaactc- caaacc cctcactcacaaagggagatggaggtccagatagggcaccctaagtcacacagaaccaggcctcctgcaccccg- ttcatt gctaatcccatagcactgggctatgagccctttgggactgggagtctccatggagtccaaccaagccttcacag- ggcagg ggtgggagggaaggggctcaggctgagtgggtttgtgtctcgcagtttcccagacagtcctggcccagctggat- gcactg ctggtcttcccaggccaagtggctcaactctcctgcacgctcagcccccagcacgtcaccatcagggactacgg- tgtgtc ctggtaccagcagcgggcaggcagtgcccctcgatatctcctctactaccgctcggaggaggatcaccaccggc- ctgctg acatccccgatcgattctcggcagccaaggatgaggcccacaatgcctgtgtcctcaccattagtcccgtgcag- cctgaa gacgacgcggattactactgctctgttggctacggctttagtccctaggggtggggtgtgagatgggtgcctcc- cctctg cctcccatttctgcccctgaccttgggtcccttttaaactttctctgagccttgcttcccctctgtaaaatggg- ttaata atattcaacatgtcaacaaca SEQ ID NO: 4 Human VpreB3 amino acid sequence (GenBank Accession No = NP_037510) macrclsfllmgtflsvsqtvlaqldallvfpgqvaqlsctlspqhvtirdygvswyqqragsapryllyyrse- edhhrp adipdrfsaakdeahnacvltispvqpeddadyycsvgygfsp SEQ ID NO: 5 Mouse VpreB1 nucleotide sequence (GenBank Accession No = NC_000082.5) agagcccagaaagcctgggagggtggtgagcaggaaccaggggtgcagtgaccctctccccaaagcagggagga- gagtgc ttcccagctggtcagggcccaggagcagtggctgtagggggcagggtgctgcaggtctggagccatggcctgga- cgtctg tcctgctcatgctgctggcctatctcacaggtaaggaaactcttggggcccagggcttctttgctcctcctatg- gccttg ctctgccccagtgatggacatgcttctatcttctcaggttgtggccctcagcccatggtgcatcagccaccatt- agcatc ttcttcccttggagccaccatccgcctctcctgtaccctgagcaacgaccataacattggcatttacagcattt- actggt accagcagaggccgggccaccctcccaggttcctgctgagatacttctcacactcagacaagcaccagggtccc- gatatc ccacctcgcttctccgggtccaaagatacgaccaggaacctggggtatctgagcatctctgaactgcagcctga- ggacga ggctgtgtattactgtgccgtggggctccggagccaggaaaagaagaggatggagagggagtgggaaggagaaa- agtcgt atacagatttgggatcttaggctctggagacattcagaccctgaactgaagacagagtttgctttgctcggcta- gtctgg tatgggaaggaggggtagaacgtgaggttttgcagagcctagaagatggaattatgcagcttttccttgttctg- cggtgt tgctatgagcccccattggaggctggattgtagaattaaagctgtttttactgaa SEQ ID NO: 6 Mouse VpreB1 amino acid sequence (GenBank Accession No = NP_058678) mawtsvllmllayltgcgpqpmvhqpplassslgatirlsctlsndhnigiysiywyqqrpghpprfllryfsh- sdkhqg pdipprfsgskdttrnlgylsiselqpedeavyycavglrsqekkrmerewegeksytdlgs SEQ ID NO: 7 Mouse VpreB2 nucleotide sequence (GenBank Accession No = NC_000082) atggcctggacgtctgtcctgctcatgctgctggcccacctcacaggtaagggaactcttggggtccagggctt- ccttgc tcctcctgtggccttgctctgccccagtgatggacatgcttctatcttctcaggttgtggccctcagcccatgg- tgcatc agccaccatcagcatcttcttcccttggagccaccatccgcctctcctgtaccctgagcaacgaccataacatt- ggcatt tacagcatttactggtaccagcagaggccgggccaccctcccaggttcctgctgagatacttctcacactcaga- caagca ccagggtcccgatatcccacctcgcttctccgggtccaaagatacggccaggaacctggggtatctgagcatct- ctgaac tgcagcctgaggacgaggctgtgtattactgcgctgtggggctccggagccacgaaaagaagagaatggagaga- gagtgg gaaggagaaaagtcgtatacagatttgggatcttag SEQ ID NO: 8 Mouse VpreB2 amino acid sequence (GenBank Accession No = NP_058679.1) mawtsvllmllahltgcgpqpmvhqppsassslgatirlsctlsndhnigiysiywyqqrpghpprfllryfsh- sdkhqg pdipprfsgskdtarnlgylsiselqpedeavyycavglrshekkrmerewegeksytdlgs SEQ ID NO: 9 Rat VpreB1 nucleotide sequence (GenBank Accession No = NM_001108845.1) ccagaaagcctgggagggtggtgagcaggaaccagtggtgcaaagcaggggcgagactgcttcctagctggtca- gggcac cggagcagtggctgtaggggtcagggtgctgcaggtctggaaccatggcctggacgtctgccctgctcatactg- ctggcc catctcacaggtacgggaactcttggggcccagagcctccttgctcctcctcttgccttgctctgccgcagtga- tgggca cgcttctattttctcaggttgtggccctcagcccgtgctgcatcagccaccatcggcctcttccttccttggaa- cctcca tccgcctcacctgtgccctgagcagcaaccataacattggcatttacagcatttactggtaccagcagaggccg- ggccac cctcccacgttcctgctgagattcttctcacactcagacaagctccagggtcccaagatcccccctcgcttctc- cggatc caaagatacagccaggaacctggggtacctgagcatctctgacctgcagccagaggacgaggctgtgtattact- gcgccg tggggcttcggagctgggaaaaggagaagaggatggagagggagtgggaagaagaaaagtagcggacagattcg- ggatct taggctctggagacattcagacctagaaccgaagacggagtttgctttgctcggctaggctggtttggggagga- ggggta gaacaccgggcttcgcagagccaggaaggtggagccagccgcttttccttgtattgcagtgttgctatgcgccc- catcgg aggctggattgtagaattaaagctgttttttttttttttgttttgtttttttgttttttgtttcttttttctta- actg SEQ ID NO: 10 Rat VpreB1 amino acid sequence (GenBank Accession No = NP_001102315.1) mawtsallillahltgcgpqpvlhqppsassflgtsirltcalssnhnigiysiywyqqrpghpptfllrffsh- sdklqg pkipprfsgskdtarnlgylsisdlqpedeavyycavglrswekekrmereweeek SEQ ID NO: 11 Rat VpreB2 nucleotide sequence (GenBank Accession No = NC_005110) atggcctggacgtctgccctgctcatactgctggcccatctcacaggtacgggaactcttggggcccagagcct- ccttgc tcctcctcttgccttgctctgccgcagtgatgggcacgcttctattttctcaggttgtggccctcagcccgtgc- tgcatc agccaccatcggcctcttccttccttggaacctccatccgcctcacctgtgccctgagcagcaaccataacatt- ggcatt tacagcatttactggtaccagcagaggccgggccaccctcccacgttcctgctgagattcttctcacactcaga- caagct ccagggtcccaagatcccccctcgcttctccggatccaaagatatagccaggaacctggggtacctgagcatct- ctgacc tgcagccagaggacgaggctgtgtattactgcgccgtggggcttcggagctgggaaaaggagaagaggatggag- agggag tgggaagaagaaaagtagcggacagattcgggatcttaggctctggagacattcagacctagaaccgaagacgg- agtttg ctttgctcggctaggctggtttggggaggaggggtagaacaccgggcttcgcagagccaggaaggtggagccag- ccgctt ttccttgtattgcagtgttgctatgcgccccatcggaggctggattgtagaattaaagctgttttttactg SEQ ID NO: 12 Rat VpreB2 amino acid sequence (GenBank Accession No = NP_001128260) mawtsallillahltgcgpqpvlhqppsassflgtsirltcalssnhnigiysiywyqqrpghpptfllrffsh- sdklqg pkipprfsgskdiarnlgylsisdlqpedeavyycavglrswekekrmereweeek SEQ ID NO: 13 Human .lamda.5 nucleotide sequence (GenBank Accession No = NG_009791) ggccacatggactggggtgcaatgggacagctgctgccagcgagagggaccagggcaccactctctagggagcc- cacact gcaagtcaggccacaaggacctctgaccctgagggccgatgaggccagggacaggccaggggggccttgaggcc- cctggt gagccaggccccaacctcaggcagcgctggcccctgctgctgctgggtctggccgtggtaacccatggcctgct- gcgccc aacagctgcatcgcagagcagggccctgggccctggagcccctggaggaagcagccggtccagcctgaggagcc- ggtggg gcaggtaaggggtgagagattccaggggatgtgggggtctgggtggcagaggcgggaaaggatgaccaagggga- gacgag ccagaggggtgaggaggaaggttaatccctggaggggagccacagacactgactttaactaaagtgtcaagatt- ttgtcc atctttgaattaatttttattgcttaatgtcatattaaaatattatttatcttgattcctgagatttcttcccc- cactta catttggcaccaaggccaatgtctccctcacctccccctagtccttggggtagggcaggactggaggcaggggc- aggacg tccacaggagtggtggccgctatccctgaaggatgcccaggcctctccctcctcctcctcccactcctcctccc- ccccct cctcttttccccttggcctatgtcacctgtccactcccaccctcactgggcaggggccactccctggagctcca- gctaag gtgtgaggggcctttcctggagtccctgggtcactagacctcagccagcatcgcctcctgaaaccagcccctag- gagaca caagcttatccagggtgcaagtgcctccaaagaagaagcccaggagaggctctagggaggccacaactccctgt- gtgacc tcagccattgccaccactctgcgtgtggtgggaggtccccagacaaagcaccaagcatcggggtccatttatga- gcattt gggacacaacagcctgttcactggtgcatgttatacccacaggcgataatcatttcaggggcagaacctccctc- tcggtg gccccataggcaggtcctgctgtgatcccctttgtgcagacggggatagagcccggagaggtgaggtgacccgt- ccgagt cactcagctcatgggcacagattctaaggcccaagctatcccctctagctctcccctgtcccatcctctaagct- gatcga gcggacacgtgcatctctgggacctgagtttccctttttctctcttttttttctttttcaaataaagtttcaca- gagttt cactcttgtcgcccagtctggagtgcaatggcgagatcttggctcattgaaacctccgtctcccaggttcaaga- cattct cctgcctcagcttccggagtagctgggattacaggcatctgccaccacacctggctaattttgtgtattttttt- ttagta aagacagagtttcaccatgttggccaggctggtctcaaactcctgacctcaggtgatccacccacctcagcctc- ccaaag tgctgggattacaggcatgagacaccacaccgggcctgagtttccccttctgcaatctgaggggccctgactgg- tgaggg ccttcagcgtcccacccacccagaggatgctggggtggctgtggtgagagctccagcagtggcagccgacctga- cccaca ccaggagcccggccatggaggcggggtcagcatggtggcaggccgggaccgggtgtcagtgtcctgcacggact- tctgag caaggagtccccatcagggtcaggctctgtgctggggctgaggtcccagaggatctagatttgccccaattcaa- gtccac aaggagcgggggccgggtgaggagacagccacatgcagggtgatgcctacagaacagagactgggatggggaag- gcccga ggggtctccacaagggacgggtgacaggtggagggagacacagataataaaaaatggtattatgttggggggct- attaat gtaagtttttatattagaatctttagaaatcttatagaaatactatgggccgggtgctgtgtctcatgcctgta- atccca gcactttaggacgccaagatgggcagatcacgaggtcaggagattgagaccatcctggccaacatggcaaaact-

ccgtct ctactaaaaatacaaaaactagctgggcgtggtggcgcgagcctatagtcccagctactcgggaggggaggcag- gagaat cgcttgaatcagggaggtggaggttgcagtgagctgagattgcaccgctgcactccagcctggacgacagaacg- agactc ccactcaaaaaaaaaaaaaggtattatgctgggggggatatgaatatgagtttttataatctttagaaatacta- tgggcc aggtgcagtggctcatgcctgtaatcccacaactttgggaggctgaggcgggattgtttgagcccaggagtttg- agatca gcctgagcaacatagcaagaccccatttctacaaaacatataaaaactagctagtcatggtggcacttgcctgt- ggtccc agctacttaggaggctggtgagaggattgcttaagcctcggaggttgaggatgcagtaagctgagatcccacca- ctgcac tccagcctgggtgacagaaggagaccctgtctcaaaaaaaaaaaaaaaaaaaaaagactgattattcctgtaga- attctg gtaaatatctcctatcaaataaatgacttttctattcatagcttattaaaagatattttcattgtttttaagaa- ataggt tgtgtatcactttttatatttagttgtaaatttatttgttttatttatttttttagagatgggggtctcactat- gttgcc tgggctggcctccaactgctgggctcaagcaatatccctgcctcagcgtccccagtagctgggactacaagcat- gcgcta ccacaccggcataattttttgtagagatgaggtttcgccatgttgcctgggctggtcttgaacccctgggctca- agccat ccacccgcctcggcctcctaaagtgctgagattacagacgtgagccaccctacctagcctgtactatttttaat- aggtct taataggttttgaatgttaattatttttaaattaatttcaaaaatcttctacaacatggatgaaacctgaagac- attata cttagtgaaataagccagacacaaaaggacaaatgtcatttgaatccacttctatgaggtacctagaataggca- aattca cttggacaaaaagtagatttgaggttagcagggtgagggggagggaagaatgggggactgtagttaacgggttt- agagtt tctgtttgggaagatgaaagagttctggagatggatggtggtgaaggttgcccaacggtgtgaatgtacttagt- gccacg gagctgtacgtttaaaaatagttaaagtggaaatattgatgctatgtataaaaatggagcggggtgtggtggct- cacacc tataatcccagcactttgagaggccaaggtgggcagatcacctgaggtcgggagttcgagaccagcctgacaaa- catgga gaaacaccgtctctactgaaaatacaaaaaattagccaagtgtgctggcacatgcctgtaatcccagctactcg- ggaggc tgaagcaggagaatcgcttgaacccaggatgtggaggttgcggtgagccaaggtggcaccattgcactccagcc- tgggcg acaagggtgaaattccatttcaaaaaaataaaaggaaatgggactgtacatagcaggagagagagggagagatc- aatatg acacttctttttttttttttttttttttgggacagtcttgctctgttgccaggctgcactccagcctgggcaac- agagcg agactctgtctcaaaaaaaaaaagaatgtctagatgcagtagctaagttcgcagaagccatttcagtgtgggag- gcgagg tgactgagtccagggactccaggtttctggctgaggggttgagaccgcccatggtcattgtgatgagatgaaga- cagaaa atgagctgggctgggggaaggtggtcatctcctctggacgtgattagtttgaggctcctgttgggtagcccact- gggcaa ggtcagtaggcaattgaggagctgagagggtttggagctgggatagactccagcctcaccatgtgggcaatagt- gggggt cacagagtgtgaaaatggctaagaaagaggtctgggtggggtctggggagtcagcagccaggcatgggccatgg- agaaac agatgccaggggaagaggaaaggggagtctcagaccccaaggggaaaagagtcactggaaagaggggccagccc- tgtgtc gcatccagcggagacaccaggtacagcagaaggaccttggacatgaccatgaggagggccttgttgaccatggc- ctggga gagatgggggtgagagcctgggggaccacaccatgtccccagcacacagtgcctggtagacaggatggatttat- ggatgg acggacaggtagatggatggacgaatggacagatgatagatggatgcaaagacagatgaatagatggacagatg- cataga tggacagatggacagatggatggacggacggatggaatgaatgatcagaaaaggcttcatgaacaaagtgagac- tgagct gcatctccatgggtagatataaaagcagaggactctcctcttgagtcaggaatgacccaatgtcctggtccagg- gaggaa gtcagcctccttgactggggacacttgtggcagatttcagaggcccttaaaatgaggccaagtgaggtggacag- gtccga gccagctgaggactcctcagccacacggcacagctgcctgaggggatgtgtcactcagggagttgctgggacct- actggg cccagcgttgccatcagcaccaacagtttcagagagggggacacacgctggggcagcacctgcctcagagaagg- gacagg cacagagacactactgggggacactactgggacactggccacccccctaccctgtgcctgggtcacagcctaca- cactgc agccctgtgcccctcactcccagcaggttcctgctccagcgcggctcctggactggccccaggtgctggccccg- ggggtt tcaatccaagcataactcagtgacgcatgtgtttggcagcgggacccagctcaccgttttaagtaagtggctct- aacttc ccaggctgtcccaccctctcctgtctctggaaaatgtgttttctctctctggggcttcttcccctctgccctcc- cagcct taagcactgacccctacctttgtccatggggcctggaggagatgtgttagtctcagggtaatggcaggaagggc- ccccac agtgggagcagccgccttcaggttccaacagcaggacacagcctggtcccagggcctgggctgggattgggcgg- ggtcag ggctcctcccctctcccagggcagatgtctgagtgagggacagaggctggttctgatgaggggccctgcagtgg- ccttag agacagtccctgggaccccaggttctaggctgagggctggatgcccatccagcctgggagggccacacgggggc- ctgggg acacaggggtcacccccaggggagaccaatggagggcacagagagggctctgggtctaggctgcagctctgtgg- cctctg ctgggtcttcagggcatggggacacagaggaacggatgaggtcccagagcccagccctcccaggacagtcacca- gaaagg agagggtctcttagtgcagagatgtgcctgtccctggagccctgtcatctctggggcctggtgtctctctgttc- atgggt cgacctcccaccttcatttgaggaagggcaccttagactcagaaggtgactagcggggagtaaacgggagtgca- gagaac tccatggctgccaggtgaagtccaggggcatcagaggctgctggggtgggcatgggggctgcggtgccccaaag- tctggg ggagcagccccaagaacccagccgatgtgaagggtcctgtggtcgggctggtggggacaggggcgacggcagag- ccccag ggtgtgtctgggtggagcccacgcttcaccaggagagctgagtgggccaggctggggcacagcctggtgcccca- ggggat gggaagctccaggccatgccaggcttgggtctccccacatcctgccagtatagttttgtgtgctgtgagggaga- ccccta gattccaaactcagactccagaaaccaggaaggagggagcacagcctgccctgggtgcacacggggaaaccgag- gctgca gaggaaagggctgggccaggacacctgggaaaggtgacttgggaagggctcctaggaaggcacagggctgtctg- ctctcc agagggctccagtggaaaggagggaatgaggagggaaggagaggccctgggtggaccaggcggccacaccatga- accctc ccagagactttagacagagagaggcgctccacaacaccccacactccctctgccatctctcaccccctcctctg- tccaca caggtcagcccaaggccaccccctcggtcactctgttcccgccgtcctctgaggagctccaagccaacaaggct- acactg gtgtgtctcatgaatgacttttatccgggaatcttgacggtgacctggaaggcagatggtacccccatcaccca- gggcgt ggagatgaccacgccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagc- agtgga ggtcccgcagaagctacagctgccaggtcatgcacgaagggagcaccgtggagaagacggtggcccctgcagaa- tgttca taggttcccagccccgaccccacccaaaggggcctggagctgcaggatcccaggggaagggtctctctctgcat- cccaag ccatccagcccttctccctgtacccagtaaaccctaaataaataccctctttgtcaaccagaaa SEQ ID NO: 14 Human .lamda.5 amino acid sequence (GenBank Accession No = NP_064455) mrpgtgqggleapgepgpnlrqrwpllllglavvthgllrptaasqsralgpgapggssrsslrsrwgrfllqr- gswtgp rcwprgfqskhnsythvfgsgtqltvlsqpkatpsytlfppsseelqankatlyclmndfypgiltytwkadgt- pitqgv emttpskqsnnkyaassylsltpeqwrsrrsyscqvmhegstvektvapaecs SEQ ID NO: 15 Mouse .lamda.5nucleotide sequence (GenBank Accession No = AC_000038) ctggaatagcttttggccaccagaggaggaacaatccttttgccgggagatctacactgcaagtgaggctagag- ttgact ttggacttgagggtcaatgaagctcagagtaggacagactctgggcactatccccaggcagtgtgaagttctcc- tcctgc tgctgctgttgggtctagtggatggtgtccaccacatactttccccaagctcagcagaaaggagcagagctgtg- ggccct ggagcttcagtgggaagcaacaggcctagcctatgggcccttcccggcaggtaagagacttgctttttggggaa- ggtacg cgtgtaggtccacggactagaggctagaatgagtgactgggaaggaaggtggctattgaggccatgggtgtgag- aaggaa ggatctgcctaagaggagggggctgtgcaaatgctagcttaaacttggtacctcgatcattgcaaggccagtgt- tctacc aatgaaccacatccatagcccacctccattttccatttattttgagaagaggtcttgctaggtttcccaggctg- atctta ctctctgtagcctatgcaagatttgaacgtcctatagctttcctagatccttcctccttcctaagtcctcttta- gtagct agaattaagactgtggtggcgcgtttgtttttgttttttgttttttgtttttttgtgtgtgtgtgtgtgtgtgt- catggt cctagttcatttagaattaaatgtgtgtgtttatcgtagtcaggtagttgcagttctagaagtttattcttagg- taatta ttaaaacatccattaaatattgtgagggtgtcttgggttgggaggaggagacaccatggccatggagatcaccc- agtcaa gaagaccttgcctgtaaagcatgaggacttaattcagactctcagcaaccattcatctactatcaagatgtttt- tactca gataaatagaaaaaaaaaaaaaaaaggtttggtgtggtggcacaccttttatatcccagcagttgggaggcaga- ggcagg cagatctttgtgagtttgaggccagcctggtctacatagtagtctcatgatagaaataaaaacaaaagaatcct- tgcaat atagtgtgcatgggtcctgggaggaagcatagggccagtaggaactgatgcacacacgcatgtatgcacacacg- cacaag cacatatgcacacacaaatattcaccagagattcctggttccaccacacccaccagagaagatataccacccag- gaattt ctgagaatctgctgggcctgatattgccatcaacactgaaaaaattcagagatggaagtaagagatggatagat- ggactt gtcgggatactggctgctcatctattttctgccaaggacatagtttatttcctgaagttctgtgtctgactcac- ccaaca ggctcctgttccagatcatcccacggggagcaggtcccaggtgctcgccccataggcttccatctaagccccag- ttttgg tatgtctttggtggtgggacccagctcacaatcctaggtaagtggttctcatggtctcatgatccagctggctc- agggaa gtccatttttgctctggggaattcttactatctgcctttcctagccttgcagtctgaactgtaaaggcagtagt- aattct aaggtaaatcacagggaaaggccctaacagcttcatctactcttctgaggcagatgcccaaaaagagatcaagg- aatgag atttttcaggccatagagaccactacacctcagcatctaaaccgaggcccagatgcccatccagactaagaaga- ccacat agtaggtgcagggacatggctggtgccatgaaccccaccttcaaagattcaggccaccctagatagaaaaccac- agaggc tgagagagaaatttggttcaatttaatcttctgctggcccatgaggtcacagagacacacaaaaggctcagagt- gatcag gttactagaaccaggtcctcccaggatgattactagaaaagaaatgtagactgtctattgttcctgagggctgg- agcctg ttgtctaacttgtccatcttcctcaaatatccttagacttagatgaggaatgaaggagcaaatggggccaagtg- aaatca ggagtaaccagagacttcctggatggtcaggggtttgctatccttcctagtctgggagatggagcctcaggaac- acagcc agtataggtcttgtgattactgtggacaaagcagtaggtccttggaggagttggaggatttttctggatggaat- ctaatc ctcggtgagataactaaatggaatctggagcacaggccccgaagcccttatacagcaggcacacctcaagacca- actctc caggaccagtcttgcagaataaatgagcagcaattctcagaggagtctctgagcactggagaagcaattcaggt- tgggga gctgccctctgcctcaccagaaggccagggtcagatcccaattcactaccaggaggcctgggttagatcccaaa- tcaggt tccagcttcaaggggctagagaattcagctggtcttagtctcagcgggggaactgagattgcaagggtctgggt- ctgggt cattttatctggaagaggaacatgttctaatgggatgctaggctgtctgctctccaggggactcaagtggtcag- aggaga agaaggaagcatccctggatggaagactgatgctgtagtgaatggccacagagctcctgataagagaaggacgc- ttcctt atcacgtgggctctcctatgctaactcttatctccttctctatctgcgcaggtcagcccaagtctgaccccttg- gtcact ctgttcctgccttccttaaagaatcttcaggccaacaaggccacactagtgtgtttggtgagcgaattctaccc- aggtac tttggtggtggactggaaggtagatggggtccctgtcactcagggtgtagagacaacccaaccctccaaacaga- ccaaca acaaatacatggtcagcagctacctgacactgatatctgaccagtggatgcctcacagtagatacagctgccgg- gtcact catgaaggaaacactgtggagaagagtgtgtcacctgctgagtgttcttagaccacaatcctccctgaagcctc- aggggc ctggatctgaagtgccagaaaaagttgttttttgttttgttttttgttttttttcccattaaccatctcactgt- ctttcc tgtgcctaatactcaataaatatcttaccaccaac SEQ ID NO: 16 Mouse .lamda.5amino acid sequence (GenBank Accession No = NP_001177254) mklrygqtlgtiprqcevlllllllglvdgvhhilspssaersravgpgasvgsnrpslwalpgrllfqiiprg- agprcs phrlpskpqfwyvfgggtqltilgqpksdplvtlflpslknlqankatlvclvsefypgtlvvdwkvdgvpvtq- gvettq pskqtnnkymvssyltlisdqwmphsryscrvthegntveksyspaecs SEQ ID NO: 17 Rat .lamda.5nucleotide sequence (GenBank Accession No = NC_005110) atgaagctcagggcaggacagacactgggcactatccccaggcaatgtgaaattctccttctgttgctgctgtt- gggcct ggtggatggtgtccaccatatactttccccaagctcagcacaaaggggcagagctgtgggccctggagcctcag- tgggaa gcagcaggtctagcctgtggacccttccaggcaggtaagagactttcttataggggaatgtatgtgtgtgggtc- catgga ctggaggctgaaatgggtgactgggaaggaaggtaaccattgaggccataggtgtgagaaggaaggatctgcct- aagagg agggggctgggcaaatgctagcttaaacttagtacctaactcatgcaaggccagtattctatcaatgagccata- tccata gcccacctcccttttccatttatttagagaagagggcttgcctgattgcctaggctgatcttactttctgtagc- ctatgc aagatttgacctcctagatccttcctccttcctaagtcctctttagtagctggaattaaggctggtaccaggtt- atttta gtgtgtcatggtcctagttcattcagaattgtgtgtgctcatcttagtcaaatagttgcagttttagaagttta- ttctta ggcaattatcaaaacatccattaaatattctaagagtgtcttgggctgggaagagtagacactggggacatgga- gaccac ccagtgaagaagactttgactgtcaagcatgaggacctagttcagactctcagcactcatgcatctgctatcaa- gataca atcacatgtttttttactcagataaatagaaaaaaattaaggtttggtgtggtggcacatcccttatatcccag- cagttg

gcaggcagaggcagatagacctttgtgagtttgaggccagtctggtttacatagtggcctcatgacagaaataa- aacaaa agaattctcacaaaatatagtgtgcatgggtcctgggatgaagcacaggaccaggaggaatacacacacacaca- cacaca cacacacacacacacacacacacacacacaaatacacatcagagattcctggttccaccacactcaccagagaa- gatata ccacccagaacttttctgagaatcagttgggcctggtgttgccatcaactggatggaaataggagatggataga- tggact tattttctgccaaggacacagttcatttcctgaagtccggtgcctacctcacccaacaggttcctgttccagat- catccc acggggagcaggtcccaggtgctggccccataggcttccatccaagtcccagttatggtacgtctttggtagtg- ggaccc agctcacaatcctaggtaagcgattcccatggtctcatgatccagctgtcttagggaagtccctttttcctctg- gggagt tcttctcacctgcctttcctagtcttgtagtctgaatggtacctttttctgtgagtgaggggaaggcaagtagt- tctaag gtaaaccacaggaaaggtcccaatagcttcagctactcttccgaggcagatgtccaaaaagggatcaggggctg- aggttt tcaggctgtagagaccactgcacttcagcatctaaactgaggcccagatgcccatccagattaagaaggccaca- tagtag gtgcagggatatgactggtgccatgattcctgcctttaaagattcaggtaaccctagatagaaaaccacagagg- ctgaga gaagaatctggcccagtttaatcttctgctggccaatgaggtcatagagacacagaaaaggcttagagtgacca- ggtaac tagaaccagctcctcccaggatgattactagaaaaaaaatgtagactgtctattgttcctggggtttctcaggc- ctggag cctgttgtctaacttgtccatctccctcaaatgtccttagagttagatgaggaatgagggagcaaatggcgcca- agtgaa atcaggagtcaccagagacttcctgggtggtcaggggtttgctatccttcctagtctgggagacggagccccag- gagcac agtcagagtaggtcttgtgattactgtggacaaagcatcaggcccttggaggagttcctaggacttttcaggat- ggaatc taatccttggtgagataactaaatagaatctggagcacaggcccgggagcccttatacagcaggcacacctcaa- gaccaa ctccccaggaccagtcttgcagaataaatgagcagtaattctcagaggaggctctgacactggagaagcaatgg- gggttg gggagctgctccctgaactcccccacccccataggaggccagcgtcagatcccaattcagattccagctcctag- tggcta gagagtacagatggccttggtcttagtggggaaactgagattgcaaggggcagggtgtgggtcatttcacctgg- aagagg aacacggtctaatggggcaccaggctgtctgctctccaggggactcaggtgggcagaggaaaagaaggaagcat- ccttga tggaacactctgagctgtagtgaatggctacagggctcctgataagaggaggatgcttccctgtcatgtgggct- ctccta tgccaactcttatccccttctctatctgcacagggcagcccaagtctgaccccttggtcactctgttcctgcct- tcctta aagaatctccaggttaagaaggcgacactagtgtgtctggtgagcgaattctacccaggtactttggtggtgga- ctggaa ggtagatgggatccctgtcactcagggtgtggagacaacccaaccctccaaacagaccaacaacaagtacgtgg- ccagca gctacctgacactgatgtctgaccaatggatgcctcacagtagatacacctgccaggtcactcatgaaggaaac- actgtg gagaagagtgtgtcacctgctgaatgttcttag SEQ ID NO: 18 Rat .lamda.5amino acid sequence (GenBank Accession No = NP_001177270) mklragqtlgtiprqceilllllllglvdgvhhilspssaqrgravgpgasvgssrsslwtlpgrflfqiiprg- agprcw phrlpsksqlwyvfgsgtqltilgqpksdplvtlflpslknlqvkkatlvclvsefypgtlvvdwkvdgipvtq- gvettq pskqtnnkyvassyltlmsdqwmphsrytcqythegntveksyspaecs SEQ ID NO: 19 5' homology arm acctggccaaactgagcatgacctttgacctagccagctcttaaacttgttctgagatcacaaaccagccagac- caaatt SEQ ID NO: 20 3' homology arm tcctcccagaatgcttccctgggtcaaacccagagccacaaaggcttccattagaccattctggtaagtgacag- agtcac SEQ ID NO: 21 5' homology arm AAAAAAAAAAAAGCCCAGCTAGCTTAGTTGGTAGAGCATGAGACTCTTAATCTCAGAGTCATGGGTTCAGGC CTCATGTTTGGCACCATCTATAGTGTGCAGTTATAAATCAAACAGTTCACGATGTGGCTGGCTAGGCACTGGC AACTGCAGTCTCACCTGCTCCCATGGTTCCCAGTTCCCACAGCTAGTTTGCTGCCAGGCTGTTCACACTTCCCA GGTCATCTACCCACTGTGGCAAGCCTGCAGAAAGCCTGCTATTGCTAGCTCAGATTCCCTTAGCCCTATAAAAT GATAACACCACAGACTTTTACTATACCATCCAGATTTATAACAGTAATTCTCCAACCC SEQ ID NO: 22 3' homology arm CAGGAGTACACCAAAATGTCTAGCCAAAATTTTTATATATGATCACTTAAATAAGACTCCTTAACATAAACCTA CATGATATACCAAGTCTTTTCTGCCAAGGCTCTGACACTATAGTTTGTCCTATTCTGGAGTTAGGTAAGCAAAG GGCTATTTAGGTGTGGATTGCAAAGAGAGAATAGCAAGACAACCTGCCCATTCTTTGCCACACCTCACTAATC AGTGTCCCTTGGAAAGCACTGTAAATATGGAGGTTTCTTTTTGTATTATGTAGTAGTGGATTTAACTTGAGGAG CCCCAAAAGGGGTCAGCAAAGCATGGGAAATCTAAGAATTTAACACTTCAGTGACTTTTAATCACCTACAGAT CCAGGAAAATAAGCCTGTCTCTT

Sequence CWU 1

1

261727DNAHomo sapiens 1gagtcagagc tctgcatgtc tgcaccatgt cctgggctcc tgtcctgctc atgctgtttg 60tctactgcac aggtgaggga acccccagat cccaaagact cctgcccctt ccttcatcct 120gccctgcccc cacggcccac atgcatctgt gtcaccaggt tgtggtcctc agccggtgct 180gcatcagccg ccggccatgt cctcggccct tggaaccaca atccgcctca cctgcaccct 240gaggaacgac catgacatcg gtgtgtacag cgtctactgg taccagcaga ggccgggcca 300ccctcccagg ttcctgctga gatatttctc acaatcagac aagagccagg gcccccaggt 360cccccctcgc ttctctggat ccaaagatgt ggccaggaac agggggtatt tgagcatctc 420tgagctgcag cctgaggacg aggctatgta ttactgtgct atgggggccc gcagctcgga 480gaaggaggag agggagaggg agtgggagga agaaatggaa cccactgcag ccaggacacg 540tgtcccttga actgaagaca gcagaggcac gcatcccctt ggagagactg tcatggaaga 600gggtggagcc gccgcccgaa gcgccgagga ggctgagcca ctcagcatct cctggtcctg 660cagtgttgct gtaaatcccc attggagact gcattaggga attaaagctg cttgtcactt 720tttgctg 7272145PRThomo sapiens 2Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr Cys Thr Gly 1 5 10 15 Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30 Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45 Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60 Pro Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly 65 70 75 80 Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95 Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110 Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg Glu 115 120 125 Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg Thr Arg Val 130 135 140 Pro 145 31701DNAhomo sapiens 3cttcccagcc ctgtgcccca aagcacctgg agcatatagc cttgcagaac ttctacttgc 60ctgcctccct gcctctggcc atggcctgcc ggtgcctcag cttccttctg atggggacct 120tcctgtcagg tgaatctttc ctgggcctca gtcacctggg tgtggggtgg ggaacagtac 180tggccccaga aggcccctgc aaggaggcaa atcgatgggg acagtagggc aggtcctggg 240aggggtattt tttttttttt tgagacggag ttttgctctt gttgcccagg ctggagtgca 300acggtgcaat ctcggctcac tgcacctctg cctcccaggt tcaagcgatt cttctgcctc 360agcgtcctga gtagctggga ttacaggcat gcgccaccac gcctggctaa ttttgtattt 420ttagtagaga caaggtttct tcatgttggt caggctggtc tcaaactcct gacctcaggt 480gatccgccca cctcagcttc ccaaagtgct aggattacag gcttgagcca ctgcacctgg 540ctgggaggcg tattcaggta gaggggtgac ctggcttcag acagctctgc ccttgacctg 600gggcaaatca cttccctgtg tgggccttgg ttttctcatt cgtgaatgac cagatcacta 660gagccctgga ctctgacttt gggtctcctt cttagttttt cacaggggaa gctattgtgg 720gttggtccct accccacagg gcctgaggca atctaacctc cctgagaggg tccctgcagc 780cagttgcctg aggctgagtt gatgtgtggg acagcccagg aggttcctgg gggtggttag 840tctgtattca gggtttggaa gagctgaagt gaagtgggcg gggaagtggg ggagaggggt 900gcagttctgc agagaaacgt gggtgggtag cagagggacc tagaggctgc tagtccaacc 960ttctgagctc tgggccttta actgaacaca gatccttgag gatatggcac taatggagat 1020ttgggggcta actccaaacc cctcactcac aaagggagat ggaggtccag atagggcacc 1080ctaagtcaca cagaaccagg cctcctgcac cccgttcatt gctaatccca tagcactggg 1140ctatgagccc tttgggactg ggagtctcca tggagtccaa ccaagccttc acagggcagg 1200ggtgggaggg aaggggctca ggctgagtgg gtttgtgtct cgcagtttcc cagacagtcc 1260tggcccagct ggatgcactg ctggtcttcc caggccaagt ggctcaactc tcctgcacgc 1320tcagccccca gcacgtcacc atcagggact acggtgtgtc ctggtaccag cagcgggcag 1380gcagtgcccc tcgatatctc ctctactacc gctcggagga ggatcaccac cggcctgctg 1440acatccccga tcgattctcg gcagccaagg atgaggccca caatgcctgt gtcctcacca 1500ttagtcccgt gcagcctgaa gacgacgcgg attactactg ctctgttggc tacggcttta 1560gtccctaggg gtggggtgtg agatgggtgc ctcccctctg cctcccattt ctgcccctga 1620ccttgggtcc cttttaaact ttctctgagc cttgcttccc ctctgtaaaa tgggttaata 1680atattcaaca tgtcaacaac a 17014123PRThomo sapiens 4Met Ala Cys Arg Cys Leu Ser Phe Leu Leu Met Gly Thr Phe Leu Ser 1 5 10 15 Val Ser Gln Thr Val Leu Ala Gln Leu Asp Ala Leu Leu Val Phe Pro 20 25 30 Gly Gln Val Ala Gln Leu Ser Cys Thr Leu Ser Pro Gln His Val Thr 35 40 45 Ile Arg Asp Tyr Gly Val Ser Trp Tyr Gln Gln Arg Ala Gly Ser Ala 50 55 60 Pro Arg Tyr Leu Leu Tyr Tyr Arg Ser Glu Glu Asp His His Arg Pro 65 70 75 80 Ala Asp Ile Pro Asp Arg Phe Ser Ala Ala Lys Asp Glu Ala His Asn 85 90 95 Ala Cys Val Leu Thr Ile Ser Pro Val Gln Pro Glu Asp Asp Ala Asp 100 105 110 Tyr Tyr Cys Ser Val Gly Tyr Gly Phe Ser Pro 115 120 5855DNAMus musculus 5agagcccaga aagcctggga gggtggtgag caggaaccag gggtgcagtg accctctccc 60caaagcaggg aggagagtgc ttcccagctg gtcagggccc aggagcagtg gctgtagggg 120gcagggtgct gcaggtctgg agccatggcc tggacgtctg tcctgctcat gctgctggcc 180tatctcacag gtaaggaaac tcttggggcc cagggcttct ttgctcctcc tatggccttg 240ctctgcccca gtgatggaca tgcttctatc ttctcaggtt gtggccctca gcccatggtg 300catcagccac cattagcatc ttcttccctt ggagccacca tccgcctctc ctgtaccctg 360agcaacgacc ataacattgg catttacagc atttactggt accagcagag gccgggccac 420cctcccaggt tcctgctgag atacttctca cactcagaca agcaccaggg tcccgatatc 480ccacctcgct tctccgggtc caaagatacg accaggaacc tggggtatct gagcatctct 540gaactgcagc ctgaggacga ggctgtgtat tactgtgccg tggggctccg gagccaggaa 600aagaagagga tggagaggga gtgggaagga gaaaagtcgt atacagattt gggatcttag 660gctctggaga cattcagacc ctgaactgaa gacagagttt gctttgctcg gctagtctgg 720tatgggaagg aggggtagaa cgtgaggttt tgcagagcct agaagatgga attatgcagc 780ttttccttgt tctgcggtgt tgctatgagc ccccattgga ggctggattg tagaattaaa 840gctgttttta ctgaa 8556142PRTMus musculus 6Met Ala Trp Thr Ser Val Leu Leu Met Leu Leu Ala Tyr Leu Thr Gly 1 5 10 15 Cys Gly Pro Gln Pro Met Val His Gln Pro Pro Leu Ala Ser Ser Ser 20 25 30 Leu Gly Ala Thr Ile Arg Leu Ser Cys Thr Leu Ser Asn Asp His Asn 35 40 45 Ile Gly Ile Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60 Pro Arg Phe Leu Leu Arg Tyr Phe Ser His Ser Asp Lys His Gln Gly 65 70 75 80 Pro Asp Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Thr Arg Asn 85 90 95 Leu Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val 100 105 110 Tyr Tyr Cys Ala Val Gly Leu Arg Ser Gln Glu Lys Lys Arg Met Glu 115 120 125 Arg Glu Trp Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser 130 135 140 7516DNAMus musculus 7atggcctgga cgtctgtcct gctcatgctg ctggcccacc tcacaggtaa gggaactctt 60ggggtccagg gcttccttgc tcctcctgtg gccttgctct gccccagtga tggacatgct 120tctatcttct caggttgtgg ccctcagccc atggtgcatc agccaccatc agcatcttct 180tcccttggag ccaccatccg cctctcctgt accctgagca acgaccataa cattggcatt 240tacagcattt actggtacca gcagaggccg ggccaccctc ccaggttcct gctgagatac 300ttctcacact cagacaagca ccagggtccc gatatcccac ctcgcttctc cgggtccaaa 360gatacggcca ggaacctggg gtatctgagc atctctgaac tgcagcctga ggacgaggct 420gtgtattact gcgctgtggg gctccggagc cacgaaaaga agagaatgga gagagagtgg 480gaaggagaaa agtcgtatac agatttggga tcttag 5168142PRTMus musculus 8Met Ala Trp Thr Ser Val Leu Leu Met Leu Leu Ala His Leu Thr Gly 1 5 10 15 Cys Gly Pro Gln Pro Met Val His Gln Pro Pro Ser Ala Ser Ser Ser 20 25 30 Leu Gly Ala Thr Ile Arg Leu Ser Cys Thr Leu Ser Asn Asp His Asn 35 40 45 Ile Gly Ile Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60 Pro Arg Phe Leu Leu Arg Tyr Phe Ser His Ser Asp Lys His Gln Gly 65 70 75 80 Pro Asp Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn 85 90 95 Leu Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val 100 105 110 Tyr Tyr Cys Ala Val Gly Leu Arg Ser His Glu Lys Lys Arg Met Glu 115 120 125 Arg Glu Trp Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser 130 135 140 9878DNARattus norvegicus 9ccagaaagcc tgggagggtg gtgagcagga accagtggtg caaagcaggg gcgagactgc 60ttcctagctg gtcagggcac cggagcagtg gctgtagggg tcagggtgct gcaggtctgg 120aaccatggcc tggacgtctg ccctgctcat actgctggcc catctcacag gtacgggaac 180tcttggggcc cagagcctcc ttgctcctcc tcttgccttg ctctgccgca gtgatgggca 240cgcttctatt ttctcaggtt gtggccctca gcccgtgctg catcagccac catcggcctc 300ttccttcctt ggaacctcca tccgcctcac ctgtgccctg agcagcaacc ataacattgg 360catttacagc atttactggt accagcagag gccgggccac cctcccacgt tcctgctgag 420attcttctca cactcagaca agctccaggg tcccaagatc ccccctcgct tctccggatc 480caaagataca gccaggaacc tggggtacct gagcatctct gacctgcagc cagaggacga 540ggctgtgtat tactgcgccg tggggcttcg gagctgggaa aaggagaaga ggatggagag 600ggagtgggaa gaagaaaagt agcggacaga ttcgggatct taggctctgg agacattcag 660acctagaacc gaagacggag tttgctttgc tcggctaggc tggtttgggg aggaggggta 720gaacaccggg cttcgcagag ccaggaaggt ggagccagcc gcttttcctt gtattgcagt 780gttgctatgc gccccatcgg aggctggatt gtagaattaa agctgttttt tttttttttg 840ttttgttttt ttgttttttg tttctttttt cttaactg 87810136PRTRattus norvegicus 10Met Ala Trp Thr Ser Ala Leu Leu Ile Leu Leu Ala His Leu Thr Gly 1 5 10 15 Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ser Ala Ser Ser Phe 20 25 30 Leu Gly Thr Ser Ile Arg Leu Thr Cys Ala Leu Ser Ser Asn His Asn 35 40 45 Ile Gly Ile Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60 Pro Thr Phe Leu Leu Arg Phe Phe Ser His Ser Asp Lys Leu Gln Gly 65 70 75 80 Pro Lys Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn 85 90 95 Leu Gly Tyr Leu Ser Ile Ser Asp Leu Gln Pro Glu Asp Glu Ala Val 100 105 110 Tyr Tyr Cys Ala Val Gly Leu Arg Ser Trp Glu Lys Glu Lys Arg Met 115 120 125 Glu Arg Glu Trp Glu Glu Glu Lys 130 135 11711DNARattus norvegicus 11atggcctgga cgtctgccct gctcatactg ctggcccatc tcacaggtac gggaactctt 60ggggcccaga gcctccttgc tcctcctctt gccttgctct gccgcagtga tgggcacgct 120tctattttct caggttgtgg ccctcagccc gtgctgcatc agccaccatc ggcctcttcc 180ttccttggaa cctccatccg cctcacctgt gccctgagca gcaaccataa cattggcatt 240tacagcattt actggtacca gcagaggccg ggccaccctc ccacgttcct gctgagattc 300ttctcacact cagacaagct ccagggtccc aagatccccc ctcgcttctc cggatccaaa 360gatatagcca ggaacctggg gtacctgagc atctctgacc tgcagccaga ggacgaggct 420gtgtattact gcgccgtggg gcttcggagc tgggaaaagg agaagaggat ggagagggag 480tgggaagaag aaaagtagcg gacagattcg ggatcttagg ctctggagac attcagacct 540agaaccgaag acggagtttg ctttgctcgg ctaggctggt ttggggagga ggggtagaac 600accgggcttc gcagagccag gaaggtggag ccagccgctt ttccttgtat tgcagtgttg 660ctatgcgccc catcggaggc tggattgtag aattaaagct gttttttact g 71112136PRTRattus norvegicus 12Met Ala Trp Thr Ser Ala Leu Leu Ile Leu Leu Ala His Leu Thr Gly 1 5 10 15 Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ser Ala Ser Ser Phe 20 25 30 Leu Gly Thr Ser Ile Arg Leu Thr Cys Ala Leu Ser Ser Asn His Asn 35 40 45 Ile Gly Ile Tyr Ser Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60 Pro Thr Phe Leu Leu Arg Phe Phe Ser His Ser Asp Lys Leu Gln Gly 65 70 75 80 Pro Lys Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Ile Ala Arg Asn 85 90 95 Leu Gly Tyr Leu Ser Ile Ser Asp Leu Gln Pro Glu Asp Glu Ala Val 100 105 110 Tyr Tyr Cys Ala Val Gly Leu Arg Ser Trp Glu Lys Glu Lys Arg Met 115 120 125 Glu Arg Glu Trp Glu Glu Glu Lys 130 135 137184DNAHomo sapiens 13ggccacatgg actggggtgc aatgggacag ctgctgccag cgagagggac cagggcacca 60ctctctaggg agcccacact gcaagtcagg ccacaaggac ctctgaccct gagggccgat 120gaggccaggg acaggccagg ggggccttga ggcccctggt gagccaggcc ccaacctcag 180gcagcgctgg cccctgctgc tgctgggtct ggccgtggta acccatggcc tgctgcgccc 240aacagctgca tcgcagagca gggccctggg ccctggagcc cctggaggaa gcagccggtc 300cagcctgagg agccggtggg gcaggtaagg ggtgagagat tccaggggat gtgggggtct 360gggtggcaga ggcgggaaag gatgaccaag gggagacgag ccagaggggt gaggaggaag 420gttaatccct ggaggggagc cacagacact gactttaact aaagtgtcaa gattttgtcc 480atctttgaat taatttttat tgcttaatgt catattaaaa tattatttat cttgattcct 540gagatttctt cccccactta catttggcac caaggccaat gtctccctca cctcccccta 600gtccttgggg tagggcagga ctggaggcag gggcaggacg tccacaggag tggtggccgc 660tatccctgaa ggatgcccag gcctctccct cctcctcctc ccactcctcc tcccccccct 720cctcttttcc ccttggccta tgtcacctgt ccactcccac cctcactggg caggggccac 780tccctggagc tccagctaag gtgtgagggg cctttcctgg agtccctggg tcactagacc 840tcagccagca tcgcctcctg aaaccagccc ctaggagaca caagcttatc cagggtgcaa 900gtgcctccaa agaagaagcc caggagaggc tctagggagg ccacaactcc ctgtgtgacc 960tcagccattg ccaccactct gcgtgtggtg ggaggtcccc agacaaagca ccaagcatcg 1020gggtccattt atgagcattt gggacacaac agcctgttca ctggtgcatg ttatacccac 1080aggcgataat catttcaggg gcagaacctc cctctcggtg gccccatagg caggtcctgc 1140tgtgatcccc tttgtgcaga cggggataga gcccggagag gtgaggtgac ccgtccgagt 1200cactcagctc atgggcacag attctaaggc ccaagctatc ccctctagct ctcccctgtc 1260ccatcctcta agctgatcga gcggacacgt gcatctctgg gacctgagtt tccctttttc 1320tctctttttt ttctttttca aataaagttt cacagagttt cactcttgtc gcccagtctg 1380gagtgcaatg gcgagatctt ggctcattga aacctccgtc tcccaggttc aagacattct 1440cctgcctcag cttccggagt agctgggatt acaggcatct gccaccacac ctggctaatt 1500ttgtgtattt ttttttagta aagacagagt ttcaccatgt tggccaggct ggtctcaaac 1560tcctgacctc aggtgatcca cccacctcag cctcccaaag tgctgggatt acaggcatga 1620gacaccacac cgggcctgag tttccccttc tgcaatctga ggggccctga ctggtgaggg 1680ccttcagcgt cccacccacc cagaggatgc tggggtggct gtggtgagag ctccagcagt 1740ggcagccgac ctgacccaca ccaggagccc ggccatggag gcggggtcag catggtggca 1800ggccgggacc gggtgtcagt gtcctgcacg gacttctgag caaggagtcc ccatcagggt 1860caggctctgt gctggggctg aggtcccaga ggatctagat ttgccccaat tcaagtccac 1920aaggagcggg ggccgggtga ggagacagcc acatgcaggg tgatgcctac agaacagaga 1980ctgggatggg gaaggcccga ggggtctcca caagggacgg gtgacaggtg gagggagaca 2040cagataataa aaaatggtat tatgttgggg ggctattaat gtaagttttt atattagaat 2100ctttagaaat cttatagaaa tactatgggc cgggtgctgt gtctcatgcc tgtaatccca 2160gcactttagg acgccaagat gggcagatca cgaggtcagg agattgagac catcctggcc 2220aacatggcaa aactccgtct ctactaaaaa tacaaaaact agctgggcgt ggtggcgcga 2280gcctatagtc ccagctactc gggaggggag gcaggagaat cgcttgaatc agggaggtgg 2340aggttgcagt gagctgagat tgcaccgctg cactccagcc tggacgacag aacgagactc 2400ccactcaaaa aaaaaaaaag gtattatgct gggggggata tgaatatgag tttttataat 2460ctttagaaat actatgggcc aggtgcagtg gctcatgcct gtaatcccac aactttggga 2520ggctgaggcg ggattgtttg agcccaggag tttgagatca gcctgagcaa catagcaaga 2580ccccatttct acaaaacata taaaaactag ctagtcatgg tggcacttgc ctgtggtccc 2640agctacttag gaggctggtg agaggattgc ttaagcctcg gaggttgagg atgcagtaag 2700ctgagatccc accactgcac tccagcctgg gtgacagaag gagaccctgt ctcaaaaaaa 2760aaaaaaaaaa aaaaagactg attattcctg tagaattctg gtaaatatct cctatcaaat 2820aaatgacttt tctattcata gcttattaaa agatattttc attgttttta agaaataggt 2880tgtgtatcac tttttatatt tagttgtaaa tttatttgtt ttatttattt ttttagagat 2940gggggtctca ctatgttgcc tgggctggcc tccaactgct gggctcaagc aatatccctg 3000cctcagcgtc cccagtagct gggactacaa gcatgcgcta ccacaccggc ataatttttt 3060gtagagatga ggtttcgcca tgttgcctgg gctggtcttg aacccctggg ctcaagccat 3120ccacccgcct cggcctccta aagtgctgag attacagacg tgagccaccc tacctagcct 3180gtactatttt taataggtct taataggttt tgaatgttaa ttatttttaa attaatttca 3240aaaatcttct acaacatgga tgaaacctga agacattata cttagtgaaa taagccagac 3300acaaaaggac aaatgtcatt tgaatccact tctatgaggt acctagaata ggcaaattca 3360cttggacaaa aagtagattt gaggttagca gggtgagggg gagggaagaa tgggggactg 3420tagttaacgg gtttagagtt tctgtttggg aagatgaaag agttctggag atggatggtg 3480gtgaaggttg cccaacggtg tgaatgtact tagtgccacg gagctgtacg tttaaaaata 3540gttaaagtgg aaatattgat gctatgtata aaaatggagc ggggtgtggt ggctcacacc 3600tataatccca gcactttgag aggccaaggt gggcagatca cctgaggtcg ggagttcgag 3660accagcctga caaacatgga gaaacaccgt ctctactgaa aatacaaaaa attagccaag 3720tgtgctggca catgcctgta atcccagcta ctcgggaggc tgaagcagga gaatcgcttg 3780aacccaggat gtggaggttg cggtgagcca

aggtggcacc attgcactcc agcctgggcg 3840acaagggtga aattccattt caaaaaaata aaaggaaatg ggactgtaca tagcaggaga 3900gagagggaga gatcaatatg acacttcttt tttttttttt ttttttttgg gacagtcttg 3960ctctgttgcc aggctgcact ccagcctggg caacagagcg agactctgtc tcaaaaaaaa 4020aaagaatgtc tagatgcagt agctaagttc gcagaagcca tttcagtgtg ggaggcgagg 4080tgactgagtc cagggactcc aggtttctgg ctgaggggtt gagaccgccc atggtcattg 4140tgatgagatg aagacagaaa atgagctggg ctgggggaag gtggtcatct cctctggacg 4200tgattagttt gaggctcctg ttgggtagcc cactgggcaa ggtcagtagg caattgagga 4260gctgagaggg tttggagctg ggatagactc cagcctcacc atgtgggcaa tagtgggggt 4320cacagagtgt gaaaatggct aagaaagagg tctgggtggg gtctggggag tcagcagcca 4380ggcatgggcc atggagaaac agatgccagg ggaagaggaa aggggagtct cagaccccaa 4440ggggaaaaga gtcactggaa agaggggcca gccctgtgtc gcatccagcg gagacaccag 4500gtacagcaga aggaccttgg acatgaccat gaggagggcc ttgttgacca tggcctggga 4560gagatggggg tgagagcctg ggggaccaca ccatgtcccc agcacacagt gcctggtaga 4620caggatggat ttatggatgg acggacaggt agatggatgg acgaatggac agatgataga 4680tggatgcaaa gacagatgaa tagatggaca gatgcataga tggacagatg gacagatgga 4740tggacggacg gatggaatga atgatcagaa aaggcttcat gaacaaagtg agactgagct 4800gcatctccat gggtagatat aaaagcagag gactctcctc ttgagtcagg aatgacccaa 4860tgtcctggtc cagggaggaa gtcagcctcc ttgactgggg acacttgtgg cagatttcag 4920aggcccttaa aatgaggcca agtgaggtgg acaggtccga gccagctgag gactcctcag 4980ccacacggca cagctgcctg aggggatgtg tcactcaggg agttgctggg acctactggg 5040cccagcgttg ccatcagcac caacagtttc agagaggggg acacacgctg gggcagcacc 5100tgcctcagag aagggacagg cacagagaca ctactggggg acactactgg gacactggcc 5160acccccctac cctgtgcctg ggtcacagcc tacacactgc agccctgtgc ccctcactcc 5220cagcaggttc ctgctccagc gcggctcctg gactggcccc aggtgctggc cccgggggtt 5280tcaatccaag cataactcag tgacgcatgt gtttggcagc gggacccagc tcaccgtttt 5340aagtaagtgg ctctaacttc ccaggctgtc ccaccctctc ctgtctctgg aaaatgtgtt 5400ttctctctct ggggcttctt cccctctgcc ctcccagcct taagcactga cccctacctt 5460tgtccatggg gcctggagga gatgtgttag tctcagggta atggcaggaa gggcccccac 5520agtgggagca gccgccttca ggttccaaca gcaggacaca gcctggtccc agggcctggg 5580ctgggattgg gcggggtcag ggctcctccc ctctcccagg gcagatgtct gagtgaggga 5640cagaggctgg ttctgatgag gggccctgca gtggccttag agacagtccc tgggacccca 5700ggttctaggc tgagggctgg atgcccatcc agcctgggag ggccacacgg gggcctgggg 5760acacaggggt cacccccagg ggagaccaat ggagggcaca gagagggctc tgggtctagg 5820ctgcagctct gtggcctctg ctgggtcttc agggcatggg gacacagagg aacggatgag 5880gtcccagagc ccagccctcc caggacagtc accagaaagg agagggtctc ttagtgcaga 5940gatgtgcctg tccctggagc cctgtcatct ctggggcctg gtgtctctct gttcatgggt 6000cgacctccca ccttcatttg aggaagggca ccttagactc agaaggtgac tagcggggag 6060taaacgggag tgcagagaac tccatggctg ccaggtgaag tccaggggca tcagaggctg 6120ctggggtggg catgggggct gcggtgcccc aaagtctggg ggagcagccc caagaaccca 6180gccgatgtga agggtcctgt ggtcgggctg gtggggacag gggcgacggc agagccccag 6240ggtgtgtctg ggtggagccc acgcttcacc aggagagctg agtgggccag gctggggcac 6300agcctggtgc cccaggggat gggaagctcc aggccatgcc aggcttgggt ctccccacat 6360cctgccagta tagttttgtg tgctgtgagg gagaccccta gattccaaac tcagactcca 6420gaaaccagga aggagggagc acagcctgcc ctgggtgcac acggggaaac cgaggctgca 6480gaggaaaggg ctgggccagg acacctggga aaggtgactt gggaagggct cctaggaagg 6540cacagggctg tctgctctcc agagggctcc agtggaaagg agggaatgag gagggaagga 6600gaggccctgg gtggaccagg cggccacacc atgaaccctc ccagagactt tagacagaga 6660gaggcgctcc acaacacccc acactccctc tgccatctct caccccctcc tctgtccaca 6720caggtcagcc caaggccacc ccctcggtca ctctgttccc gccgtcctct gaggagctcc 6780aagccaacaa ggctacactg gtgtgtctca tgaatgactt ttatccggga atcttgacgg 6840tgacctggaa ggcagatggt acccccatca cccagggcgt ggagatgacc acgccctcca 6900aacagagcaa caacaagtac gcggccagca gctacctgag cctgacgccc gagcagtgga 6960ggtcccgcag aagctacagc tgccaggtca tgcacgaagg gagcaccgtg gagaagacgg 7020tggcccctgc agaatgttca taggttccca gccccgaccc cacccaaagg ggcctggagc 7080tgcaggatcc caggggaagg gtctctctct gcatcccaag ccatccagcc cttctccctg 7140tacccagtaa accctaaata aataccctct ttgtcaacca gaaa 718414213PRTHomo sapiens 14Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro 1 5 10 15 Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly Leu Ala 20 25 30 Val Val Thr His Gly Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser Arg 35 40 45 Ala Leu Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu Arg 50 55 60 Ser Arg Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly Pro 65 70 75 80 Arg Cys Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr His 85 90 95 Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala 100 105 110 Thr Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 115 120 125 Asn Lys Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro Gly Ile 130 135 140 Leu Thr Val Thr Trp Lys Ala Asp Gly Thr Pro Ile Thr Gln Gly Val 145 150 155 160 Glu Met Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175 Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr 180 185 190 Ser Cys Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205 Pro Ala Glu Cys Ser 210 153315DNAMus musculus 15ctggaatagc ttttggccac cagaggagga acaatccttt tgccgggaga tctacactgc 60aagtgaggct agagttgact ttggacttga gggtcaatga agctcagagt aggacagact 120ctgggcacta tccccaggca gtgtgaagtt ctcctcctgc tgctgctgtt gggtctagtg 180gatggtgtcc accacatact ttccccaagc tcagcagaaa ggagcagagc tgtgggccct 240ggagcttcag tgggaagcaa caggcctagc ctatgggccc ttcccggcag gtaagagact 300tgctttttgg ggaaggtacg cgtgtaggtc cacggactag aggctagaat gagtgactgg 360gaaggaaggt ggctattgag gccatgggtg tgagaaggaa ggatctgcct aagaggaggg 420ggctgtgcaa atgctagctt aaacttggta cctcgatcat tgcaaggcca gtgttctacc 480aatgaaccac atccatagcc cacctccatt ttccatttat tttgagaaga ggtcttgcta 540ggtttcccag gctgatctta ctctctgtag cctatgcaag atttgaacgt cctatagctt 600tcctagatcc ttcctccttc ctaagtcctc tttagtagct agaattaaga ctgtggtggc 660gcgtttgttt ttgttttttg ttttttgttt ttttgtgtgt gtgtgtgtgt gtgtcatggt 720cctagttcat ttagaattaa atgtgtgtgt ttatcgtagt caggtagttg cagttctaga 780agtttattct taggtaatta ttaaaacatc cattaaatat tgtgagggtg tcttgggttg 840ggaggaggag acaccatggc catggagatc acccagtcaa gaagaccttg cctgtaaagc 900atgaggactt aattcagact ctcagcaacc attcatctac tatcaagatg tttttactca 960gataaataga aaaaaaaaaa aaaaaggttt ggtgtggtgg cacacctttt atatcccagc 1020agttgggagg cagaggcagg cagatctttg tgagtttgag gccagcctgg tctacatagt 1080agtctcatga tagaaataaa aacaaaagaa tccttgcaat atagtgtgca tgggtcctgg 1140gaggaagcat agggccagta ggaactgatg cacacacgca tgtatgcaca cacgcacaag 1200cacatatgca cacacaaata ttcaccagag attcctggtt ccaccacacc caccagagaa 1260gatataccac ccaggaattt ctgagaatct gctgggcctg atattgccat caacactgaa 1320aaaattcaga gatggaagta agagatggat agatggactt gtcgggatac tggctgctca 1380tctattttct gccaaggaca tagtttattt cctgaagttc tgtgtctgac tcacccaaca 1440ggctcctgtt ccagatcatc ccacggggag caggtcccag gtgctcgccc cataggcttc 1500catctaagcc ccagttttgg tatgtctttg gtggtgggac ccagctcaca atcctaggta 1560agtggttctc atggtctcat gatccagctg gctcagggaa gtccattttt gctctgggga 1620attcttacta tctgcctttc ctagccttgc agtctgaact gtaaaggcag tagtaattct 1680aaggtaaatc acagggaaag gccctaacag cttcatctac tcttctgagg cagatgccca 1740aaaagagatc aaggaatgag atttttcagg ccatagagac cactacacct cagcatctaa 1800accgaggccc agatgcccat ccagactaag aagaccacat agtaggtgca gggacatggc 1860tggtgccatg aaccccacct tcaaagattc aggccaccct agatagaaaa ccacagaggc 1920tgagagagaa atttggttca atttaatctt ctgctggccc atgaggtcac agagacacac 1980aaaaggctca gagtgatcag gttactagaa ccaggtcctc ccaggatgat tactagaaaa 2040gaaatgtaga ctgtctattg ttcctgaggg ctggagcctg ttgtctaact tgtccatctt 2100cctcaaatat ccttagactt agatgaggaa tgaaggagca aatggggcca agtgaaatca 2160ggagtaacca gagacttcct ggatggtcag gggtttgcta tccttcctag tctgggagat 2220ggagcctcag gaacacagcc agtataggtc ttgtgattac tgtggacaaa gcagtaggtc 2280cttggaggag ttggaggatt tttctggatg gaatctaatc ctcggtgaga taactaaatg 2340gaatctggag cacaggcccc gaagccctta tacagcaggc acacctcaag accaactctc 2400caggaccagt cttgcagaat aaatgagcag caattctcag aggagtctct gagcactgga 2460gaagcaattc aggttgggga gctgccctct gcctcaccag aaggccaggg tcagatccca 2520attcactacc aggaggcctg ggttagatcc caaatcaggt tccagcttca aggggctaga 2580gaattcagct ggtcttagtc tcagcggggg aactgagatt gcaagggtct gggtctgggt 2640cattttatct ggaagaggaa catgttctaa tgggatgcta ggctgtctgc tctccagggg 2700actcaagtgg tcagaggaga agaaggaagc atccctggat ggaagactga tgctgtagtg 2760aatggccaca gagctcctga taagagaagg acgcttcctt atcacgtggg ctctcctatg 2820ctaactctta tctccttctc tatctgcgca ggtcagccca agtctgaccc cttggtcact 2880ctgttcctgc cttccttaaa gaatcttcag gccaacaagg ccacactagt gtgtttggtg 2940agcgaattct acccaggtac tttggtggtg gactggaagg tagatggggt ccctgtcact 3000cagggtgtag agacaaccca accctccaaa cagaccaaca acaaatacat ggtcagcagc 3060tacctgacac tgatatctga ccagtggatg cctcacagta gatacagctg ccgggtcact 3120catgaaggaa acactgtgga gaagagtgtg tcacctgctg agtgttctta gaccacaatc 3180ctccctgaag cctcaggggc ctggatctga agtgccagaa aaagttgttt tttgttttgt 3240tttttgtttt ttttcccatt aaccatctca ctgtctttcc tgtgcctaat actcaataaa 3300tatcttacca ccaac 331516209PRTMus musculus 16Met Lys Leu Arg Val Gly Gln Thr Leu Gly Thr Ile Pro Arg Gln Cys 1 5 10 15 Glu Val Leu Leu Leu Leu Leu Leu Leu Gly Leu Val Asp Gly Val His 20 25 30 His Ile Leu Ser Pro Ser Ser Ala Glu Arg Ser Arg Ala Val Gly Pro 35 40 45 Gly Ala Ser Val Gly Ser Asn Arg Pro Ser Leu Trp Ala Leu Pro Gly 50 55 60 Arg Leu Leu Phe Gln Ile Ile Pro Arg Gly Ala Gly Pro Arg Cys Ser 65 70 75 80 Pro His Arg Leu Pro Ser Lys Pro Gln Phe Trp Tyr Val Phe Gly Gly 85 90 95 Gly Thr Gln Leu Thr Ile Leu Gly Gln Pro Lys Ser Asp Pro Leu Val 100 105 110 Thr Leu Phe Leu Pro Ser Leu Lys Asn Leu Gln Ala Asn Lys Ala Thr 115 120 125 Leu Val Cys Leu Val Ser Glu Phe Tyr Pro Gly Thr Leu Val Val Asp 130 135 140 Trp Lys Val Asp Gly Val Pro Val Thr Gln Gly Val Glu Thr Thr Gln 145 150 155 160 Pro Ser Lys Gln Thr Asn Asn Lys Tyr Met Val Ser Ser Tyr Leu Thr 165 170 175 Leu Ile Ser Asp Gln Trp Met Pro His Ser Arg Tyr Ser Cys Arg Val 180 185 190 Thr His Glu Gly Asn Thr Val Glu Lys Ser Val Ser Pro Ala Glu Cys 195 200 205 Ser 172993DNARattus norvegicus 17atgaagctca gggcaggaca gacactgggc actatcccca ggcaatgtga aattctcctt 60ctgttgctgc tgttgggcct ggtggatggt gtccaccata tactttcccc aagctcagca 120caaaggggca gagctgtggg ccctggagcc tcagtgggaa gcagcaggtc tagcctgtgg 180acccttccag gcaggtaaga gactttctta taggggaatg tatgtgtgtg ggtccatgga 240ctggaggctg aaatgggtga ctgggaagga aggtaaccat tgaggccata ggtgtgagaa 300ggaaggatct gcctaagagg agggggctgg gcaaatgcta gcttaaactt agtacctaac 360tcatgcaagg ccagtattct atcaatgagc catatccata gcccacctcc cttttccatt 420tatttagaga agagggcttg cctgattgcc taggctgatc ttactttctg tagcctatgc 480aagatttgac ctcctagatc cttcctcctt cctaagtcct ctttagtagc tggaattaag 540gctggtacca ggttatttta gtgtgtcatg gtcctagttc attcagaatt gtgtgtgctc 600atcttagtca aatagttgca gttttagaag tttattctta ggcaattatc aaaacatcca 660ttaaatattc taagagtgtc ttgggctggg aagagtagac actggggaca tggagaccac 720ccagtgaaga agactttgac tgtcaagcat gaggacctag ttcagactct cagcactcat 780gcatctgcta tcaagataca atcacatgtt tttttactca gataaataga aaaaaattaa 840ggtttggtgt ggtggcacat cccttatatc ccagcagttg gcaggcagag gcagatagac 900ctttgtgagt ttgaggccag tctggtttac atagtggcct catgacagaa ataaaacaaa 960agaattctca caaaatatag tgtgcatggg tcctgggatg aagcacagga ccaggaggaa 1020tacacacaca cacacacaca cacacacaca cacacacaca cacacacaca aatacacatc 1080agagattcct ggttccacca cactcaccag agaagatata ccacccagaa cttttctgag 1140aatcagttgg gcctggtgtt gccatcaact ggatggaaat aggagatgga tagatggact 1200tattttctgc caaggacaca gttcatttcc tgaagtccgg tgcctacctc acccaacagg 1260ttcctgttcc agatcatccc acggggagca ggtcccaggt gctggcccca taggcttcca 1320tccaagtccc agttatggta cgtctttggt agtgggaccc agctcacaat cctaggtaag 1380cgattcccat ggtctcatga tccagctgtc ttagggaagt ccctttttcc tctggggagt 1440tcttctcacc tgcctttcct agtcttgtag tctgaatggt acctttttct gtgagtgagg 1500ggaaggcaag tagttctaag gtaaaccaca ggaaaggtcc caatagcttc agctactctt 1560ccgaggcaga tgtccaaaaa gggatcaggg gctgaggttt tcaggctgta gagaccactg 1620cacttcagca tctaaactga ggcccagatg cccatccaga ttaagaaggc cacatagtag 1680gtgcagggat atgactggtg ccatgattcc tgcctttaaa gattcaggta accctagata 1740gaaaaccaca gaggctgaga gaagaatctg gcccagttta atcttctgct ggccaatgag 1800gtcatagaga cacagaaaag gcttagagtg accaggtaac tagaaccagc tcctcccagg 1860atgattacta gaaaaaaaat gtagactgtc tattgttcct ggggtttctc aggcctggag 1920cctgttgtct aacttgtcca tctccctcaa atgtccttag agttagatga ggaatgaggg 1980agcaaatggc gccaagtgaa atcaggagtc accagagact tcctgggtgg tcaggggttt 2040gctatccttc ctagtctggg agacggagcc ccaggagcac agtcagagta ggtcttgtga 2100ttactgtgga caaagcatca ggcccttgga ggagttccta ggacttttca ggatggaatc 2160taatccttgg tgagataact aaatagaatc tggagcacag gcccgggagc ccttatacag 2220caggcacacc tcaagaccaa ctccccagga ccagtcttgc agaataaatg agcagtaatt 2280ctcagaggag gctctgacac tggagaagca atgggggttg gggagctgct ccctgaactc 2340ccccaccccc ataggaggcc agcgtcagat cccaattcag attccagctc ctagtggcta 2400gagagtacag atggccttgg tcttagtggg gaaactgaga ttgcaagggg cagggtgtgg 2460gtcatttcac ctggaagagg aacacggtct aatggggcac caggctgtct gctctccagg 2520ggactcaggt gggcagagga aaagaaggaa gcatccttga tggaacactc tgagctgtag 2580tgaatggcta cagggctcct gataagagga ggatgcttcc ctgtcatgtg ggctctccta 2640tgccaactct tatccccttc tctatctgca cagggcagcc caagtctgac cccttggtca 2700ctctgttcct gccttcctta aagaatctcc aggttaagaa ggcgacacta gtgtgtctgg 2760tgagcgaatt ctacccaggt actttggtgg tggactggaa ggtagatggg atccctgtca 2820ctcagggtgt ggagacaacc caaccctcca aacagaccaa caacaagtac gtggccagca 2880gctacctgac actgatgtct gaccaatgga tgcctcacag tagatacacc tgccaggtca 2940ctcatgaagg aaacactgtg gagaagagtg tgtcacctgc tgaatgttct tag 299318209PRTRattus norvegicus 18Met Lys Leu Arg Ala Gly Gln Thr Leu Gly Thr Ile Pro Arg Gln Cys 1 5 10 15 Glu Ile Leu Leu Leu Leu Leu Leu Leu Gly Leu Val Asp Gly Val His 20 25 30 His Ile Leu Ser Pro Ser Ser Ala Gln Arg Gly Arg Ala Val Gly Pro 35 40 45 Gly Ala Ser Val Gly Ser Ser Arg Ser Ser Leu Trp Thr Leu Pro Gly 50 55 60 Arg Phe Leu Phe Gln Ile Ile Pro Arg Gly Ala Gly Pro Arg Cys Trp 65 70 75 80 Pro His Arg Leu Pro Ser Lys Ser Gln Leu Trp Tyr Val Phe Gly Ser 85 90 95 Gly Thr Gln Leu Thr Ile Leu Gly Gln Pro Lys Ser Asp Pro Leu Val 100 105 110 Thr Leu Phe Leu Pro Ser Leu Lys Asn Leu Gln Val Lys Lys Ala Thr 115 120 125 Leu Val Cys Leu Val Ser Glu Phe Tyr Pro Gly Thr Leu Val Val Asp 130 135 140 Trp Lys Val Asp Gly Ile Pro Val Thr Gln Gly Val Glu Thr Thr Gln 145 150 155 160 Pro Ser Lys Gln Thr Asn Asn Lys Tyr Val Ala Ser Ser Tyr Leu Thr 165 170 175 Leu Met Ser Asp Gln Trp Met Pro His Ser Arg Tyr Thr Cys Gln Val 180 185 190 Thr His Glu Gly Asn Thr Val Glu Lys Ser Val Ser Pro Ala Glu Cys 195 200 205 Ser 1980DNAArtificial Sequence5 homology arm 19acctggccaa actgagcatg acctttgacc tagccagctc ttaaacttgt tctgagatca 60caaaccagcc agaccaaatt 802080DNAArtificial Sequence3 homology arm 20tcctcccaga atgcttccct gggtcaaacc cagagccaca aaggcttcca ttagaccatt 60ctggtaagtg acagagtcac 8021348DNAArtificial Sequence5 homology arm 21aaaaaaaaaa aagcccagct agcttagttg gtagagcatg agactcttaa tctcagagtc 60atgggttcag gcctcatgtt tggcaccatc tatagtgtgc agttataaat caaacagttc 120acgatggctg gctaggcact ggcaactgca gtctcacctg ctcccatggt tcccagttcc 180cacagctagt ttgctgccag gctgttcaca cttcccaggt catctaccca ctgtggcaag 240cctgcagaaa gcctgctatt gctagctcag ttcccttagc cctataaaat gataacacca 300cagactttta ctataccatc cagatttata acagtaattc tccaaccc 34822389DNAArtificial Sequence3 homology arm 22caggagtaca ccaaaatgtc tagccaaaat ttttatatat gatcacttaa ataagactcc 60ttaacataaa cctacatgat ataccaagtc ttttctgcca aggctctgac actatagttt 120gtcctatctg gagttaggta agcaaagggc tatttaggtg tggattgcaa agagagaata 180gcaagacaac ctgcccattc tttgccacac ctcactaatc agtgtccctt ggaaagcact 240gtaaatatgg aggtttcttt ttgtattatg tagtgtggat ttaacttgag gagccccaaa 300aggggtcagc aaagcatggg

aaatctaaga atttaacact tcagtgactt ttaatcacct 360acagatccag gaaaataagc ctgtctctt 3892321RNAartificialRNA oligo 23agacgugugc ucuuccgauc u 212421DNAartificialprimer 24gaagacattt gggaaggact g 212534DNAartificialprimer 25gtgactggag ttcagacgtg tgctcttccg atct 342655DNAartificialprimermisc_feature(34)..(35)n is a, c, g, or t 26acactctttc cctacacgac gctcttccga tctnngggaa ggactgactc tctga 55

Claims

1. A method for producing an antibody specific for a selected antigen, said antibody comprising an immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region, the method comprising isolating from a cultured cell expressing said IgH chain or fragment thereof comprising said human IgH chain variable region, one or more of the group consisting of: (i) said IgH chain or fragment thereof comprising said human IgH chain variable region; and (ii) nucleic acid encoding said IgH chain or fragment thereof comprising said human IgH chain variable region; wherein said human IgH chain variable region is of a transgenic mouse contacted with said selected antigen or epitope thereof, for a time and under conditions which permit the antigen-contacted mouse to produce antibody specific for said antigen; wherein said transgenic mouse comprises an IgH chain transgene, the IgH 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; the genome further comprising (i) a human VpreB gene capable of expressing a human VpreB, and (ii) a mouse or rat .lamda.5 gene; wherein the transgenic mouse is capable of expressing a chimaeric surrogate light chain comprising human VpreB and mouse or rat .lamda.5 for pairing with the heavy chain.

2. The method of claim 1, wherein the constant region is a mouse constant region.

3. The method of claim 1, wherein the constant region and the .lamda.5 gene are mouse constant region and mouse .lamda.5 gene, respectively.

4. The method of claim 2, wherein the transgenic mouse is a strain selected from the group consisting of a 129-derived mouse strain, a C57Bl/6 derived mouse strain and a JM8 derived mouse strain.

5. The method of claim 2, wherein the constant region is of a mouse 129 strain constant region or of a C57Bl/6 strain constant region, and the .lamda.5 gene is a mouse 129 strain or a mouse C57Bl/6 strain.

6. The method of claim 1, wherein the constant region gene and the .lamda.5 gene are endogenous genes of said transgenic mouse.

7. The method of claim 1, wherein the genome is homozygous for said transgene, human VpreB gene and mouse or rat .lamda.5 gene.

8. The method of claim 1, wherein endogenous antibody heavy chain expression in said mouse is inactive.

9. The method of claim 1, wherein endogenous antibody light chain expression in said mouse is inactive.

10. The method of claim 1, wherein the heavy chain transgene is devoid of a CHI gene segment and the genome comprises no functional antibody light chain locus.

11. The method of claim 1, wherein the heavy chain transgene is devoid of a gamma CHI gene segment and the genome comprises no functional antibody light chain locus.

12. The method of claim 1, wherein the heavy chain transgene is devoid of a mu CHI gene segment and the genome comprises no functional antibody light chain locus.

13. The method of claim 1, wherein the constant region is a mu constant region.

14. The method of claim 1, wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1.

15. The method of claim 1, wherein said genome does not comprise a non-human vertebrate species VpreB1 and/or VpreB2 gene.

16. The method of claim 1, wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene.

17. The method of claim 16, wherein non-functional V and/or J gene segments are omitted.

18. The method of claim 16, wherein the lambda V region or VJ region comprises a human VpreB gene and its associated promoter.

19. The method of claim 16, wherein the insertion is an insertion into an antibody light chain locus.

20. The method of claim 16, wherein the insertion replaces endogenous V.lamda. in whole or in part.

21. The method of claim 1, wherein the human VpreB gene is not within the endogenous VpreB-.lamda.5 locus.

22. The method of claim 1, wherein the expression of the human VpreB gene is under endogenous control.

23. The method of claim 1, wherein the human VpreB gene is operably linked to one or more DNaseI hypersensitive sites.

24. The method of claim 1, wherein the human VpreB gene is present in the genome in any one of 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies.

25. The method of claim 1, wherein all .lamda.5 sequences in the genome are mouse .lamda.5 sequences.

26. The method of claim 22, wherein the human VpreB gene is operably linked to an endogenous promoter.

27. The method of claim 1, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain variable gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

28. The method of claim 1, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

29. The method of claim 1, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of one or more or all heavy chain variable region gene segments selected from the group consisting of VH6-1, VH1-3, VH3-7, VH1-8, VH3-9, VH3-11, JH1 and JH6, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

30. The method of claim 27, wherein use of one or more or all of VH6-1, VH1-3, VH3-9 and JH1 is higher in the repertoire of said mouse than in the repertoire of the control.

31. The method of claim 27, wherein use of one or more or all of VH3-7, VH1-8, VH3-11 and JH6 is lower in the repertoire of said mouse than in the repertoire of the control.

32. The method of claim 4, wherein said transgenic mouse is a C57Bl/6--129/Sv hybrid strain.

33. The method of claim 13, wherein said mu constant region is an endogenous mu constant region.

34. The method of claim 16, wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and all the J regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene.

35. The method of claim 19, where the insertion is an insertion into an endogenous lambda locus upstream of the endogenous lambda constant region.

36. The method of claim 26, wherein said endogenous promoter is an endogenous VpreB promoter (e.g., a VpreB1 promoter).

37. The method of claim 28, wherein the transgenic mouse expresses a repertoire of Ig heavy chain variable regions that differs by a probability of less than 0.05 in a binomial distribution test, from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

38. The method of claim 1, wherein the genome of said transgenic mouse comprises (a) said antibody heavy chain transgene; and (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 endogenous antibody heavy and light chain expression is inactive.

39. The method of claim 38, wherein the genome of said transgenic mouse is homozygous for said heavy and light chain transgenes.

40. A method of promoting B cell development in a non-human vertebrate comprising an immunoglobulin heavy (IgH) chain comprising a human IgH chain variable region, wherein said non-human vertebrate is a mouse or a rat, wherein said non-human vertebrate comprises an IgH chain transgene, the IgH transgene comprising 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, optionally wherein the genome is homozygous for said IgH chain transgene; the method comprising: (i) inactivating endogenous heavy chain expression in said vertebrate; (ii) providing in the genome of said vertebrate: (a) a human VpreB gene capable of expressing a human VpreB, and (b) a mouse or rat .lamda.5 gene; wherein the transgenic mouse is capable of expressing a chimaeric surrogate light chain comprising human VpreB and mouse or rat .lamda.5, wherein the transgenic mouse is capable of expressing a pre-B-cell receptor comprising said chimaeric surrogate light chain and an IgH mu chain comprising said human variable region, wherein said pre-B-cell receptor promotes B cell development of cells bearing said IgH mu chain in said vertebrate.

41. The method of claim 40, wherein said non-human vertebrate comprising an IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain is a mouse constant region.

42. The method of claim 40, wherein said non-human vertebrate comprising an IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain and the .lamda.5 gene are mouse constant region and mouse .lamda.5 gene, respectively.

43. The method of claim 41, wherein said mouse is a strain selected from the group consisting of a 129-derived mouse strain, a C57Bl/6 derived mouse strain and a JM8 derived mouse strain.

44. The method of claim 41, wherein the constant region of said antibody heavy chain is of a mouse 129 strain constant region or of a C57Bl/6 strain constant region, and the .lamda.5 gene is a mouse 129 strain or a mouse C57Bl/6 strain.

45. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the constant region gene and the .lamda.5 gene are endogenous genes of said transgenic mouse.

46. The method of claim 40, wherein the genome is homozygous for said transgene, human VpreB gene and mouse or rat .lamda.5 gene.

47. The method of claim 1, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein endogenous antibody heavy chain expression in said mouse is inactive.

48. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein endogenous antibody light chain expression in said mouse is inactive.

49. The method of claim 40, wherein the heavy chain transgene is devoid of a CHI gene segment and the genome comprises no functional antibody light chain locus.

50. The method of claim 40, wherein the heavy chain transgene is devoid of a gamma CHI gene segment and the genome comprises no functional antibody light chain locus.

51. The method of claim 40, wherein the heavy chain transgene is devoid of a mu CHI gene segment and the genome comprises no functional antibody light chain locus.

52. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the constant region of said antibody heavy chain is a mouse mu constant region.

53. The method of claim 40, wherein the human VpreB gene has a nucleotide sequence that is at least 85% identical to SEQ ID NO:1.

54. The method of claim 40, wherein said genome does not comprise a non-human vertebrate species VpreB1 and/or VpreB2 gene.

55. The method of claim 40, wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene.

56. The method of claim 55, wherein non-functional V and/or J gene segments are omitted.

57. The method of claim 55, wherein the lambda V region or VJ region comprises a human VpreB gene and its associated promoter.

58. The method of claim 55, wherein the insertion is an insertion into an antibody light chain locus.

59. The method of claim 55, wherein the insertion replaces endogenous V.lamda. in whole or in part.

60. The method of claim 40, wherein the human VpreB gene is not within the endogenous VpreB-.lamda.5 locus.

61. The method of claim 40, wherein the expression of the human VpreB gene is under endogenous control.

62. The method of claim 40, wherein the human VpreB gene is operably linked to one or more DNaseI hypersensitive sites.

63. The method of claim 40, wherein the human VpreB gene is present in the genome in any one of 2, 3, 4, 5, 6, 7, 8 9 or 10 copies.

64. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein all .lamda.5 sequences in the genome are mouse .lamda.5 sequences.

65. The method of claim 61, wherein the human VpreB gene is operably linked to an endogenous promoter.

66. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the heavy chain variable region repertoire of a control mouse in the proportion expressed as a percentage of use of heavy chain variable gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

67. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

68. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, wherein said transgenic mouse expresses a repertoire of Ig heavy chain variable regions that significantly differs from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of one or more or all heavy chain variable region gene segments selected from the group consisting of VH6-1, VH1-3, VH3-7, VH1-8, VH3-9, VH3-11, JH1 and JH6, wherein the control and said transgenic mouse genomes are of the same background strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

69. The method of claim 66, wherein use of one or more or all of VH6-1, VH1-3, VH3-9 and JH1 is higher in the repertoire of said mouse than in the repertoire of the control.

70. The method of claim 66, wherein use of one or more or all of VH3-7, VH1-8, VH3-11 and JH6 is lower in the repertoire of said mouse than in the repertoire of the control.

71. The method of claim 43, wherein said transgenic mouse is a C57Bl/6-129/Sv hybrid strain.

72. The method of claim 52, wherein said mu constant region is an endogenous mu constant region.

73. The method of claim 55, wherein said genome comprises an insertion of human lambda V region or lambda VJ region comprising all of the V regions and all the J regions and intervening sequences, wherein the lambda V region or VJ region comprises a human VpreB gene.

74. The method of claim 58, where the insertion is an insertion into an endogenous lambda locus upstream of the endogenous lambda constant region.

75. The method of claim 65, wherein said endogenous promoter is an endogenous VpreB promoter (e.g., a VpreB1 promoter).

76. The method of claim 67, wherein the transgenic mouse expresses a repertoire of Ig heavy chain variable regions that differs by a probability of less than 0.05 in a binomial distribution test, from the IgH heavy chain variable region repertoire of a control vertebrate in the proportion expressed as a percentage of use of heavy chain JH gene segments, wherein the control and said transgenic mouse genomes are of the same background mouse strain and both comprise said antibody heavy chain transgene, the transgenes being identical in the control and said mouse, and wherein the control does not express a human VpreB.

77. The method of claim 40, wherein said non-human vertebrate comprising said IgH chain transgene is a mouse, and wherein the genome of said transgenic mouse comprises (a) said antibody heavy chain transgene; and (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 endogenous antibody heavy and light chain expression is inactive.

78. The method of claim 77, wherein the genome of said transgenic mouse is homozygous for said heavy and light chain transgenes.